Cell Signalling Biology Michael J. Berridge � Module 5 � OFF Mechanisms 5 �1

Module 5

OFF Mechanisms

Synopsis

Signalling pathways are composed of the ON mechanisms that generate internal signals and the OFFmechanism that remove these signals as cells recover from stimulation. Most attention will be focusedon how second messengers and their downstream effectors are inactivated. The second messengerscyclic AMP and cyclic GMP are inactivated by phosphodiesterase (PDE). Inositol trisphosphate (InsP3)metabolism is carried out by both inositol trisphosphatase and inositol phosphatases. Diacylglycerol(DAG) metabolism occurs through two enzyme systems, DAG kinase and DAG lipase.

In the case of Ca2 + signalling, recovery is carried out by the Ca2 + pumps and exchangers thatremove Ca2 + from the cytoplasm. The mitochondria also play an important role in Ca2 +

homoeostasis.Many of these second messengers activate downstream effectors through protein phosphorylation,

and these activation events are reversed by corresponding protein phosphatases.

Protein phosphatasesIt has been estimated that the human genome encodes ap-proximately 2000 protein kinases that phosphorylate anenormous number of intracellular proteins, many of whichfunction in cell signalling. There is an equally impressivearray of protein phosphatases that are responsible for re-moving these regulatory phospho groups. These proteinphosphatases can be divided into two main groups: theprotein tyrosine phosphatases (PTPs) and the protein ser-ine/threonine phosphatases.

Protein tyrosine phosphatases (PTPs)It has been estimated that tyrosine phosphorylation ac-counts for less than 0.1% of all the protein phosphoryla-tion in cells. Nevertheless, this small amount of phos-phorylation is critical because it is involved in some veryimportant signalling systems, and particularly those con-cerned with regulating cell growth and development. Thefact that the level of tyrosine phosphorylation increases10–20-fold when cells are stimulated by growth factorsor undergo oncogenic transformation highlights the im-portance of protein tyrosine phosphatases (PTPs) in sig-nal transduction. Protein tyrosine phosphatase structureand function reveals that these enzymes belong to a largeheterogeneous family that functions to dephosphorylatephosphotyrosine residues with a high degree of spatialand temporal precision. The PTP superfamily can be di-vided into classical protein tyrosine phosphatases anddual-specificity phosphatases (DSPs).

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Please cite as Berridge, M.J. (2012) Cell Signalling Biology;doi:10.1042/csb0001005

Protein tyrosine phosphatase structure and functionThe protein tyrosine phosphatase (PTP) superfamily is aheterogeneous group of enzymes with widely divergentstructures (Module 5: Figure tyrosine phosphatase super-family). They can be divided into the classical PTPs andthe dual-specificity phosphatases (DSPs). The former canbe divided further into the non-transmembrane PTPs andthe receptor-type PTPs. What all the phosphatases have incommon is a signature motif (H-C-X-X-G-X-X-R) loc-ated in the PTP domain that is responsible for its catalyticactivity. The different structural elements [e.g. Src homo-logy 2 (SH2), PDZ and immunoglobulin-like domains]that flank this PTP domain function to regulate enzymeactivity and to position the enzyme in the right locationnear its specific substrates. These structural elements aredescribed in more detail when the individual enzymes aredescribed.

All PTPs utilize the same catalytic mechanism duringwhich the phosphate on the substrate is first transferred tothe cysteine residue in the signature motif before being hy-drolysed by water to release the phosphate anion (Module5: Figure tyrosine phosphatase catalysis). This role of thecysteine residue in the phosphyltransfer reaction is an ex-ample of one of the oxidation-sensitive processes that istargeted by the redox signalling . Some of the reactive oxy-gen species (ROS) messenger functions depend upon thisinhibition of PTPs (Module 2: Figure ROS formation andaction).

These families of enzymes that hydrolyse phosphotyr-osine residues have a critical cysteine residue in the act-ive site, which is particularly susceptible to oxidation.During redox signalling, this cysteine is oxidized, res-ulting in a decrease in the activity of the PTPs. Sincethe latter are normally expressed in great excess over the

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Cell Signalling Biology Michael J. Berridge � Module 5 � OFF Mechanisms 5 �2

Module 5: Figure tyrosine phosphatase superfamily

Summary of the protein tyrosine phosphatase (PTP) superfamily.There is a very large family of protein tyrosine phosphatases (PTPs). Some are located in the cytoplasm, whereas others are so called receptor-typePTPs. See the text for further details. Reproduced from Curr. Opin. Cell Biol., Vol. 13, Tonks, N.K. and Neel, B.G., Combinatorial control of the specificityof protein tyrosine phosphatases, pp. 182–195. Copyright (2001), with permission from Elsevier; see Tonks and Neel 2001.

corresponding kinases, an oxidation-induced inhibition ofphosphatase activity would greatly enhance the flow ofinformation down those signalling cascades that rely ontyrosine phosphorylation, such as the MAP kinase sig-nalling pathway and the Ca2 + signalling pathway. In thecase of the latter, a positive-feedback mechanism operatesbetween the ROS and Ca2 + signalling systems (Module 2:Figure ROS effects on Ca2 + signalling).

Classical protein tyrosine phosphatasesThe classical protein tyrosine phosphatases are composedof two main groups, the non-transmembrane protein tyr-osine phosphatases and the receptor-type protein tyrosinephosphatases (Module 5: Figure tyrosine phosphatase su-perfamily).

Non-transmembrane protein tyrosine phosphatasesThe non-transmembrane protein tyrosine phosphatases(PTPs) are a heterogeneous family that share similar PTPdomains, but have additional elements that determine boththeir location and their function within the cell. Thefollowing are some of the major members of the non-transmembrane PTPs:

• Protein tyrosine phosphatase 1B (PTP1B)• T cell protein tyrosine phosphatase (TC-PTP)• Src homology 2 (SH2) domain-containing protein tyr-

osine phosphatase-1 (SHP-1)• Src homology 2 (SH2) domain-containing protein tyr-

osine phosphatase-2 (SHP-2)

Protein tyrosine phosphatase 1B (PTP1B)Protein tyrosine phosphatase 1B (PTP1B) has a typicalprotein tyrosine phosphatase (PTP) domain at the N-terminus and a regulatory region at the C-terminus. Thelatter contains a hydrophobic region that targets the en-zyme to the cytoplasmic surface of the endoplasmic retic-ulum (ER). Despite this localization to the ER, some ofthe main substrates of PTP1B are the tyrosine kinase re-ceptors, e.g. the epidermal growth factor (EGF) receptorand insulin receptor, and the non-receptor tyrosine kinasec-Src. PTP1B also acts on components of the JAK/STATsignalling pathway, such as STAT5a and STAT5b.

PTP1B plays a role in stabilizing cadherin complexesby dephosphorylating the phosphotyrosine residues onβ-catenin. In order to bind to cadherin, PTP1B must bephosphorylated on Tyr-152 by the non-receptor proteintyrosine kinase Fer (Module 6: Figure classical cadherinsignalling).

T cell protein tyrosine phosphatase (TC-PTP)T cell protein tyrosine phosphatase (TC-PTP) has a sim-ilar structure to PTP1B, but operates on a different set ofsubstrates. It exists as two alternatively spliced forms thatdiffer with regard to the structure of the C-terminus. TC-48 has a hydrophobic domain resembling that of PTP1Band is similarly located in the endoplasmic reticulum (ER).On the other hand, TC-45 lacks the hydrophobic residuebut has a nuclear localization signal (NLS) that directs itinto the nucleus. When cells are stimulated with epidermalgrowth factor (EGF), the TC-45 leaves the nucleus and

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Cell Signalling Biology Michael J. Berridge � Module 5 � OFF Mechanisms 5 �3

Module 5: Figure tyrosine phosphatase catalysis

The catalytic mechanism of protein tyrosine phosphatases.The signature motif, located at the bottom of a deep catalytic cleft, con-tains three important residues (cysteine, aspartate and glycine) that arenecessary for the catalytic process. A. The peptide containing the phos-photyrosine (pTyr) residue enters the cleft where the cysteine residueinitiates a nucleophilic attack. The aspartate residue has a critical role inprotonating the phenolate leaving group in the substrate. B. Once thephosphate has been transferred to the cysteinyl group, the substrateleaves the enzyme, and the final step is to hydrolyse the phosphate.The co-ordination of a water molecule to the glycine residue favours thehydrolysis of the phosphoryl residue. The nucleophilicity of the watermolecule is increased by the abstraction of a proton by the aspartateresidue. Once the phosphate has been removed, the active site is readyto hydrolyse another phosphotyrosine residue. Reproduced from Pan-nifer, A.D.B., Flint, A.J., Tonks, N.K. and Barford, D. (1998) Visualisation ofthe cysteinyl-phosphate intermediate of a protein-tyrosine phosphataseby X-ray crystallography. J. Biol. Chem. 273:10454–10462, with permis-sion from the American Society for Biochemistry and Molecular Biology;see Pannifer et al. 1998.

interacts with the EGF receptor complex, where one of itstargets appears to be Shc.

Src homology 2 (SH2) domain-containing proteintyrosine phosphatase-1 (SHP-1)As their name implies, the Src homology 2 (SH2) domain-containing protein tyrosine phosphatases (SHPs) have twoN-terminal SH2 domains (Module 6: Figure modular pro-tein domains). There are two SHPs (SHP-1 and SHP-2),which have similar structures (Module 5: Figure structureof the SHPs). These SHPs must not be confused with theSH2 domain-containing inositol phosphatases (SHIPs),which form a subgroup of the Type II inositol poly-phosphate 5-phosphatases, even though these two typesof phosphatases often end up exerting very similar effectson cells.

Even though SHP-1 and SHP-2 are highly related struc-turally, they have very different functions. The primaryfunction of SHP-1 is to inhibit signalling pathways that usetyrosine phosphorylation to transmit information. Many

of its actions are directed against signalling systems inhaematopoietic cells. It attaches itself to the signalling com-plexes via its SH2 domains, thereby enabling the proteintyrosine phosphatase (PTP) domain to dephosphorylatethe phosphotyrosine residues involved in the process ofsignal transduction. Alternatively, SHP-1 is drawn intothese signalling complexes through an attachment to vari-ous inhibitory receptors, particularly those that act to in-hibit antigen and integrin receptor signalling. For example,SHP-1 is associated with the FcγRIII receptors that inhibitthe FcεRI receptors in mast cells (Module 11: Figure mastcell inhibitory signalling).

SHP-1 participates in an important feedback loop thatexists between the reactive oxygen species (ROS) andCa2 + signalling pathways (Module 2: Figure ROS effectson Ca2 + signalling).

Src homology 2 (SH2) domain-containing proteintyrosine phosphatase-2 (SHP-2)Even though Src homology 2 (SH2) domain-containingprotein tyrosine phosphatase-2 (SHP-2) has a close struc-tural resemblance to its related family member SHP-1, ithas a very different function. Instead of exerting an inhib-itory effect, it usually has a positive effect on the activityof various growth factor receptors such as the epidermalgrowth factor (EGF), fibroblast growth factor (FGF), in-sulin, and perhaps also the platelet-derived growth factor(PDGF) and integrin receptors.

Receptor-type protein tyrosine phosphatasesReceptor-type protein tyrosine phosphatase (RPTPs) havea transmembrane domain that retains them within theplasma membrane. Even though these enzymes are de-scribed as receptor-type, the nature of the ligand is poorlydefined. Many of them have features of cell adhesion mo-lecules and may thus be activated by cell-surface moleculesembedded in neighbouring cells. This seems to be the casefor RPTPμ and RPTPκ, which form homophilic inter-actions as they bind to identical molecules on opposingcells. The following are some of the major members of theRPTPs:

• CD45• Protein tyrosine phosphatase α (PTPα)• Leucocyte common antigen-related (LAR)

CD45CD45 is a typical transmembrane protein tyrosine phos-phatase (PTP) (Module 5: Figure tyrosine phosphatase su-perfamily). It has a highly glycosylated extracellular do-main, and the cytoplasmic region has two PTP domains,but the second is catalytically inactive. CD45 has a criticalfunction in T cell signalling, where it contributes early inthe signalling cascade by activating Lck, which is a T-cellreceptor transducer (Module 9: Figure TCR signalling).It acts by dephosphorylating the phosphate on Tyr-505,which opens up the molecular structure of Lck so that itcan begin to phosphorylate ζ-associated protein of 70 kDa(ZAP-70). Similarly, CD45 functions in B-cell antigen re-ceptor (BCR) activation by stimulating Lyn (Module 9:Figure B-cell activation).

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Cell Signalling Biology Michael J. Berridge � Module 5 � OFF Mechanisms 5 �4

Module 5: Figure structure of the SHPs

SH2 SH2

SH2 SH2

PTP PTP

Y Y

Y Y

SHP-1 SHP-2

Prolyl-richdomain

Structural organization of the Src homology 2 (SH2)-domain-containing protein tyrosine phosphatases (SHP-1 and SHP-2).The two mammalian Src homology 2 (SH2) domain-containing protein tyrosine phosphatases (SHPs) have very similar structures. The main featuresare the PTP domain and the two N-terminal SH2 domains. The C-terminal region has two tyrosine (Y) residues, which in the case of SHP-2 areseparated by a prolyl-rich domain.

Protein tyrosine phosphatase α (PTPα)Protein tyrosine phosphatase α (PTPα) functions in theactivation of the non-receptor Src family, where it removesthe inhibitory phosphotyrosine residue.

Leucocyte common antigen-related (LAR)The leucocyte common antigen-related (LAR) protein tyr-osine phosphatase (PTP) has a number of specific devel-opmental functions, such as a role in the terminal differ-entiation of alveoli in the mammary gland, as well as indevelopment within the forebrain and hippocampus.

Dual-specificity phosphatases (DSPs)As their name implies, these dual-specificity phos-phatases (DSPs) are unusual in that they can de-phosphorylate both phosphotyrosine (pTyr) and phos-phoserine/phosphothreonine (pSer/pThr) residues. Thefollowing are some of the major members of the dual-specificity phosphatase family:

• Cdc25• Mitogen-activated protein kinase (MAPK) phos-

phatases (MKPs)

Mitogen-activated protein kinase (MAPK) phosphatases(MKPs)The family of mitogen-activated protein kinase (MAPK)phosphatases (MKPs) contains ten members (Module 2:Table MAPK signalling toolkit) that have specific func-tions in reversing the phosphorylation events responsiblefor the MAP kinase signalling pathway. One of the last

events of this signalling cascade is the phosphorylation ofthe MAPKs by the dual-specificity MAPK kinases, whichadd phosphates to both tyrosine and threonine residues.During the recovery phase, these phosphates are removedby the MAPK phosphatases (Module 5: Figure dual-spe-cificity MKP).

