mcb cell signaling lectures 1 & 2 ken blumer dept
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Lecture 1 General Concepts of Signal Transduction Cell Communication Types of Receptors Molecular Signaling Receptor Binding Scatchard Analysis Competitive Binding