Some of the MAPK phosphatases are expressed con-stitutively, whereas others are actively induced when cellsare stimulated, thus setting up a negative-feedback loop.An example of such a negative-feedback loop is evidentfor the extracellular-signal-regulated kinase (ERK) sig-nalling pathway (Module 2: Figure ERK signalling). An-other characteristic of these phosphatases is that they areoften highly specific for particular targets. A good exampleof this specificity is illustrated by MAPK phosphatase-3 (MKP-3), which acts specifically to dephosphorylateERK2.

Certain neurons, such as the medium spiny neuronsin the striatum, express a striatal-enriched protein tyr-osine phosphatase (STEP), which plays a highly specificrole in regulating the neuronal MAPK signalling path-way (Module 10: Figure medium spiny neuron signalling).In response to N-methyl-aspartate (NMDA) stimulation,the increase in Ca2 + acts on calcineurin (CaN) to dephos-phorylate and activate STEP, which then limits the dura-tion of phospho-ERK signalling. By contrast, elevationsin Ca2 + induced by voltage-operated channels (VOCs)or the release of internal Ca2 + have no effect, indicat-ing a tight association between NMDA receptors andSTEP.

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Cell Signalling Biology Michael J. Berridge � Module 5 � OFF Mechanisms 5 �5

Module 5: Figure dual-specificity MKP

TY

RESPONSE

Erk2

MKP-3PTP

KinaseTY

P

PKinase

TYKinase

PTPKIM

KIM

Dual specific MAPK kinase

MAPK SIGNALLING PATHWAY

P

P

P

P

Mode of action of dual-specificity mitogen-activated protein kinase (MAPK) phosphatase.Extracellular-signal-regulated kinase 2 (RK2), which is one of the main components of mitogen-activated protein kinase (MAPK) signalling, isphosphorylated on threonine (T) and tyrosine (Y) by MEK1/2, a dual-specificity MAPK kinase (Module 2: Figure ERK signalling). These phosphorylationevents are reversed by MAPK phosphatase-3 (MKP-3). The specificity of the interaction between ERK2 and MKP-3 depends upon the latter havinga kinase interaction motif (KIM) that binds to a specific site on ERK. This interaction enables the protein tyrosine phosphatase (PTP) domain todephosphorylate the two phosphorylated residues on ERK2, thus curtailing its ability to stimulate downstream responses.

Cdc25The human genome contains three CDC25 dual-specificity enzymes (Cdc25A, Cdc25B and Cdc25C)(Module 9: Table cell cycle toolkit). This enzyme wasfirst described as a regulator of the cell cycle in studieson yeast cells, and still retains its yeast nomenclature. Thethree human isoforms also act to regulate the cell cycleby controlling both the entry into S phase (Cdc25A) andthe entry into mitosis (Cdc25B and C) (Module 9: Figurecell cycle signalling mechanisms). The level of Cdc25A in-creases in late G1 and remains high throughout the rest ofthe cell cycle. The level of Cdc25B is increased during Sphase to activate the entry into mitosis, and returns to alow level after mitosis is complete. The level of Cdc25Cremains high throughout the cell cycle. All three isoformshave a similar C-terminal catalytic region, whereas the N-terminus, which has the regulatory regions, is somewhatvariable. The activity of the Cdc25 isoforms is regulated byboth activating and inhibitory phosphorylation. All threeisoforms contain a phosphorylation site, which controlsthe binding of 14-3-3 protein that then inhibits the en-zyme. This inhibitory site is phosphorylated by enzymesthat are activated by cell stress, such as DNA damage. Thisstress-induced inhibition of the Cdc25 isoforms is thus animportant mechanism for both G1 and G2/M cell cyclearrest.

The expression of Cdc25A is controlled by E2F. OnceCdc25A is expressed in the cytoplasm, it is available toactivate cyclin-dependent kinase 2 (CDK2) to initiate theprocess of DNA synthesis. The activity of Cdc25A is verysensitive to DNA damage, which activates the checkpointkinases 1 and 2 (CHK1 and CHK2) to phosphorylateSer-123, which then promotes ubiquitination and rapiddegradation. CHK1 is also responsible for phosphorylat-ing Thr-507, which facilitates its interaction with 14-3-3 protein, which keeps the enzyme inactive until it isrequired.

Cdc25B, which plays an important role in the way cyclinB controls mitosis, is activated at the G2/M transition(Module 9: Figure mitotic entry). Like the other Cdc25isoforms, Cdc25B is kept quiescent by phosphorylatingSer-323 that provides a binding site for 14-3-3 protein. Thissite is phosphorylated by the p38 pathway and providesa mechanism whereby this component of the mitogen-activated protein kinase (MAPK) signalling pathway canarrest the cell cycle (Module 2: Figure MAPK signalling).

The Cdc25C enzyme is kept quiescent through phos-phorylation of Ser-216, which provides a binding sitefor 14-3-3 protein. During entry into mitosis, this in-hibitory phosphate is removed and this enables thePolo-like kinases (Plks) to phosphorylate other sites inthe regulatory region that enables Cdc25C to begin to

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Cell Signalling Biology Michael J. Berridge � Module 5 � OFF Mechanisms 5 �6

dephosphorylate CDK1-activating kinases (Module 9:Figure cell cycle signalling mechanisms).

Protein serine/threonine phosphatasesThere are a very large number of kinases that contributeto the ON reactions of cell signalling by phosphorylat-ing both serine and threonine residues on target pro-teins. By contrast, there is a relatively small group of pro-tein serine/threonine phosphatases that remove these ser-ine and threonine phosphates, thus reversing the activ-ity of the kinases as part of the OFF reaction. Theserine/threonine phosphatase classification reveals thatmost of these kinases belong to either the phosphoproteinphosphatase (PPP) family or the Mg2 + -dependent proteinphosphatase (PPM) family.

Serine/threonine phosphatase classificationThere are two major families of serine/threonine phos-phatases (Module 5: Table serine/threonine phosphatasesclassification). With regard to signalling, the followingmembers of the PPP family are particularly abundant andimportant with regard to cell signalling:

• Protein phosphatase 1 (PP1)• Protein phosphatase 2A (PP2A)• Protein phosphatase 2B (PP2B)• Pleckstrin homology domain leucine-rich repeats pro-

tein phosphatase (PHLPP)

Protein phosphatase 1 (PP1)Three genes code for the protein phosphatase 1 (PP1) cata-lytic subunit (PP1C), which give rise to four isoforms(PP1α, PP1β, PP1γ1 and PP1γ2). Despite this limitednumber of catalytic subunits, PP1 performs a large numberof functions operating in many different cellular locations.It owes this versatility to the fact that it can interact witha large number of regulatory and inhibitory proteins. Theactivity of PP1 is inhibited by inhibitor 1 (I-1). The func-tion of the PP1 regulatory/targeting and inhibitory pro-teins are summarized in Module 5: Table PP1 regulatoryand inhibitory subunits and proteins.

The regulatory subunits determine the substrate spe-cificity and variable intracellular locations of PP1, whichfunctions in the control of many cellular processes:

• The myosin phosphatase targeting subunit 1 (MYPT1)functions to localize PP1 to the myosin filaments inthe contractile ring that controls cytokinesis during celldivision (Module 9: Figure cytokinesis).

• PP1 plays an important role in the control of glyco-gen metabolism. In liver cells, GL targets PP1 to gly-cogen, where it functions to dephosphorylate glycogensynthase and phosphorylase (Module 7: Figure glyco-genolysis and gluconeogenesis). The glycogen-targetingsubunit GM in skeletal muscle also directs PP1 to the sur-face of glycogen granules, where it has a similar function.In skeletal muscle, it also targets PP1 to the sarcoplasmicreticulum (Module 5: Figure PP1 targeting to glycogen),where it functions to dephosphorylate the sarco/endo-plasmic reticulum Ca2 + -ATPase (SERCA) pump inhib-

Module 5: Table serine/threonine phosphatase classificationClassification of the protein serine/threonine phosphatasesPhosphatase CommentPPP family

PP1 (protein phosphatase 1) There are three PP1 genes thatgive rise to four isoforms; PP1has multiple regulatorycomponents Module 5: TablePP1 regulatory, targeting andinhibitory subunits andproteins

PP2A (protein phosphatase2A)

An abundant and ubiquitousphosphatase that has multiplescaffolding and regulatorysubunits (Module 5: FigurePP2A holoenzyme)

PP2B (calcineurin) A Ca2 + -sensitive proteinphosphatase (Module 4:Figure calcineurin)

PP4 (protein phosphatase 4) May function in nuclear factor κB(NF-κB) signalling and histonedeacetylase 3 (HDAC3)dephosphorylation

PP5 (protein phosphatase 5) May function in control of cellgrowth

PP6 (protein phosphatase 6) May function in G1/S transition ofcell cycle

PP7 (protein phosphatase 7) Located in retinal and brainPPM family The PPM family contains

approximately nine humangenes; little is known aboutmost of these enzymes exceptfor PP2C and Ppm2

PP2C (Pmp1) Prototypic member of the PPMfamily; implicated indephosphorylation ofcyclin-dependent kinases(CDKs), regulation of RNAsplicing and control of p53activity

Ppm2 Pyruvate dehydrogenasephosphatase

Pleckstrin homology domainleucine rich repeats proteinphosphatase (PHLPP)

PHLPP1PHLPP2

The protein serine/threonine phosphatases are divided into twomain families, the main phosphoprotein phosphatase (PPP)family and the smaller Mg2 + -dependent protein phosphatase(PPM) family.

itor phospholamban (Module 5: Figure phospholambanmode of action).

• PP1 controls smooth muscle relaxation (Module 7: Fig-ure smooth muscle cell E-C coupling).

• Activity of the striatal-enriched phosphatase (STEP),which dephosphorylates ERK in neurons, is regulatedby PP1 (Module 10: Figure medium spiny neuron sig-nalling).

• PP1 contributes to the regulation of the Na+ -K+ -2Cl−

cotransporter 1 (NKCC1) (Module 3: Figure cationchloride cotransporter).

• Cell volume regulation in response to hypotonicity de-pends upon the dephosphorylation of the K+ -Cl− co-transporter 1 (KCC1) the by PP1 (Module 3: Figure cellvolume regulation).

• The phosphorylation of AMPA receptors is regulatedby PP1 and inhibitor 1 (I1) (Module 3: Figure AMPAreceptor phosphorylation).

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Cell Signalling Biology Michael J. Berridge � Module 5 � OFF Mechanisms 5 �7

Module 5: Figure PP1 targeting to glycogen

P

P

PP

P

PPP

PP1

PP1 PP1

G in skeletalmuscle

G in liverM

L

PP1

Synthase

Phospholamban

Sarcoplasmicreticulum

Glycogensynthesis

UDPglucose

Glucose-1-P

Glycogen

Phosphorylase

Glycogenolysis

Function of the regulatory proteins GM and GL in targeting protein phosphatase 1 (PP1) to both glycogen and the sarcoplasmic reticulum.The regulatory protein GM in skeletal muscle or GL in liver has two targeting domains. One is located in the middle of the molecule (shown ingreen) that directs protein phosphatase 1 (PP1) to glycogen, where it can dephosphorylate the enzymes glycogen synthase and phosphorylasethat control glycogen synthesis and glycogenolysis respectively. The other is a transmembrane region (shown in blue) in the C-terminal region thatdirects PP1 towards the sarcoplasmic reticulum, where it acts to dephosphorylate phospholamban, which functions to regulate the activity of thesarco/endo-plasmic reticulum Ca2 + -ATPase 2 (SERCA2a) pump (Module 5: Figure phospholamban mode of action).

Dopamine and cyclic AMP-regulated phosphoprotein ofapparent molecular mass 32 kDa (DARPP-32)DARPP-32 is a dopamine and cAMP-regulatedphosphoprotein of apparent molecular mass 32 kDa, whichfunctions as a molecular switch to regulate the activity ofprotein phosphatase 1 (PP1). As its name implies, it is regu-lated by protein kinase A (PKA)-dependent phosphoryla-tion and is localized in dopamine-sensitive neurons suchas the medium spiny neurons found in the dorsal stri-atum and nucleus accumbens. DARPP-32 may functionas a node to co-ordinate the activity of the dopamine andglutamate signalling pathways (Module 10: Figure mediumspiny neuron signalling). This integration of two separateneural signalling pathways may underlie the neural plasti-city that occurs during drug addiction.

Protein phosphatase 2A (PP2A)Protein phosphatase 2A (PP2A) is one of the most abund-ant of the serine/threonine protein phosphatases: it is es-timated to make up about 0.3% of total cellular protein.It is a highly versatile enzyme in that it can operate inmany different cellular regions. This versatility dependsupon the protein phosphatase 2A (PP2A) holoenzyme or-ganization, which is a trimeric structure consisting of thescaffolding protein A subunit of protein phosphatase 2A,a regulatory B subunit and a catalytic C subunit (Module5: Figure PP2A holoenzyme). There are a large numberof regulatory B subunits, which are responsible for direct-ing the holoenzyme to different cellular locations. Protein

phosphatase 2A (PP2A) function depends primarily onits role in reversing the phosphorylation events that arepart of many signalling pathways, and particularly thosecontrolling processes such as development, differentiation,morphogenesis and cell proliferation. Its inhibitory role incell proliferation has led to its classification as a tumoursuppressor.

A mutation arising from expansion of a CAG trinuc-leotide repeat of the Bβ gene (Module 5: Table PP2A sub-units) is the cause of autosomal dominant spinocerebellarataxia type 12 (SCA12). A role for PP2A in cancer hasemerged from the relationship between protein phos-phatase 2A (PP2A) and tumour suppression.

Protein phosphatase 2A (PP2A) holoenzymeorganizationProtein phosphatase 2A (PP2A) is a highly versatile en-zyme that dephosphorylates a diverse array of proteinslocated in many different cellular locations. It owes thisversatility to a large family of regulatory B proteins, whichare part of the PP2A molecular toolkit (Module 5: TablePP2A subunits). The holoenzyme is a trimer composed ofa PP2A scaffolding A subunit, which binds to a regulat-ory B subunit and a catalytic C subunit (Module 5: FigurePP2A holoenzyme). Given that there are two A subunits,two C subunits and at least 13 B subunits, many com-binations are possible, resulting in multiple heterotrimericholoenzymes. Much of the versatility of this enzyme de-pends on the large number of B regulatory subunits thathave subtly different properties, especially with regard to

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Cell Signalling Biology Michael J. Berridge � Module 5 � OFF Mechanisms 5 �8

Module 5: Table PP1 regulatory, targeting and inhibitory sub-units and proteinsThe regulatory, targeting and inhibitory subunits and proteins of pro-tein phosphatase 1 (PP1).Subunit or protein Cellular location and functionPP1 regulatory and targeting

subunits and proteinsGlycogen targeting

GM Directs protein phosphatase 1(PP1) to glycogen particles inskeletal and heart muscle;controls glycogen metabolism(Module 5: Figure PP1targeting to glycogen)

GL Directs PP1 to glycogen particlesin liver; controls glycogenmetabolism; distributed widely,but high in liver and muscle

R5 Also known as protein targeting toglycogen (PTG) (Module 6:Figure glycogen scaffold).

Myosin/actin targetingMYPT1 (myosin

phosphatase targetingsubunit)

Directs PP1 to myofibrils insmooth muscle cells andnon-muscle cells; also knownas myosin-binding subunit(MBS); controls smoothmuscle relaxation (Module 7:Figure smooth muscle cell E-Ccoupling)

MYPT2 Directs PP1 to myofibrils inskeletal muscle, where itcontrols contraction; alsofound in heart and brain.

Plasma membrane andcytoskeleton targetingNeurabin I Neuronal plasma membrane and

actin cytoskeleton; functions inneurite outgrowth and synapsemorphology

Spinophilin (Neurabin II) Widespread location on plasmamembrane and actin; attachesPP1 to ryanodine receptors(Module 3: Figure ryanodinereceptor structure)

A-kinase-anchoringprotein 220 (AKAP220)

Brain and testis, where it islocated on cytoskeleton toco-ordinate protein kinase A(PKA) and PP1 signalling.

Yotiao (a splice variant ofAKAP350)

Located in the neuronalpostsynaptic density (Module10: Figure postsynapticdensity), where it modulatessynaptic transmission

PP1 inhibitory proteinsI-1 (Inhibitor 1) This inhibitor of PP1 is widely

distributedI-2 (Inhibitor 2)DARPP-32 (dopamine and

cAMP-regulatedphosphoprotein ofapparent molecular mass32 kDa)

This inhibitor of PP1 is found inbrain and kidney

The function of PP1 is determined by its associated proteins thatregulate its activity and are responsible for targeting it to itsspecific sites of action. (Information reproduced and adaptedfrom Cohen 2002.)

their ability to direct holoenzymes to different cellular re-gions and substrates. Some of the roles of the B subunit indetermining PP2A function are summarized in Module 5:Table PP2A subunits, but many of the targeting functionsare still being elucidated.

Module 5: Table PP2A subunitsSubunit composition of protein phosphatase 2A (PP2A).PP2A subunits Cellular location and functionPP2A scaffolding A subunits

Aα

Aβ

PP2A regulatory BsubunitsB family

Bα Neuronal cell bodies and nucleus;linked to microtubules and is atau phosphatase

Bβ Abundant in brain and testis; inbrain, it is in the cell body(excluding the nucleus) andextends into axons anddendrites; linked to microtubulesand is a tau phosphatase;mutated in spinocerebellar ataxiatype 12 (SCA12)

Bγ Abundant in brain and testisBδ

B’ familyB’α Skeletal muscle and cardiac cells;

targets PP2A to the apoptoticprotein Bcl-2

B’β BrainB’γ Skeletal muscle and cardiac cells;

directs PP2A to L-type CaV1.2channel to reverse protein kinaseA (PKA)-dependentphosphorylation (Module 3:Figure CaV1.2 L-type channel)

B’δ BrainB’ε Brain and testis

B’’ familyPR48 Located in the nucleus, where it

interacts with Cdc6 in thepre-replication complexes duringDNA synthesis

PR59 Interacts with p107, aretinoblastoma (Rb)-relatedprotein that can arrest the cellcycle by dephosphorylating thetranscription factor E2F

PR72PR130 Directs PP2A to the signalling

complex assembled onA-kinase-anchoring protein 350(AKAP350) localized on thecentrosome; PR130 links PP2Ato the ryanodine receptor(Module 3: Figure ryanodinereceptor structure)

PP2A catalytic subunitsCα

Cβ

A large number of genes are used to encode the scaffolding,regulatory and catalytic subunits that are used to make up thediverse array of protein phosphatase 2A (PP2A) holoenzymes(Module 5: Figure PP2A holoenzyme).

Protein phosphatase 2A (PP2A) functionThe primary role of protein phosphatase 2A (PP2A) is todephosphorylate many of the phosphoproteins that func-tion in cell signalling pathways:

• PP2A can modulate the MAP kinase signalling path-way both positively and negatively. With regard to theformer, it can dephosphorylate some of the inhibit-ory sites on Raf-1. In addition, it can inhibit the sig-nalling cascade by reversing some of the phosphoryla-tion events downstream of Raf-1 (Module 2: FigureERK signalling).

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Cell Signalling Biology Michael J. Berridge � Module 5 � OFF Mechanisms 5 �9

Module 5: Figure PP2A holoenzyme

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Assembly of the protein phosphatase 2A (PP2A) holoenzyme.The protein phosphatase 2A (PP2A) holoenzyme is assembled from three subunits that have different functions. The molecular framework is providedby the scaffolding subunit (A), which is made up of 15 non-identical repeats, which are organized into a hook-shaped molecule. These repeats areconnected by inter-repeat loops (shown in blue). Each repeat has two α-helices that are connected by intra-repeat loops (shown in orange), whichline up to provide a cradle to bind the other subunits. Loops 1–10 are responsible for binding one of the regulatory B subunits, which belong tothree families (B, B’ and B’’). There are two PP2A catalytic subunits (Cα and Cβ) and one of these attaches to loops 11–15. This recruitment of thecatalytic subunit into the holoenzyme depends upon carboxymethylation of Leu-309 by leucine carboxmethyltransferase (LCMT) and is reversed by aphosphatase methylesterase (PME-1). Once assembled, the holoenzyme functions to dephosphorylate a wide range of phosphorylated substrates.

• Some of the key phosphorylation events of the canonicalWnt/β-catenin pathway are reversed by PP2A (Module2: Figure Wnt canonical pathway).

• Protein kinase A (PKA)-dependent phosphorylation ofthe L-type CaV1.2 channel is reversed by PP2A (Module3: Figure CaV1.2 L-type channel).

• PKA-dependent phosphorylation of the type 2 ryanod-ine receptor (RYR2) in cardiac cells is reversed by PP2A(Module 3: Figure ryanodine receptor structure).

• PP2A interacts with the scaffolding protein A-kinase-anchoring protein 79 (AKAP79), which is associatedwith synapse-associated protein 97 (SAP97), to comeinto close contact with the GluR1 subunit of the AMPAreceptor (Module 10: Figure postsynaptic density).

• The phosphorylation status of the neuron-specificmicrotubule-associated protein tau, which has beenimplicated in Alzheimer’s disease, is regulated by thePP2A holoenzyme carrying the Bβ regulatory subunit(Module 5: Table PP2A subunits).

• PP2A functions in Myc degradation.

Protein phosphatase 2A (PP2A) and tumour suppressionOne of the important actions of protein phosphatase 2A(PP2A) is to regulate cell proliferation, where it normallyacts to reverse the protein phosphorylation of the prolifer-

ation signalling pathways driven by various growth factors(Module 9: Figure proliferation signalling network). Forexample, PP2A contributes to Myc degradation, which isan important regulator of cell proliferation and is oftenamplified in many human cancers. Modifications of PP2Aeither through mutation of its subunits or by interactionswith viral proteins can cause cancer (Module 5: FigurePP2A modifications and cancer). The negative effects oncell growth have led to the concept of PP2A functioning asa tumour suppressor. Some of the most convincing evid-ence for this comes from the finding that simian virus 40(SV40) small T antigen and polyoma virus small T andmiddle T antigens bind to the scaffolding A subunit, res-ulting in a decrease in phosphatase activity.

Protein phosphatase 2B (PP2B)Protein phosphatase 2B (PP2B) is known more com-monly as calcineurin (CaN), which is a Ca2 + -activated ser-ine/threonine phosphatase (Module 4: Figure calcineurin).

Protein phosphatase 4 (PP4)Not much is known about protein phosphatase 4 (PP4).Like the other serine/threonine phosphatases, PP4 is madeup of a catalytic subunit (PP4C) that interacts with variousregulatory subunits (R1, R2, R3 and α4). In addition, it caninteract with signalling proteins such as nuclear factor κB

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Cell Signalling Biology Michael J. Berridge � Module 5 � OFF Mechanisms 5 �10

Module 5: Figure PP2A modifications and cancer

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Modifications of protein phosphatase 2A (PP2A) by mutations and interactions with viral proteins can cause cancer.Protein phosphatase 2A (PP2A) is considered to be a tumour suppressor because cancers can develop or be exacerbated when the activity of thisenzyme is reduced either by mutations of the subunits or by interactions with viral proteins:

1. Mutations of the scaffolding A subunit, which then fails to bind the B and C subunits, have been identified in a number of human cancers (breast,colon, lung and skin).

2. Truncation of the B subunit, which prevents it from interacting with the catalytic C subunit, has been implicated in metastasis.3. Tumour-promoting viruses act by binding to the scaffolding A subunit to displace the regulatory B subunit.

(NF-κB) and histone deacetylase 3 (HDAC3). There isincreasing evidence that PP4 may have a highly specificrole in modulating a variety of signalling mechanisms. Forexample, it can activate NF-κB by dephosphorylating Thr-43. It may play a role in histone acetylation and chromatinremodelling by dephosphorylating HDAC3.

Pleckstrin homology domain leucine-rich repeats proteinphosphatase (PHLPP)There are two pleckstrin homology domain leucine-richrepeats protein phosphatases (PHLPP1 and PHLPP2)which are characterized by having an N-terminal PH do-main followed by multiple leucine-rich repeats (LRR) andthen a PP2C phosphatase domain. A splice variant iso-form PHLPP1β, which is also known as suprachiasmaticnucleus circadian oscillatory protein (SCOP), has been im-plicated in long-term memory formation.

One of the main functions of PHLPP is to dephos-phorylate the hydrophobic motif of protein kinase B(PKB) to inhibit the activity of this enzyme. The abil-ity of PHLPP to inhibit PKB has been implicated inendocannabinoid-induced insulin resistance (Module 12:Figure insulin resistance).

PHLPP can also interact with K-Ras resulting in a de-crease in the MAP kinase signalling pathway and thismechanism might play a role in the regulation of neuronalprotein synthesis required for long-term memory forma-tion.

Phosphodiesterase (PDE)The OFF mechanism of the cyclic AMP signalling path-way and the cyclic GMP signalling pathway is carried outby phosphodiesterases (PDEs) that inactivate the two cyc-lic nucleotide second messengers (cyclic AMP and cyc-lic GMP). The PDEs belong to a large family compris-ing 11 PDE gene families (Module 5: Table PDE familyproperties). This extensive PDE family share one thingin common: they all hydrolyse cyclic nucleotide secondmessengers, but in other respects, they are very differentwith regard to substrate specificity, kinetic properties, reg-ulation and cellular distribution. Much of this variabilityresides in the N-terminal region, which has different do-mains that determine the unique characteristics of eachfamily member (Module 5: Figure PDE domains). In thelight of this enormous family diversity, it is difficult tomake too many generalizations, so each family memberis considered separately. Most information is available forPDE1–PDE6:

• PDE1 is a Ca2 + -sensitive cyclic AMP phosphodi-esterase.

• PDE2 is a cyclic GMP-stimulated cyclic AMP phos-phodiesterase.

• PDE3 is a cyclic GMP-inhibited cyclic AMP phosphod-iesterase.

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Cell Signalling Biology Michael J. Berridge � Module 5 � OFF Mechanisms 5 �11

Module 5: Table PDE family propertiesSummary of the organization and properties of the 11 phosphodiesterase (PDE) families.

Number of Commonly usedPDE family Gene splice variants Regulatory domain, role Phosphorylation Substrate(s) inhibitorPDE1 1A, 1B, 1C 9 CaM, activation PKA cGMP, cAMP KS-505PDE2 2A 3 GAF, activation Unknown cAMP, cGMP EHNAPDE3 3A, 3B 1 each Transmembrane domains,

membrane targetingPKB cAMP Milrinone

PDE4 4A, 4B, 4C, 4D >20 UCR1, UCR2, unclear ERK, PKA cAMP RolipramPDE5 5A 3 GAF, unclear PKA, PKG cGMP Sildenafil Dipyrimadole,

ZaprinastPDE6 6A, 6B, 6C 1 each GAF, activation PKC, PKA cGMP Dipyrimadole, ZaprinastPDE7 7A, 7B 6 Unknown Unknown cAMP None identifiedPDE8 8A, 8B 6 PAS, unknown Unknown cAMP None identifiedPDE9 9A 4 Unknown Unknown cGMP None identifiedPDE10 10A 2 GAF, unknown Unknown cAMP, cGMP None identifiedPDE11 11A 4 GAF, unknown Unknown cAMP, cGMP None identified

Some of the phosphodiesterase (PDE) families have more than one gene, and complexity is enhanced further by numerous splicevariants. The different PDEs have variable substrate specificities: some hydrolyse either cyclic AMP or cyclic GMP, whereas others havedual specificity. Reproduced from Handbook of Cell Signaling, Vol. 2, Glick, J.L. and Beavo, J.A., Phosphodiesterase families,pp. 431–435. Copyright (2003), with permission from Elsevier; see Glick and Beavo 2003.

Module 5: Figure PDE domains

Domain structure of the phosphodiesterase (PDE) family that functions to hydrolyse and inactivate the second messenger cyclic AMP.Many of the phosphodiesterase (PDE) family members are characterized by having paired regulatory domains in the N-terminal regulatory region.PDE1 has Ca2 + /calmodulin-binding domains; PDE2, PDE5, PDE6, PDE10 and PDE11 have cyclic GMP-binding GAF domains; PDE4 has up-stream conserved regulatory regions 1 and 2 (UCR1 and UCR2). Reproduced from Handbook of Cell Signaling, Vol. 2, Glick, J.L. and Beavo, J.A.,Phosphodiesterase families, pp. 431–435. Copyright (2003), with permission from Elsevier; see Glick and Beavo 2003.

• PDE4 is a cyclic AMP phosphodiesterase.• PDE5 is a cyclic GMP-specific phosphodiesterase sens-

itive toViagra.• PDE6 is the cyclic GMP phosphodiesterase in photore-

ceptors.

PDE1The characteristic feature of PDE1 is that it is activated byCa2 + . This Ca2 + sensitivity depends on the Ca2 + sensorcalmodulin (CaM), which binds to two CaM-binding do-mains located in the regulatory N-terminal region of PDE1(Module 5: Figure PDE domains). The PDE1 family con-sists of three genes.

PDE1APDE1A, which has five splice variants, has a higher affin-ity for cyclic GMP (Km approximately 5 μM) than cyclicAMP (Km approximately 110 μM). Phosphorylation ofPDE1A1 and PDE1A2 by protein kinase A (PKA) resultsin a decrease in its sensitivity to Ca2 + activation.

PDE1BPDE1B, which has two splice variants, has a higher affinityfor cyclic GMP (Km approximately 2.7 μM) than for cyclicAMP (Km approximately 24 μM). This isoform is stronglyexpressed in the brain. Phosphorylation of PDE1B byCa2 + /calmodulin-dependent protein kinase II (CaMKII)results in a decrease in its sensitivity to Ca2 + activation.

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Cell Signalling Biology Michael J. Berridge � Module 5 � OFF Mechanisms 5 �12

PDE1CPDE1C, which has five splice variants, has a high affinityfor both cyclic GMP and cyclic AMP (Km approximately1 μM). The PDE1C2 splice variant is located in olfactorysensory cilia, where it functions to regulate the role ofcyclic AMP in transducing odorant stimuli (Module 10:Figure olfaction) It is also expressed in β-cells, where itfunctions to regulate glucose-induced insulin secretion.

PDE2PDE2 is a cyclic AMP phosphodiesterase that can bestimulated by cyclic GMP. PDE2 exists as a single gene(PDE2A) that has three splice variants that determineits subcellular distribution, with PDE2A1 being soluble,whereas PDE2A2 and PDE2A3 are particulate. The mem-brane location of PDE2A2 may depend upon a trans-membrane segment in the N-terminal region, whereasPDE2A3 appears to associate with membranes throughan N-terminal myristoylation site.

PDE2 is strongly expressed in the brain and is also foundin skeletal muscle, heart, liver, adrenal glomerulosa andpancreatic cells.

Although PDE2 is a dual-specificity enzyme capable ofhydrolysing both cyclic AMP and cyclic GMP, the enzymeseems to favour cyclic AMP because cyclic GMP acts asan allosteric regulator that greatly enhances the ability ofPDE2 to hydrolyse cyclic AMP. It is for this reason thatthis enzyme is referred to as a cyclic GMP-stimulated cyc-lic AMP PDE.

The ability of cyclic GMP to enhance the hydrolysisof cyclic AMP may account for the signalling cross-talkthat occurs in some cells. For example, the nitric ox-ide (NO)/cyclic GMP-induced reduction in L-type Ca2 +

channel activity in cardiac cells may depend upon cyclicGMP stimulating PDE2, thereby reducing the level of cyc-lic AMP that normally regulates these channels. Anotherexample is found in zona glomerulosa cells, where atrialnatriuretic factor (ANF) may inhibit the secretion of al-dosterone by using cyclic GMP to increase the activity ofPDE2 to reduce the level of cyclic AMP, which drives therelease of this steroid (Module 7: Figure glomerulosa cellsignalling).

PDE3There are two genes encoding PDE3, which is a cyclicGMP-inhibited cyclic AMP phosphodiesterase. They arecharacterized by having six putative transmembrane seg-ments in the N-terminal region (Module 5: Figure PDEdomains), which seem to be responsible for targeting thisenzyme to cell membranes. These two family members(PDE3A and PDE3B) have different functions and cellu-lar locations.

PDE3APDE3A is located in blood platelets, smooth muscle cellsand cardiac myocytes.

PDE3BPDE3B is found in brown and white fat cells, pancreaticβ-cells and liver cells, all of which are cells that function in

energy metabolism. This isoform is particularly import-ant as an effector for the action of insulin in antagoniz-ing the catecholamine-dependent lipolysis and release offatty acids from white fat cells (Module 7: Figure lipolysisand lipogenesis). Insulin acts through the PtdIns 3-kinasesignalling pathway to increase the enzymatic activity ofPDE3B, and the resulting decline in the activity of cyc-lic AMP leads to a decrease in lipid hydrolysis. A similarmechanism operates in liver cells to carry out the anti-glycogenolytic action of insulin (Module 7: Figure livercell signalling). Insulin-like growth factor I (IGF-I) andleptin may reduce insulin secretion in response to GLP-1by stimulating the activity of phosphodiesterase PDE3B,thereby reducing the level of cyclic AMP (Module 7: Fig-ure β-cell signalling).

Insulin resistance and obesity may arise from a reducedexpression of PDE3B.

PDE4PDE4 functions only to hydrolyse cyclic AMP. It consistsof four genes with approximately 20 splice variants, whichfall into three main categories: long, short and super-short.Much of this variation depends upon the expression oftheir characteristic upstream conserved regions 1 and 2(UCR1 and UCR2) in the N-terminal regulatory region(Module 5: Figure PDE domains). The long isoforms haveboth UCR1 and UCR2, the short isoforms lack UCR1,whereas the super-short isoforms lack UCR1 and have atruncated UCR2.

The activity of the various PDE4 isoforms can be reg-ulated through a feedback loop operated through proteinkinase A (PKA) and by inputs from other signalling path-ways, such as MAP kinase signalling. The ability of PKA tomodulate the activity of PDE4 is facilitated by the fact thatthey are both associated on the same scaffolding proteinmuscle A-kinase-anchoring protein (mAKAP).

PDE4APDE4A is located in the soma of olfactory neurons, in con-trast with PDE1C2, which is in the cilium. PDE4A1 as-sociates with membranes through a hydrophobic domainin the N-terminal region, whereas PDE4A5 is located atthe plasma membrane, where it associates with proteinscontaining SH3 domains.

PDE4BPDE4B plays an important role in inflammatory re-sponses, because PDE4B− / − mice display a large decreasein their ability to release tumour necrosis factor α (TNFα)in response to lipopolysaccharide (LPS). The cyclic AMPsignalling pathway functions in the modulation of inflam-matory responses. It has an anti-inflammatory role in mac-rophages, and this inhibitory effect is usually dampened bythe up-regulation of PDE4B (Module 11: Figure macro-phage signalling). PDE4B has an important role in regulat-ing the contractile activity of uterine smooth muscle cells.The antidepressant Rolipram inhibits PDE4B.

Mutations in PDE4B have been linked to schizophrenia.

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Cell Signalling Biology Michael J. Berridge � Module 5 � OFF Mechanisms 5 �13

Module 5: Figure PDE5 functional states

A model depicting different functional states of PDE5.PDE5 functions as a dimer with the two subunits connected through a region that includes the allosteric cyclic GMP-binding GAF domains. During thecourse of enzyme activation, cyclic GMP appears to bind first to the catalytic site (Km of 1–6 μM), which induces a conformational change that thenenhances the affinity of cyclic GMP binding to the GAF domains. This binding to the regulatory region induces a further conformational change toexpose the serine residue, which is then phosphorylated by cyclic GMP-dependent protein kinase (cGK). This phosphorylated state is the most activeform of the enzyme. This thus represents a complex feedback loop whereby cyclic GMP promotes its own hydrolysis by binding allosterically to theenzyme and by promoting its phosphorylation by stimulating cGK. Reproduced from Handbook of Cell Signaling, Vol. 2, Francis, S.H. and Corbin,J.D., Phosphodiesterase-5, pp. 447–451. Copyright (2003), with permission from Elsevier; see Francis and Corbin 2003.

PDE4DPDE4D may play some role asthma. PDE4D− / − micehave been found to lack normal muscarinic responses, res-ulting in a loss of airway hyperreactivity.

PDE5There is a single cyclic GMP-specific phosphodiesterase(PDE5) gene with three splice variants. It is a cyclic GMP-specific phosphodiesterase, which has a unique feature inthat it is also regulated by cyclic GMP binding to the tan-dem GAF domains in the regulatory region (Module 5:Figure PDE domains). Binding of cyclic GMP to theseGAF domains is necessary for protein kinase A (PKA)or cyclic GMP-dependent protein kinase (cGK) to phos-phorylate a single site in the N-terminal region, whichthen results in an increase in both the rate of catalysis andcyclic GMP-binding affinity of the catalytic site. This com-plex combination of regulation through both the allostericbinding of cyclic GMP and phosphorylation by cGK canresult in different functional states of the enzyme (Module5: Figure PDE5 functional states).

PDE5 plays a major role in regulating the cyclic GMPsignalling pathway in various cells, such as smooth musclecells (Module 7: Figure smooth muscle cell cGMP sig-nalling), blood platelets, renal tissue (proximal and collect-ing ducts), cerebellar Purkinje cells and pancreatic ducts.

In the case of corpus cavernosum smooth muscle cells,which regulate penile erection (Module 7: Figure corpuscavernosum), PDE5 is the target for Viagra , a drug usedto treat male erectile dysfunction.

PDE6PDE6 is a highly specialized enzyme that is the primary ef-fector of visual transduction in vertebrate photoreceptors(Module 10: Figure phototransduction overview).

The stability of PDE6 is regulated by aryl hydrocar-bon receptor-interacting protein-like 1 (AIPL1). Just howAIPL1 functions is not entirely clear, but it appears tofunction as a specific chaperone required for PDE6 bio-synthesis and stability. Leber congenital amaurosis (LCA),which is an early onset human retinopathy, has been linkedto mutations in the AIPL1 gene.

Ca2 + pumps and exchangersA variety of pumps and exchangers are responsible for re-moving Ca2 + from the cytoplasm (Module 5: Figure Ca2 +

uptake and extrusion). The most obvious function of suchpumps is therefore to enable cells to recover from Ca2 + -induced signalling events. However, such pumps have twoother important functions. Firstly, they ensure that the in-ternal stores are kept loaded with signal Ca2 + by pumpingCa2 + into the sarcoplasmic reticulum (SR) of muscle cellsor the endoplasmic reticulum (ER) of non-muscle cells.Pumps are also important for loading Ca2 + into the Golgi.Secondly, they maintain the resting level of Ca2 + . The con-stant leakage of Ca2 + into the cell down the very large con-centration gradients facing the cytoplasm, both from theoutside and from the internal stores, is expelled by pumpsto ensure that the resting Ca2 + concentration is held con-stant at approximately 100 nM. A pump classification re-veals that there are five different mechanisms responsiblefor carrying out these functions of recovery, maintainingthe Ca2 + stores and the resting level of Ca2 + :

• Plasma membrane Ca2 + -ATPase (PMCA)• Sodium/calcium exchangers (NCX and NCKX)• Sarco/endo-plasmic reticulum Ca2 + -ATPase (SERCA)• Mitochondrial uniporter• Secretory-pathway Ca2 + -ATPase (SPCA)

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Two of these pumps (PMCA and NCX) are locatedon the plasma membrane, whereas the others are loc-ated on internal organelles. The organization and distri-bution of Ca2 + pumps determines the properties of Ca2 +

pumps, which are adapted to carry out different homoeo-static functions. The PMCA pump family consists of fourgenes with diversity enhanced by alternative splicing attwo sites. The SERCA family has three genes, and altern-ative splicing gives at least six different isoforms. Likewise,the NCX has a family of three genes, and alternative spli-cing gives rise to numerous isoforms. The way in whichalternative splicing can enhance diversity is illustrated forSERCA2a and its isoforms, which have not only differentproperties, but also different distributions. In summary,the molecular organization gives rise to a diverse reper-toire of pumps from which cells can select out that com-bination of pumps that exactly meets their Ca2 + signallingrequirements.

The molecular structure of the Ca2 + pumps is designedto transfer Ca2 + ions across membranes against very largeelectrochemical gradients. The exception to this is the mi-tochondrial uniporter, which is not a pump in the strictsense, but it is a channel that allows Ca2 + to flow fromthe cytoplasm into the mitochondrial matrix. The plasmamembrane Ca2 + -ATPase (PMCA) molecular structureand that of the SERCA pump are very similar with re-gard to their main domains. They have ten transmembranedomains, with both the N-terminal and C-terminal endsfacing the cytoplasm. The NCX and NCKX molecularstructure consists of nine and 11 transmembrane domainsrespectively. They both have a large cytoplasmic loop con-necting transmembrane domains 5 and 6 (Module 5: Figuresodium/calcium exchangers).

The different structural domains have specific functions,which have been well described for the sarco/endo-plasmicreticulum Ca2 + -ATPase (SERCA) pump structure andmechanism. There is less information on the NCX pumpmechanism, where the energy to pump Ca2 + is de-rived from the flow of Na+ down its electrochemicalgradient.

Ca2 + pump regulation plays a critical role in enablingpumps to deal with large variations in the intracellular levelof Ca2 + .

Alterations in the way cells pump Ca2 + have beenlinked to a variety of diseases. For example, Darier’s dis-ease is an autosomal skin disorder that results from a lossof one copy of the SERCA2 gene. Brody disease resultsfrom a defect in the SERCA1a pump that is respons-ible for relaxing skeletal muscle. Hailey--Hailey disease iscaused by an inactivating mutation of the secretory Ca2 + -ATPase.

Pump classificationCells use different types of Ca2 + pumps, twotypes [plasma membrane Ca2 + -ATPase (PMCA) andNa+ /Ca2 + exchanger (NCX)] are located on the plasmamembrane while the sarco/endo-plasmic reticulum Ca2 + -ATPase (SERCA) and the uniporter are located on internalorganelles (Module 2: Figure Ca2 + signalling toolkit).

Plasma membrane pumpsThe plasma membrane Ca2 + -ATPase (PMCA) located onthe plasma membrane extrudes Ca2 + from the cell usingenergy derived from the hydrolysis of ATP.

The sodium/calcium exchangers (NCX and NCKX),which consist of two families, the Na+ /Ca2 + exchanger(NCX) and the Na+ /Ca2 + − K+ exchanger (NCKX), arelocated on the plasma membrane and extrude Ca2 + in ex-change for Na+ . The energy for Ca2 + extrusion is derivedfrom the influx of Na+ , which enters the cell down itselectrochemical gradient.

Organellar pumpsThe sarco/endo-plasmic reticulum Ca2 + -ATPase(SERCA) located on either the sarcoplasmic retic-ulum (SR) of muscle cells or on the endoplasmic reticulum(ER) of non-muscle cells uses the energy derived fromthe hydrolysis of ATP to pump Ca2 + from the cytoplasminto the internal store.

The secretory-pathway Ca2 + -ATPase (SPCA) is loc-ated on the Golgi, where it functions to maintain the levelof Ca2 + within the lumen in order to maintain processessuch as glycosylation, proteolytic processing and proteintrafficking.

The mitochondrial uniporter is not strictly a pump, butis included because it functions in mitochondrial Ca2 + up-take to remove Ca2 + from the cytoplasm. The uptake ofCa2 + through the uniporter is driven by the large trans-membrane potential that is maintained across the inner mi-tochondrial membrane (Module 5: Figures mitochondrialCa2 + signalling). Mitochondria modulate Ca2 + signallingby operating as a ‘buffer’ in that they rapidly sequesterCa2 + during the course of a response, and then return itto the cytoplasm through a mitochondrial Na+ /Ca2 + ex-changer as the concentration of Ca2 + returns towards itsresting level.

Properties of Ca2 + pumpsThe different Ca2 + pumping mechanisms have very dif-ferent properties with regard to their affinity for Ca2 + andthe rate at which they can transport this ion across mem-branes (Module 5: Figure Ca2 + uptake and extrusion):

Low-affinity, high-capacity pumpsThe Na+ /Ca2 + exchanger (NCX), the Na+ /Ca2 + –K+

exchanger (NCKX) and the mitochondrial uniporter, forexample, have low affinities for Ca2 + , but have very highcapacities, and this enables them to function early in therecovery process, since they can rapidly remove the largequantities of Ca2 + that are released into the cytoplasmduring signalling. The high capacity of NCX and NCKXis based on the rapid turnover rate of the exchanger, whichcan carry out 1000 to 5000 reactions/s.

High-affinity, low-capacity pumpsOn the other hand, the plasma membrane Ca2 + -ATPase(PMCA), sarco/endo-plasmic reticulum Ca2 + -ATPase

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Cell Signalling Biology Michael J. Berridge � Module 5 � OFF Mechanisms 5 �15

Module 5: Figure Ca2 + uptake and extrusion

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Functional organization of Ca2 + pumps.The Na+ /Ca2 + exchanger (NCX) and the mitochondrial uniporter are particularly effective at pumping Ca2 + when the cytosolic Ca2 + concentrationis high, in that they combine a low affinity for Ca2 + with a high capacity. The plasma membrane Ca2 + -ATPase (PMCA) and sarco/endo-plasmicreticulum Ca2 + -ATPase (SERCA) pumps have much lower capacities, but their much higher affinities enable them to reduce the level of Ca2 + backto its resting level. The different extrusion mechanisms thus co-operate with each other to regulate the level of Ca2 + over a large dynamic range.

(SERCA) and secretory-pathway Ca2 + -ATPase (SPCA)pumps have lower capacities, but their higher affinitiesmean that they can continue to pump at lower Ca2 + levels,thus enabling them to maintain the internal stores and theresting level. The SPCA is unusual in that it can pumpMn2 + equally as well as Ca2 + . The PMCA and SERCApumps have low capacities, because the ATP-dependentconformational process that occurs during the pumpingmechanism occurs at a low rate (approximately 150 re-actions/s). These two pumps belong to the P2 subfamilyof P-type ion transport ATPases that are characterized bythe formation of an aspartyl phosphate during the reactioncycle of the pump mechanism.

Organization and distribution of Ca2 + pumpsCa2 + pumps have molecular structures designed to trans-fer Ca2 + ions across membranes against very large electro-chemical gradients. This pumping problem has been solvedin different ways. The diversity of Ca2 + pumps dependsupon the existence of multigene families (Module 5: TableCa2 + pumping toolkit) within which additional diversityis generated by alternative splicing. This diversity createsmany isoforms with subtle variations, not only in theirpumping properties but also in their Ca2 + pump regula-tion. An important consequence of all this diversity is thateach cell has access to an enormous repertoire from whichit can select out those pumps with properties exactly suitedto their particular signalling requirements.

Plasma membrane Ca2 + -ATPase (PMCA)The plasma membrane Ca2 + -ATPase (PMCA) gene fam-ily contains four closely related genes (PMCA1–PMCA4)with numerous alternatively spliced forms denoted by thelower-case letters of the alphabet (Module 5: Table Ca2 +

pumping toolkit). Expression of these various splice iso-forms is a regulated event in that they change in a consistentway during both development and differentiation. Thereare indications that changes in the level of Ca2 + may influ-ence the expression of these splice isoforms. For example,an elevation in the level of Ca2 + in cerebellar granule cellsresults in the up-regulation of PMCA1a, PMCA2 andPMCA3, but a down-regulation of PMCA4a. The maindomain structure of the PMCAs reveals the presence often transmembrane (TM) domains with two large cytosolicloops between TM2 and TM3 and between TM4 and TM5(Module 5: Figure PMCA domain structure). The latteris particularly significant, because it contains the aspartylphosphorylation site (P). These two loops have importantfunctions in Ca2 + pump regulation of PMCA activity. ThePMCA isoforms 1 and 4 are widely expressed (Module 5:Table Ca2 + pumping toolkit), whereas isoforms 2 and 3 aremainly restricted to the brain and skeletal muscle. Withinthe brain, there are regional differences in the expressionof these isoforms, e.g. PMCA2 is high in cerebellar Purk-inje cells and cochlear hair cells, whereas PMCA3 is foundmainly in the choroid plexus. A mutation in PMCA2 res-ults in hearing loss.

One consequence of pump diversity is that cells haveaccess to pumps that transport at different rates. Cells that

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Cell Signalling Biology Michael J. Berridge � Module 5 � OFF Mechanisms 5 �16

Module 5: Figure PMCA domain structure

Domain structure of the plasma membrane Ca2 + -ATPase (PMCA).A. The sites marked A and C are the main sites where alternative splicing occurs to create at least 20 different isoforms. These two splice sites occurin the two large cytoplasmic loops, and are thus likely to influence the way in which these two loops regulate pump activity. B. In the absence ofcalmodulin (CaM), the autoinhibitory C-terminal region is thought to bend around to inhibit enzymatic activity. In the presence of Ca2 + , CaM bindsto the CaM-binding domain (CaMBD), and this regulatory chain is pulled away, resulting in an increase in pump activity. Reproduced from Strehler,E.E. and Zacharias, D.A. (2001) Role of alternative splicing in generating isoform diversity among plasma membrane calcium pumps. Physiol. Rev.81:21–50; used with permission from The American Physiological Society; see Strehler and Zacharias 2001.

have to generate rapid transients have the fastest pumps.For example, PMCA3f (skeletal muscle) and PMCA2a(stereocilia) are the fastest, whereas PMCA4b (Jurkat cells)is the slowest.

In kidney tubule cells, the PMCA1b plays an import-ant role in the reabsorption of Ca2 + by the paracellulartransport pathway (Module 7: Figure kidney Ca2 + reab-sorption).

Plasma membrane Ca2 + -ATPase (PMCA) molecularstructureDespite the large molecular diversity within the plasmamembrane Ca2 + -ATPase (PMCA) family, the overallstructure of all the members is very similar. They haveten transmembrane domains with both the N-terminal andC-terminal regions facing the cytosol (Module 5: FigurePMCA domain structure). Most of the extracellular andintracellular loops that link the transmembrane domainsare relatively short, except for two of the four loops thatface the cytosol. The largest cytoplasmic loop connectingTM4 and TM5 is of particular significance because it con-tains two important sites for the pump cycle. The first siteis the nucleotide-binding domain, where the ATP binds tothe pump molecule. The second site is the phosphorylationdomain, which contains the invariant aspartate residue thatis phosphorylated during the conformational changes thatoccur during each pump cycle (Module 5: Figure SERCApump cycle).

The location of the PMCA pumps may be determinedby binding to a family of PDZ domain-containing pro-

teins. Such an interaction may occur through the PDZinteraction domains located in the C-terminal region.

Sarco/endo-plasmic reticulum Ca2 + -ATPase (SERCA)The sarco/endo-plasmic reticulum Ca2 + -ATPase(SERCA) family of pumps contains three genes withnumerous alternatively spliced isoforms (Module 5:Table Ca2 + pumping toolkit). The role of SERCAis to pump Ca2 + back into the endoplasmic reticulum(ER)/sarcoplasmic reticulum (SR) (Module 5: Figure Ca2 +

uptake and extrusion). There have been considerable ad-vances in the understanding of the sarco/endo-plasmicreticulum Ca2 + -ATPase (SERCA) pump structure andmechanism of Ca2 + transfer across the ER/SR membrane.

Inactivating mutations of SERCA1 are the cause ofBrody disease. Expression of SERCA2 is regulated bymiR-152. A mutation of the SERCA2 pump causesDarier’s disease.

Sarco/endo-plasmic reticulum Ca2 + -ATPase (SERCA)pump structure and mechanismThe resolution of the sarco/endo-plasmic Ca2 + -ATPase(SERCA) pump structure has provided a detailed scenarioof how this pump might work. For the plasma membraneCa2 + -ATPase (PMCA) and SERCA pumps, some of thetransmembrane domains make up the Ca2 + -binding siteused to transfer Ca2 + across the membrane. ATP providesthe energy to drive this transfer. Like the PMCA pump, theSERCA pump is a member of the P-type pumps, so calledbecause the pump is energized by ATP phosphorylatingan aspartate residue. This phosphorylation event inducesthe conformational change necessary to drive Ca2 + across

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Cell Signalling Biology Michael J. Berridge � Module 5 � OFF Mechanisms 5 �17

Module 5: Table Ca2 + pumping toolkitSummary of genomic organization, spliced isoform and distribution of Ca2 + pumps and exchangers.Component Spliced isoform DistributionPlasma membrane Ca2 + -ATPase (PMCA) pumps

PMCA1 (the human gene is located on 12q21–q23) PMCA1a Excitable cells: brain, skeletal muscle, heart and kidneyPMCA1b Ubiquitous; a housekeeper pumpPMCA1c Skeletal muscle, heartPMCA1d Skeletal muscle, heartPMCA1e BrainPMCA1x Ubiquitous; a housekeeper pump

PMCA2 (the human gene is located on 3p25–p26) PMCA2a Brain, heart, uterusPMCA2b WidespreadPMCA2c TestisPMCA2w Brain, kidney, uterusPMCA2x Brain, heartPMCA2yPMCA2z Brain, heart

PMCA3 (the human gene is located on Xq28) PMCA3a Brain, spinal cord, testisPMCA3b Adrenal, brain, skeletal musclePMCA3cPMCA3d BrainPMCA3e Skeletal musclePMCA3f Brain, skeletal musclePMCA3xPMCA3z

PMCA4 (the human gene is located on 1q25–q32) PMCA4a WidespreadPMCA4b Ubiquitous; a housekeeper pumpPMCA4x WidespreadPMCA4z Heart, testis

Sarco/endo-plasmic reticulum Ca2 + -ATPase (SERCA)pumpsSERCA1 SERCA1a Fast twitch skeletal muscle

SERCA1b Fast twitch skeletal muscleSERCA2 SERCA2a Cardiac and slow twitch skeletal muscle

SERCA2b Ubiquitous; a housekeeper pump in smooth muscleand many other cells

SERCA2c Heart and skeletal muscleSERCA3 SERCA3a Mast cells, lymphocytes, platelets, monocytes,

vascular endothelial cells and cerebellar Purkinjecells

SERCA3b Haematopoietic cells; blood plateletsSERCA3c Haematopoietic cells; Blood plateletsSERCA3d Heart and skeletal muscleSERCA3e Pancreas and lungSERCA3f Heart and skeletal muscle

Secretory-pathway Ca2 + -ATPase (SPCA) pumpsSPCA1 SPCA1a–SPCA1d Ubiquitous; located in the Golgi.SPCA2

Na+ /Ca2 + exchangers (NCXs)NCX1 Heart, kidneyNCX2 NeuronsNCX3 Neurons

Na+ /Ca2 + –K+ exchangers (NCKXs)NCKX1 Rod photoreceptors (Module 10: Figure

phototransduction) and plateletsNCKX2 Brain, cone photoreceptorsNCKX3 Brain, aorta, uterus and intestineNCKX4 Brain, aorta, lung and thymus

With regard to the distribution, the list is not complete, but includes those tissues where the different isoforms are strongly expressed.(For a detailed description of the nomenclature and distribution of spliced variants, see Strehler and Zacharias 2001 for the PMCA pumpsand Schnetkamp 2004 for NCKX.)

the membrane. The following sequence of events occursduring the SERCA pump cycle (Module 5: Figure SERCApump cycle):

1. The resting E1 state is energized by binding ATP tounveil two Ca2 + -binding sites that face the cytoplasm(E1.ATP).

2. Ca2 + enters the two binding sites to form theE1.ATP.2Ca2 + complex.

3. The binding of Ca2 + strongly activates the ATPaseactivity of the pump, resulting in the release of ADPand the transfer of phosphate to an aspartate residue to

form a high-energy phosphorylated intermediate (E1–P∼2Ca2 + ).

4. The energy stored in this phosphorylated intermediateis used to induce the conformational change to the E2–P.2Ca2 + state during which Ca2 + moves across thebilayer.

5. In the lower-energy E2–P.2Ca2 + state, the binding siteshave a reduced affinity for Ca2 + , which is free to diffuseinto the lumen.

6. Hydrolysis of the E2–P phosphoenzyme enables thepump to return to the resting E1 state, ready to beginanother cycle.

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Cell Signalling Biology Michael J. Berridge � Module 5 � OFF Mechanisms 5 �18

Module 5: Figure SERCA pump cycle

P

P

P

P

ATP

ADP

ATP

ATP

1

2

3

5

6

E ATP.2Ca.

E .ATP

E

2+1

2

1

1

E

1E . 2Ca4

E . 2+

2+

2 2Ca

SERCAPUMPCYCLE

Cytosol

ER/SR lumen

CaCa2+

2+

The sarco/endo-plasmic reticulum Ca2 + -ATPase (SERCA) pump cycle.The pump cycle consists of a series of biochemical reactions during which the pump switches between two major conformational states: an E1 statewhen the Ca2 + -binding site faces the cytoplasm, and an E2 state where the binding sites have switched to the opposite side and Ca2 + is released tothe lumen. During each cycle, two Ca2 + ions are pumped for each ATP hydrolysed. This switch between the E1 and E2 states represents one cycle,and this occurs at a frequency of about 150 reactions/s. The way in which ATP powers each pump cycle is described in the text.

The next question to consider is the molecular basis ofthis pump cycle. How are the individual steps of the pumpcycle (Module 5: Figure SERCA pump cycle) related tothe molecular structure of SERCA?A feature of SERCAstructure is characterized by a number of distinct domains(Module 5: Figure SERCA1a pump), which have clearlydefined roles at different stages of the pump cycle.

ATP binding and the loading of Ca2 + on to the bindingsiteThe first steps in the pump cycle (Steps 1 and 2 in Module5: Figure SERCA pump cycle) is ATP binding and theloading of Ca2 + on to the external binding sites. This ATPbinds to the N domain, which forms a cap sitting over the Pdomain (Module 5: Figure SERCA1a pump). The resultingconformational change within the M (transmembrane) do-mains opens up two Ca2 + -binding sites. The first point tonotice is that the N domain is somewhat removed from thetransmembrane domains where Ca2 + translocates throughthe membrane. The transmission of such a conformationalchange has to be mediated through long-range allostericinteractions. The pathway for transmitting such molecularchanges is still not clear, but there are several possibilities.One possibility is that the A domain (also referred to as thetransducer domain) may play a role. Another possibilityis that information might be transmitted through the longrod-like central helix of M5 that extends from the insideof the membrane right up to the underside of the P do-

main. This seems to be a likely mechanism, because M5,together with M4 and M6, are the transmembrane domainsthat form the Ca2 + -binding pocket. Note that the N andP domains are formed from the loop that connects to M4and M5. It is suggested that the conformational changescause a disruption of M4 and M6 helices, and this opensup a pocket for the two Ca2 + ions to bind.

Phosphorylation of the aspartate residue and Ca2 +

translocation through the membraneA critical phase in the transport process is the interac-tion between the N and P domains during which the ter-minal phosphate group of ATP is transferred to Asp-351.The problem here is that the ATP-binding site on theN domain appears from the structure to be located faraway from this phosphorylation site. Somehow the twodomains have to move in order for the two sites to ap-proach close enough for the phosphorylation to occur.Once the energized E1∼P state is formed, another con-formational change transmitted through the mechanismsdiscussed earlier brings about the movement of the M do-mains so that the Ca2 + -binding pocket is altered to face thelumen to allow Ca2 + to enter the endoplasmic reticulum(ER). This remarkable molecular machine is beautifullydesigned to efficiently couple the site of energy conver-sion (the phosphorylation domain) to the translocationmechanism in the membrane.

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Cell Signalling Biology Michael J. Berridge � Module 5 � OFF Mechanisms 5 �19

Module 5: Figure SERCA1a pump

Functional operation of the sarco/endo-plasmic reticulum Ca2 + -ATPase 1a (SERCA1a) pump.The main domains of the sarco/endo-plasmic reticulum Ca2 + -ATPase 1a (SERCA1a) pump concerned with pumping are the nucleotide-bindingdomain (N), the phosphorylation domain (P), the actuator domain (A) and the transmembrane domains (M1–M10). The molecular events involvingthese domains are described in the text by reference to the transition states of the pump cycle. Reproduced by permission from Macmillan PublishersLtd: Nature, MacLennan, D.H. and Green, N.M. (2000) Structural biology: pumping ions. 405:633–634. Copyright (2000); http://www.nature.com; seeMacLennan and Green 2000.

Ca2 + pump regulationOne of the hallmarks of Ca2 + pumps is their regulation,which enables them to adapt to changing circumstances.The most direct form of regulation is for Ca2 + to regulateits own activity, and this is particularly apparent for theplasma membrane Ca2 + -ATPase (PMCA) pump. Ca2 +

acts through calmodulin (CaM) to stimulate the pump.When the pump is activated through the Ca2 + /CaM mech-anism, the CaM remains bound for some time after Ca2 +

signalling has ceased, thus allowing the pump to have a‘memory’ so that it can respond more quickly to anotherCa2 + transient.

The Ca2 + pumps are sensitive to hormonal regula-tion, with control being exerted through various reg-ulators such as phospholamban (PLN) and sarcolipin(SLN). PLN is particularly important, as it is sensitiveto various signalling pathways operating through secondmessengers such as cyclic AMP and Ca2 + itself. Suchregulation is critical for regulating cardiac contractility(Module 7: Figure ventricular Ca2 + signalling), where thestrength of contraction is controlled by cyclic AMP, whichacts by phosphorylating PLN to remove its inhibitor ef-fect on the sarco/endo-plasmic reticulum Ca2 + -ATPase(SERCA) pump.

Phospholamban (PLN)Phospholamban (PLN) is the primary regulator of thesarco/endo-plasmic reticulum Ca2 + -ATPase (SERCA)pump. It has an important function in cardiac cells, whereit regulates the SERCA2a isoform (Step 7 in Module 7:Figure ventricular Ca2 + signalling), but it is also activein smooth muscle cells (Module 7: Figure smooth musclecell spark). PLN is a transmembrane protein containing 52amino acids (Module 5: Figure phospholamban and sarcol-ipin). It exists within the membrane in a number of states,with the unphosphorylated monomeric state being the onethat binds to cardiac SERCA2a to inhibit its activity. PLNhas a number of function states (Steps 1 to 6 in Module 5:Figure phospholamban mode of action):

1. A PLN pentamer, which forms when five PLNmonomers come together, is stabilized by leucine–isoleucine zipper interactions.

2. Monomeric unphosphorylated PLN is the active formthat binds to SERCA2a.

3. PLN binds in a groove running up both the trans-membrane and cytosolic regions of SERCA2a (Module5: Figure SERCA pump structure). PLN exerts its

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Cell Signalling Biology Michael J. Berridge � Module 5 � OFF Mechanisms 5 �20

Module 5: Figure phospholamban and sarcolipin

The structural organization of the sarco/endo-plasmic reticulum Ca2 + -ATPase SERCA pump regulators phospholamban (PLN) and sarcolipin(SLN).Phospholamban (PLN) has two main regions: a C-terminal α-helix thatembeds the molecule in the sarcoplasmic reticulum (SR) membrane andan N-terminal cytoplasmic region. The latter has an α-helical region thatis connected to the membrane region by a short β-turn. Sarcolipin (SLN)resembles PLN, except that it lacks most of the cytoplasmic domain.The high degree of homology, both conserved residues (grey/green) andidentical residues (red), within the transmembrane domain indicates thatthis region is of particular significance in mediating the ability of theseproteins to interact with, and inhibit, SERCA pumps. This inhibitory effectof PLN is reversed by protein kinase A (PKA) and Ca2 + /calmodulin-dependent protein kinase II (CaMKII) phosphorylating Ser-16 and Thr-17respectively. Reproduced by permission from Macmillan Publishers Ltd:Nat. Rev. Mol. Cell Biol., MacLennan, D.H. and Kranias, E.G. (2003) Phos-pholamban: a crucial regulator of cardiac contractility. 4:566–577. Copy-right (2003); http://www.nature.com/nrm; see MacLennan and Kranias2003.

inhibitory action by regulating the Ca2 + affinity ofthe SERCA2a pump.

4. The SERCA2a pump increases its Ca2 + pumping activ-ity when the inhibitory effect of PLN is removedfollowing its phosphorylation by protein kinase A(PKA) or Ca2 + /calmodulin-dependent protein kinaseII (CaMKII).

5. Inactive PLN is phosphorylated on either Ser-16 (byPKA) or Thr-17 (by CaMKII) (Module 5: Figure phos-pholamban and sarcolipin).

6. The phosphorylated PLN is converted back into itsactive inhibitory form following dephosphorylation byprotein phosphatase PP1 (Module 5: Figure PP1 target-ing to glycogen).

Studies on transgenic mice have established that PLNfeatures significantly in the relationship between Ca2 +

signalling and cardiac hypertrophy. There are also two

examples of inherited human dilated cardiomyopathy thathave been traced to mutations in PLN.

Sarcolipin (SLN)Sarcolipin (SLN) resembles phospholamban (PLN) withregard to its transmembrane region, but its cytoplasmicregion has been truncated (Module 5: Figure phosphalam-ban and sarcolipin). SLN is strongly expressed in fast-twitch skeletal muscle, but is low in heart. Like PLN, SLNfunctions to regulate sarco/endo-plasmic reticulum Ca2 + -ATPase (SERCA) activity and appears to bind to a similargroove in the SERCA molecule.

Secretory-pathway Ca2 + -ATPase (SPCA)The Golgi contains both a sarco/endo-plasmic reticulumCa2 + -ATPase (SERCA) pump and a secretory-pathwayCa2 + -ATPase (SPCA) that is responsible for pumpingCa2 + into the Golgi stacks. There is a variable distribu-tion of these two pumps within the Golgi. The SERCAis found in the early parts of the Golgi, thus reflecting itsorigin from the endoplasmic reticulum (ER). The SPCAis restricted to the trans-Golgi region. The SPCA pumpsCa2 + into the Golgi using mechanisms similar to those de-scribed above for the SERCA pumps. Unlike the SERCApumps, however, the SPCA is able to pump Mn2 + equallyas well as Ca2 + .

There are two SPCA isoforms (SPCA1 and SPCA2).The ATP2C1 gene that encodes SPCA1 is mutated inHailey-Hailey disease.

Sodium/calcium exchangers (NCX and NCKX)The Na+ /Ca2 + exchangers play a critical role in Ca2 +

signalling because they provide a mechanism for rapidlyextruding Ca2 + from cells (Module 5: Figure Ca2 + up-take and extrusion). These exchangers are particularly im-portant in excitable cells such as cardiac cells, neuronsand sensory neurons. However, they are also expressedon various non-excitable cells. There are two families ofexchangers, the Na+ /Ca2 + exchanger (NCX) and theNa+ /Ca2 + –K+ exchanger (NCKX) families (Module 5:Table Ca2 + pumping toolkit).

Na+ /Ca2 + exchanger (NCX)The Na+ /Ca2 + exchanger (NCX) was the first exchangerto be discovered. It functions to extrude Ca2 + from the cellin exchange for Na+ (Module 5: Figure sodium/calciumexchangers). The energy from the Na+ gradient across theplasma membrane is used to drive Ca2 + out of the cellagainst the large electrochemical gradient. There is no dir-ect role for ATP, but it does have an indirect role because itpowers the ouabain-sensitive Na+ pump that establishesthe Na+ gradient. In kidney tubule cells, the NCX playsan important role in the reabsorption of Ca2 + by the para-cellular transport pathway (Module 7: Figure kidney Ca2 +

reabsorption).The NCX has nine transmembrane (TM) segments

with a large cytoplasmic loop connecting TM5 and TM6.The regions shaded in yellow in Module 5: Figure so-dium/calcium exchangers contain α repeats (α1 and α2),which are highly homologous with two similar regions in

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Module 5: Figure phospholamban mode of action

P

1 2 3 4 5 6

Cytosol

PLN

SERCA2a

SR lumen

Ca2+

Ca2+

PKAPP1CAMKII

Phospholamban (PLN) operates within the plane of the sarcoplasmic reticulum (SR) membrane to regulate the activity of the sarco/endo-plasmicreticulum Ca2 + -ATPase 2a (SERCA2a) pump.Phospholamban (PLN) operates in the plane of the sarcoplasmic reticulum, where it acts to regulate the activity of the sarco/endo-plasmic reticulumCa2 + -ATPase 2a (SERCA2a) pump. PLN has five distinct functional states, as described in the text.

the Na+ /Ca2 + –K+ exchanger (NCKX). These are theonly two regions that show close homology, and thiswould fit with the notion that α1 and α2 play a role inthe binding and transport of cations.

NCX can operate in two modes, depending on the elec-trochemical potential, which determines the directionalityof the Na+ flux that drives the movement of Ca2 + . In theforward mode, Ca2 + is extruded from the cell, whereas inthe reverse mode, Ca2 + is brought into the cell. This re-verse mode may play an important role during excitation–contraction (E-C) coupling in heart cells, where the rapidbuild-up of Na+ during the course of the action potentialresults in a local build-up of this ion, which then begins toflow out in exchange for Ca2 + . This influx of Ca2 + willcontribute to the trigger Ca2 + entering through the L-typechannel, and thus could facilitate the excitation processes.The NCX1 isoform has an important role in ventricularcell Ca+ release (Module 7: Figure ventricular Ca2 + sig-nalling).

There is some debate concerning the exact stoichiometryof NCX. Most measurements suggest that three Na+ ionsare transported for each Ca2 + ion, which means that theexchanger is electrogenic.

Na+ /Ca2 + –K+ exchanger (NCKX)The Na+ /Ca2 + –K+ exchangers (NCKXs) were first dis-covered in rod photoreceptors. There is a family of theseexchangers with different distributions in both excitableand non-excitable cells (Module 5: Table Ca2 + pump-

ing toolkit). They differ from the Na+ /Ca2 + exchangers(NCXs) in that they extrude both Ca2 + and K+ in ex-change for Na+ (Module 5: Figure sodium/calcium ex-changers). While some of their structural features resemblethose found in NCX, there clearly are marked differences.The regions of closest homology are the two regions of α

repeats (α1 and α2 shown in yellow), which play a role inbinding cations during the exchange reaction.

The function of these exchangers has been defined bestin photoreceptors, where they are the primary mechanismfor extruding Ca2 + during the process of phototransduc-tion (see Step 5 in Module 10: Figure phototransductionoverview). The organization of the NCKX1 isoform in thephotoreceptor is of interest because it appears to be com-plexed to the cyclic nucleotide-gated channel (CNGC)that is responsible for the cyclic GMP-dependent entry ofCa2 + . Furthermore, this entry channel/exchanger com-plex also appears to be linked to two proteins (peripherinand Rom-1) located in the rim of the intracellular disc. Thefunctional significance of linking the plasma membrane tothe internal disc through this protein complex is unknown.

MitochondriaMitochondria distributed throughout the cytoplasm havemany functions. They generate ATP, they shape Ca2 + sig-nals and respond to Ca2 + signals by increasing the produc-tion of ATP, they generate reactive oxygen species (ROS)

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Cell Signalling Biology Michael J. Berridge � Module 5 � OFF Mechanisms 5 �22

Module 5: Figure SERCA pump structure

Interaction between phospholamban (PLN) and the sarco/endo-plasmic reticulum Ca2 + -ATPase (SERCA) pump.Reproduced by permission from Macmillan Publishers Ltd: Nat. Rev. Mol.Cell Biol., MacLennan, D.H. and Kranias, E.G. (2003) Phospholamban:a crucial regulator of cardiac contractility. 4:566–577. Copyright (2003);http://www.nature.com/nrm; see MacLennan and Kranias 2003.

and under extreme conditions, they release factors such ascytochrome c to induce apoptosis. Their primary functionis the generation of ATP by oxidative phosphorylation. Inaddition, they also play a critical role in a number of otheraspects of cell signalling, particularly Ca2 + signalling. Mi-tochondria contribute to the dynamics of Ca2 + signalling(Module 5: Figure Ca2 + uptake and extrusion) by par-ticipating in the OFF reactions that remove Ca2 + fromthe cytoplasm during the recovery phase (Module 2: Fig-ure Ca2 + transient mechanisms). The mitochondrial Ca2 +

uptake process is responsible for the mitochondrial mod-ulation of Ca2 + signals. Mitochondria function as Ca2 +

buffers capable of shaping both the amplitude and the spa-tiotemporal profile of Ca2 + signals. Mitochondrial Ca2 +

release mechanisms return Ca2 + back into the cytosol,where it can be sequestered by the endoplasmic reticulum(ER)/sarcoplasmic reticulum (SR). Indeed, there is a closefunctional relationship between the mitochondria and theER/SR.

An endoplasmic reticulum (ER)/mitochondrial Ca2 +

shuttle, which is important for intracellular Ca2 + dynam-ics and cell signalling, can be both beneficial and deleter-ious. With regard to the former, an increased Ca2 + con-centration within the mitochondrial matrix stimulates en-

zymes associated with the tricarboxylic acid (TCA) cycle,resulting in an increase in ATP production. Therefore thereis a two-way relationship between cytosolic Ca2 + signalsand mitochondrial function. In addition to the mitochon-drial modulation of Ca2 + signals, mentioned above, thereis a reciprocal Ca2 + modulation of mitochondrial func-tion. For example, the uptake of Ca2 + acts to stimulatethe oxidative processes that produce ATP. This increasein oxidation also enhances mitochondrial reactive oxy-gen species (ROS) formation (Module 2: Figure sites ofROS formation) that contributes to the redox signallingpathway. In addition, an alteration in the normal ebb andflow of Ca2 + through the mitochondria can be deleteriouswhen an abnormally high load of Ca2 + is transferred fromthe ER/SR to the mitochondrion. This excessive uptake ofCa2 + into the mitochondria can activate the formationof the mitochondrial permeability transition pore (MTP),which results in the release of proteins such as cytochromec, which induce the caspase cascade that contributes tothe apoptotic signalling network. Indeed, a large num-ber of cell death signals appear to operate through theER/mitochondrial Ca2 + shuttle.

Generation of ATPThe mitochondrion is often referred to as the ‘powerhouse’of the cell because of its ability to generate ATP. However,this role in energy transformation is intimately connectedwith its other role, which is to modulate Ca2 + signalling(Module 5: Figure mitochondrial Ca2 + signalling). Mito-chondria are powered by pyruvate, which enters the tri-carboxylic acid (TCA) cycle to produce NADH, whichprovides the electrons that travel down the electron trans-port chain, during which protons are ejected into the cyto-plasm. Oxygen is the final electron acceptor, but approx-imately 25% of the oxygen consumed is incompletely re-duced and appears as the superoxide radical (O2 − •), andthis mitochondrial reactive oxygen species (ROS) forma-tion can contribute to the redox signalling pathway. Theremoval of H+ creates the large membrane potential of–180 mV, which is used to energize both ATP synthesis bythe ATP synthase and the uptake of Ca2 + by the uniporter.This uptake of Ca2 + has a maximum velocity that is verymuch larger than the Ca2 + exchanger that returns Ca2 +

to the cytoplasm, which means that mitochondria can rap-idly accumulate large amounts of Ca2 + , much of which isbound to mitochondrial buffers or it precipitates as crystalsof calcium phosphate. These buffers ensure that the con-centration of Ca2 + within the matrix does not rise muchabove 1 μM. During the recovery phase of Ca2 + signals,the accumulated Ca2 + is returned to the cytoplasm by themitochondrial Na+ /Ca2 + exchanger. Under exceptionalcircumstances, when Ca2 + overwhelms the mitochondria,the mitochondrial permeability transition pore (MTP) isactivated to speed up the release of Ca2 + . Ca2 + has twomain signalling functions within the mitochondrial matrix:it activates the MTP and it also stimulates the TCA cycle toenhance the formation of ATP. One of the consequences ofthe latter is an increase in the production of O2

− •, whichacts synergistically with Ca2 + to activate the MTP.

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Cell Signalling Biology Michael J. Berridge � Module 5 � OFF Mechanisms 5 �23

Module 5: Figure sodium/calcium exchangers

1 12 23 34 45 566

7 78 89 9 10 11

N1 1

N

3Na 4Na

3Na 4Na

1Ca 1Ca 1K

1Ca 1Ca

+ + +

+

2+ 2+ +

2+ 2+

C

C

NCX NCKX

X

XIP

2+Ca regulation Splice site Splice site

3Na

3Na

1Ca

1Ca

+

+

2+

Reversemode

+

X

2+

X

Structure and function of sodium/calcium exchangers.The organization and operation of the Na+ /Ca2 + exchanger (NCX) is shown on the left and the Na+ /Ca2 + –K+ exchanger (NCKX) is shown on theright. There is a large central cytoplasmic loop, part of which has a site where alternative splicing occurs. NCX also has a Ca2 + regulatory site and abinding site for the exchanger inhibitory peptide (XIP). Both exchangers have two regions (α1 and α2, shown in yellow) containing α repeats that arehighly homologous and are thought to be the sites where the cations bind to the exchangers. See the text for further details.

Module 5: Figure mitochondrial Ca2 + signalling

H+

H+

H+

H+

V= -180mV

Ca2+

Ca

Ca

2+

Caphosphate

2+

2+

Na+

MTP

PTAsynthase

aC/ aNexchanger

Caretropinu

2+

pyruvate

pyruvate

Acetyl CoA

NADH

e-

O2

H O2

Citricacidcycle

+

+ +

+

ADP

MICU1

ATP

O2

Buffers

2++

Relationship between mitochondrial metabolism and Ca2 + signalling.Mitochondria are energized by pyruvate, which enters the mitochondrion where it is metabolized by the tricarboxylic acid (TCA) cycle (or citric acidcycle) to produce the reducing equivalents that are transferred to the electron transport chain located in the inner mitochondrial membrane. Thesubsequent extrusion of protons creates a large (–150 to –180 mV) negative membrane potential across this inner membrane. This large protongradient is used to energize both ATP synthesis and the uptake of Ca2 + . Protons flowing back through the oligomycin-sensitive ATP synthase drivethe synthesis of ATP.

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Cell Signalling Biology Michael J. Berridge � Module 5 � OFF Mechanisms 5 �24

Module 5: Figure mitochondrial Ca2 + oscillations

Mitochondrial tracking of cytosolic Ca2 + transients in hepatocytes.Hepatocytes stimulated with phenylephrine (PE) generate repetitive Ca2 + transients (lower trace, [Ca2 + ]c). The Ca2 + concentration in the mitochondria(upper trace, [Ca2 + ]m) closely tracked the cytosolic oscillation. Reproduced from Biochim. Biophys. Acta, Vol. 1366, Robb-Gaspers, L.D., Rutter,G.A., Burnett, P., Hajnoczky, G., Denton, R.M. and Thomas, A.P. (1998) Coupling between cytosolic and mitochondrial calcium oscillations: role in theregulation of hepatic metabolism, pp. 17–32. Copyright (1998), with permission from Elsevier; see Robb-Gaspers et al. 1998.

Mitochondrial Ca2 + uptakeIn resting cells, the concentration of Ca2 + within the mi-tochondrial matrix is 80–200 nM, which is close to the levelin the cytoplasm. When the cytosolic level of Ca2 + beginsto rise, Ca2 + enters the mitochondrion through a Ca2 +

mitochondrial uniporter driven by the same proton gradi-ent that is used to power ATP synthesis (Module 5: Figuremitochondrial Ca2 + signalling). A mitochondrial Ca2 +

release mechanism then returns Ca2 + from the mitochon-drial matrix back into the cytoplasm. This ebb and flow ofCa2 + through the mitochondrion is dramatically demon-strated when cells are generating repetitive Ca2 + spikes(Module 5: Figure mitochondrial Ca2 + oscillations).

Mitochondrial uniporterA uniporter located in the inner mitochondrial membraneis responsible for taking up Ca2 + from the cytoplasm(Module 5: Figure mitochondrial Ca2 + signalling). Sincethe uniporter functions as a channel, it is able to take upCa2 + over a wide range of concentrations. It can takeup Ca2 + slowly at the normal global levels of Ca2 + (ap-proximately 500 nM). As the concentration continues torise, uptake increases in a steeply Ca2 + concentration-dependent manner, with half-maximal activation occurringat around 15 μM. Mitochondria can accumulate as muchas 25–50% of the Ca2 + released from the endoplasmic re-ticulum (ER). This ability to sequester Ca2 + quickly atsuch high concentrations means that the mitochondria areparticularly effective at accumulating Ca2 + when they lieclose to Ca2 + channels, where the elementary events gen-erate very high local concentrations of Ca2 + . Under theseconditions, the uptake of Ca2 + is so fast that it can tem-porarily collapse the mitochondrial membrane potential,and such depolarizations have been recorded in neurons

following Ca2 + entry through voltage-operated channels(VOCs).

The uptake of Ca2 + through the uniporter appears tobe regulated by mitochondrial calcium uptake 1 (MICU1),which has two E-F hands that may function to monitorthe external level of Ca2 + .

Mitochondrial Ca2 + releaseThe Ca2 + that is taken up by mitochondria during sig-nalling is released back to the cytoplasm by various effluxpathways:

Na+ -dependent Ca2 + effluxLike so many functions in the mitochondrion, this modeof efflux is driven by the negative membrane potential.Ca2 + is extruded from the mitochondrion by means ofa Na+ /Ca2 + exchanger, which has a stoichiometry ofthree Na+ ions for one Ca2 + ion. The Na+ that entersdown the electrochemical gradient is exchanged for Ca2 +

(Module 5: Figure mitochondrial Ca2 + signalling). Phar-macological agents such as amiloride, diltiazem, bepridiland CGP37157 inhibit the exchanger.

Na+ -independent Ca2 + effluxMitochondria appear to have a Na+ -independent Ca2 +

release mechanism that is rather slow and may play a rolein extruding Ca2 + under resting conditions. Extrusion isdependent on the transmembrane potential, and may de-pend upon an H+ /Ca2 + antiporter.

Endoplasmic reticulum (ER)/mitochondrialCa2 + shuttleThe function of the endoplasmic reticulum (ER) is in-timately connected with that of the mitochondria. The

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Cell Signalling Biology Michael J. Berridge � Module 5 � OFF Mechanisms 5 �25

Module 5: Figure ER/mitochondrial shuttle

ATP

ATP

ATP

Cytochrome c

Apoptosis inducingfactor(AIF)

CypD

ADP

ADP

ADP

2+

2+

[Ca ]

[Ca ]

Lumen

Matrix

SERCA

MFN2

MFN1 or MFN2

Ca2+

Ca2+

Ca2+

InsP R3

H

Na

+

+

+

Endoplasmicreticulum

Mitochondrion

MTP

Uniporter

TNA

VDAC

OMM

IMM

Excesscalcium

Bak/Bax Bcl-2 ER-mitochondrialcalcium shuttle

XIAP SMAC/DIABLO_

_

1UCIM

The endoplasmic reticulum (ER)/mitochondrial Ca2 + shuttle.The endoplasmic reticulum (ER) and the mitochondria are functionally linked through their participation in Ca2 + signalling. During Ca2 + signalling,a small bolus of Ca2 + is periodically released to the cytoplasm by the inositol 1,4,5-trisphosphate receptors (InsP3Rs). While most of the Ca2 + isreturned to the ER by the sarco/endo-plasmic reticulum Ca2 + -ATPase (SERCA) pump, a proportion enters the mitochondria through the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane (OMM), and then through the uniporter in the inner mitochondrial membrane(IMM). Mitochondrial calcium uptake 1 (MICU1) may regulate the uniporter. The Ca2 + in the mitochondrial matrix is then returned to the ER through aseries of exchangers, channels and pumps. Firstly, Ca2 + leaves the matrix through a Na+ /Ca2 + exchanger and then enters the cytoplasm throughthe VDAC. Once in the cytoplasm, it can be taken back into the ER by the SERCA pumps.

concept of an ER/mitochondrial Ca2 + shuttle has emergedfrom the fact that the ER and the mitochondria forma highly dynamic interconnected network that functionsboth to generate and to modulate Ca2 + signals (Module5: Figure ER/mitochondrial shuttle). The close associ-ation between these two organelles is maintained by themitofusins (MFNs). The Ca2 + stored within the ER lu-men is released into the cytoplasm by inositol 1,4,5-tri-sphosphate receptors (InsP3Rs) and ryanodine receptors(RYRs) to provide the cytosolic Ca2 + signal to activatemany cellular processes. During the recovery phase, thiscytosolic Ca2 + can be dealt with in different ways. It canbe returned directly to the ER by the sarco/endo-plasmicreticulum Ca2 + -ATPase (SERCA) pump. Alternatively,Ca2 + is taken up by the mitochondrion and then returnedto the ER through the ER/mitochondrial shuttle. The mi-tochondria assist with recovery phase by rapidly seques-tering some of the released Ca2 + and then later returningit to the ER. During normal signalling there is thereforea continuous ebb and flow of Ca2 + between these twoorganelles. The normal situation is for most of the Ca2 +

to reside within the lumen of the ER, except during Ca2 +

signalling, when a variable proportion passes through themitochondria. At equilibrium, the bulk of internal Ca2 +

is therefore in the ER, where it not only functions as areservoir of signal Ca2 + , but also plays an essential role inprotein processing within the ER.

During various forms of stress, the normal distribu-tion of Ca2 + is altered and can result in stress signallingand apoptosis. For example, a decrease in the ER con-tent of Ca2 + can initiate the endoplasmic reticulum (ER)stress signalling pathway (Module 2: Figure ER stress sig-nalling). If the Ca2 + that is lost from the ER is takenup by the mitochondria, it can result in opening of themitochondrial permeability transition pore (MTP), col-lapse of the mitochondrial membrane potential and therelease of factors [e.g. cytochrome c, apoptosis-inducingfactors (AIFs) and second mitochondrial-derived activatorof caspases (SMAC), which is also known as the directIAP-binding protein with low pI (DIABLO)] that activ-ate the caspase cascade responsible for apoptosis (Module5: Figure ER/mitochondrial shuttle). SMAC/DIABLOfunctions to inhibit X-chromosome-linked inhibitor ofapoptosis protein (XIAP), which is a potent inhibitorof caspases 3, 7 and 9. A build-up of matrix Ca2 + willalso increase the production of reactive oxygen species(ROS), which contribute to the activation of the MTPthat is responsible for Ca2 + -induced apoptosis. The Bcl-2superfamily control of Ca2 + signalling might dependupon an alteration of this ER/mitochondrial shuttle. Thiscontrol seems to be exerted through Bcl-2 that bindsto the InsP3R to reduce the release of Ca2 + (Module5: Figure ER/mitochondrial shuttle). This protective ef-fect of Bcl-2 is neutralized by Bak and Bax through

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Cell Signalling Biology Michael J. Berridge � Module 5 � OFF Mechanisms 5 �26

Module 5: Figure mitochondria and Ca2 + entry

Mitochondrial modification of Ca2 + entry.In this study on Jurkat leukaemic T cells, thapsigargin (TG) added in a Ca2 + -free solution (0 Ca) emptied the internal Ca2 + store by blockingthe sarco/endo-plasmic reticulum Ca2 + -ATPase (SERCA) pumps. This empty store then activated a store-operated channel (SOC), as evident bythe marked increase in Ca2 + that occurred when 2 mM Ca2 + was added back to the bathing medium (black bar). In cells that had been treatedwith carbonyl cyanide m-chlorophenylhydrazone (CCCP) to inhibit the mitochondria (dotted line), the initial peak was the same, indicating that theentry channel was fully activated, but this response then declined to a much lower level. This lower plateau probably results from the fact that themitochondria lying close to the entry channels remove Ca2 + , thereby negating its inhibitory effect on entry (Module 9: Figure T-cell Ca2 + signalling).Reproduced from The Journal of Cell Biology, 1997, vol. 137, pp. 633–648 by copyright permission of The Rockefeller University Press; see Hothet al. 1997.

their ability to bind Bcl-2, thus pulling it away from theInsP3R.

Mitochondrial permeability transition pore (MTP)When describing mitochondrial Ca2 + release mechanisms,it is important to include the mitochondrial permeabil-ity transition pore (MTP), which is particularly importantwith regard to the activation of apoptosis. The MTP issometimes referred to as the permeability transition pore(PTP). The MTP is the focal point of many apoptotic sig-nals, including Ca2 + , reactive oxygen species (ROS), andpossibly also members of the Bcl-2 superfamily. One ofthe signals activating the MTP is an abnormal accumula-tion of Ca2 + in the mitochondria, which results in collapseof the mitochondrial membrane potential and the releaseof factors that activate the caspase cascade (Module 5: Fig-ure ER/mitochondrial shuttle). Mitochondria thus play apivotal role in regulating apoptosis because they lie at thecentre of a complex web of interactions that link apoptoticsignals to the caspase cascade (Module 11: Figure apop-tosis).

The MTP is a non-selective channel with a very highconductance (pore radius 1–1.3 nm) capable of releasingboth metabolites and ions. The sudden release of pro-tons completely depolarizes the potential with immedi-ate cessation of most mitochondrial functions, togetherwith a catastrophic release of essential components suchas cytochrome c and apoptosis-inducing factors (AIFs)that then go on to activate apoptosis (Module 5: FigureER/mitochondrial shuttle). As such, the mitochondrionhas a central role in the process of apoptosis, and there ismuch interest concerning the nature of this channel andhow it is activated.

The MTP is composed of components that have nor-mal functions within the mitochondrion. The pore in theinner mitochondrial membrane (IMM) is the adenine nuc-leotide translocase (ANT) that normally functions as agated port mediating the entry of ADP and the release ofATP. Under certain conditions, especially a high level ofCa2 + within the matrix, the translocase opens up to formthe non-selective pore. Bongkrekic acid, which binds toANT, is a potent inhibitor of apoptosis. This ANT is asso-ciated with the voltage-dependent anion channel (VDAC),which normally functions to enhance the permeability ofthe outer membrane. Pore opening requires cyclophilin-D (CyP-D), which might act to control the assembly ofthe MTP. CyP-D is a mitochondrial isoform of a familyof cyclophilins that are sensitive to cyclosporin A (CsA),which not only functions as an immunosuppressant, butalso is a potent inhibitor of apoptosis.

A large number of factors control the opening of theMTP. Two key factors are an increase in matrix Ca2 +

concentration and oxidation of dithiols located on ANT.Opening of the MTP seems to require both oxidative stressand an increase in Ca2 + (Module 5: Figure mitochondrialCa2 + signalling). It seems that overloading of the mito-chondria is not in itself deleterious, unless it occurs in thepresence of other factors, such as a change in the redox stateor a decline in the level of ATP. Some of the free radicalsthat open MTP are produced within the mitochondrion,because some of the oxygen is incompletely reduced andappears as the superoxide radical (O2

− •). The O2− • can

then oxidize the vic-thiols on ANT to facilitate the con-formational change that opens the pore (Module 5: Figuremitochondrial Ca2 + signalling). This oxidative mechanismis normally prevented by the highly reduced state within

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Cell Signalling Biology Michael J. Berridge � Module 5 � OFF Mechanisms 5 �27

Module 5: Figure chromaffin cell Ca2 + transients

Mitochondrial modulation of the shape of Ca2 + transients in adrenalchromaffin cells.In response to a 500 ms depolarization (first arrow) to activate Ca2 +

entry, there was a rapid increase in Ca2 + concentration that recoveredrapidly to give a very sharp transient (first arrow). When this stimulus wasrepeated in the presence of carbonyl cyanide m-chlorophenylhydrazone(CCCP) to inhibit the mitochondria, the transient had a much smalleramplitude and a much longer recovery period. This experiment demon-strates how mitochondria accelerate the recovery phase by soaking upCa2 + . Reproduced from The Journal of Cell Biology, 1997, vol. 136,pp. 833–844 by copyright permission of The Rockefeller University Press;see Babcock et al. 1997.

the mitochondrial matrix that is maintained by high levelsof glutathione (GSH).

Mitochondrial modulation of Ca2 + signalsMitochondria can take up large quantities of Ca2 + veryrapidly and thus can modulate various aspects of Ca2 +

signalling. In addition to functioning as a Ca2 + buffer,mitochondria can also modulate the flow of Ca2 + throughboth the entry and release channels.

Mitochondria function as immobile buffers. The Ca2 +

that is taken up during the course of a Ca2 + signal is thenreleased back into the cytoplasm, where it is either returnedto the endoplasmic reticulum (ER) or pumped out of thecell. The level of Ca2 + within the mitochondrial matrixis held constant by means of buffers and by formationof a calcium phosphate precipitate (Module 5: Figure mi-tochondrial Ca2 + signalling). During prolonged periodsof stimulation, large amounts of Ca2 + are taken up by themitochondrion, and this is then gradually unloaded duringperiods of rest. In the case of nerve terminals, for example,it can take up to 10 min for the mitochondrial level of Ca2 +

to return to its resting level following a period of intensestimulation.

This ability of Ca2 + to sequester large amounts of Ca2 +

can markedly modify both the shape and the amplitudeof cytosolic Ca2 + signals. An example of the former isthe ability of mitochondria to enhance Ca2 + signals bydampening out the negative feedback effects that nor-mally limit the activity of Ca2 + channels, as occurs inT cells (Module 9: Figure T-cell Ca2 + signalling). When

Module 5: Figure cytosolic and mitochondrial Ca2 + transients

Relationship between cytosolic Ca2 + transients and mitochondrialmetabolism in hepatocytes.Cytosolic Ca2 + oscillations ([Ca2 + ]c), which were induced by phenyleph-rine, markedly enhanced mitochondrial metabolism, as monitored bymeasuring the level of NAD(P)H. The three cells depicted here had verydifferent spike frequencies. Cell A had a low frequency and illustrateshow a single spike resulted in a marked increase in NAD(P)H, which thenrecovered very much more slowly than the Ca2 + transient. In cells B andC, which were spiking at higher frequencies, the repetitive Ca2 + spikesoccurred before recovery was complete, thus giving either a sawtoothprofile (cell B) or a maintained elevation of mitochondrial metabolism (cellC). Reproduced from Biochim. Biophys. Acta, Vol. 1366, Robb-Gaspers,L.D., Rutter, G.A., Burnett, P., Hajnoczky, G., Denton, R.M. and Thomas,A.P. (1998) Coupling between cytosolic and mitochondrial calcium oscil-lations: role in the regulation of hepatic metabolism, pp. 17–32. Copyright(1998), with permission from Elsevier; see Robb-Gaspers et al. 1998.

mitochondria are inhibited, the entry of external Ca2 + ismarkedly reduced (Module 5: Figure mitochondria andCa2 + entry).

Mitochondria can also modify the shape of Ca2 + tran-sients, which depend upon the sequential activation ofON and OFF reactions (Module 2: Figure Ca2 + transi-ent mechanisms). The sharpness of the transients depends

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 5 � OFF Mechanisms 5 �28

Module 5: Figure mitochondrial motility

Milton

Camicrodomain

Endoplasmic reticulum

Microtubule

Low calcium

Kinesinmotor

High calcium

2+

Miro

MiltonMiro

Milt

onM

iro

MiltonMiro

Motor detaches from the microtubuleto deposit mitochndria in microdomainswhere calcium levels are elevated

Control of mitochondrial motility by Ca2 + : a working hypothesis.When Ca2 + concentrations are low, mitochondria are pulled along microtubules by kinesin motors. Proteins such as Milton and Miro attachmitochondria to the kinesin motor. The latter is important for Ca2 + control, because it contains two EF-hands. The microdomains of Ca2 + formedaround channels like the inositol 1,4,5-trisphosphate receptor (InsP3R) or the ryanodine receptor (RYR) activate Miro, which then induces the motorsto detach from the microtubules thus depositing the microchondria in regions of high Ca2 + concentration.

not only on how quickly Ca2 + is introduced into the cyto-plasm, but also on how quickly it is removed by the variousOFF reactions. The mitochondria play an important rolein the kinetics of the recovery phase because this becomesconsiderably prolonged when their activity is inhibited(Module 5: Figure chromaffin cell Ca2 + transients).

Ca2 + modulation of mitochondrial functionAerobic ATP synthesis by the mitochondria is tightlyregulated. There is a direct control mechanism exercisedthrough the ATP/ADP ratio that automatically increasesmetabolism when the level of ATP declines. In addition,Ca2 + functions as a catabolic signal in that it activatespyruvate dehydrogenase, oxoglutarate dehydrogenase andisocitrate dehydrogenase, key regulatory enzymes of thetricarboxylic acid (TCA) cycle (Module 5: Figure mito-chondrial Ca2 + signalling). When Ca2 + builds up withinthe mitochondrion, it activates the TCA cycle, that thenincreases the supply of reducing equivalents and hence anincrease in ATP formation (Module 5: Figure cytosolicand mitochondrial Ca2 + transients). This feedback mech-anism is an example of the interaction between metabolicmessengers and cell signalling pathways (Module 2: Fig-ure metabolic signalling). Such an interaction may explainhow pyruvate can markedly enhance cardiac cell Ca2 + sig-nalling (Module 2: Figure pyruvate and Ca2 + signalling).

The increase in mitochondrial metabolism will also en-hance the formation of the superoxide radical (O2

− •), andthis mitochondrial reactive oxygen species (ROS) forma-tion can contribute to redox signalling. In addition, O2

− •

formation within the mitochondria can act synergisticallywith Ca2 + to open the mitochondrial permeability trans-ition pore (MTP).

Mitochondrial motilityMitochondria are not static in cells. They move around tocellular regions where metabolic demands are high. Thismovement is particularly evident in neurons, where theirenergy demands are widely dispersed because of their com-plex morphology (Module 10: Figure neuronal morpho-logy). For example, during gene transcription, energy isrequired at the soma, but when information is being pro-cessed at the synapses on the spines and dendrites, energydemand will shift from the cell body to the periphery.

Mitochondria travel around the cell attached to the mi-crotubules (MTs) and are propelled by plus end-directedkinesin and minus end-directed dynein motor proteins(Module 5: Figure mitochondrial motility). The way inwhich these motors are controlled to direct mitochon-dria to different regions in the cell is still somewhat ofa mystery. However, there is clear experimental evidencethat the movement of mitochondria is rapidly inhibitedby increases in intracellular Ca2 + . One way in whichCa2 + inhibits mitochondrial movement depends on themitochondrial Rho-GTPase (Miro) protein family (Miro1 and Miro 2). These Rho-GTPases have two EF-handCa2 + -binding domains. A current hypothesis is that Miroand associated proteins, such as Milton, might be part ofa complex that attaches the mitochondria to the kinesinmotor, which is the primary anterograde mitochondrial

C©2012 Portland Press Limited www.cellsignallingbiology.org

Cell Signalling Biology Michael J. Berridge � Module 5 � OFF Mechanisms 5 �29

motor (Module 4: Figure kinesin cargo transport in neur-ons). In regions of high Ca2 + , Miro functions as the sensorresponsible for detecting Ca2 + and this results in the mo-tor detaching from the microtubule (Module 5: Figure mi-tochondrial motility). The motor domain of kinesin inter-acts with Miro when the latter is bound to Ca2 + . Such amechanism could explain how mitochondria accumulatein regions where there is intense activity, since this is alsolikely to be where there are microdomains of Ca2 + .

Mitochondrial Rho-GTPase (Miro)The mitochondrial Rho-GTPase (Miro) protein family hastwo members (Miro 1 and Miro 2). They are GTPase-activating proteins (GAPs) that have two EF-hand Ca2 + -binding domains. They function together with Milton toform a complex that attaches the mitochondria to the kin-esin motor such as kinesin-3 (Module 5: Figure mitochon-drial motility).

Mitofusins (MFNs)The mitofusins (MFNs) are dynamin-like GTPases thatfunction in mitochondrial fusion. They also have an ad-ditional function in holding together the mitochondriaand the endoplasmic reticulum, which is of critical im-portance for the operation of the endoplasmic retic-ulum (ER)/mitochondrial Ca2 + shuttle (Module 5: Fig-ure ER/mitochondrial shuttle). The ER membrane hasMFN2 that forms dimeric anti-parallel complexes witheither MFN2 or MFN1 located in the outer membrane ofthe mitochondrion.

Charcot-Marie-Tooth disease 2A is caused by mutationsin MFN2.

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C©2012 Portland Press Limited www.cellsignallingbiology.org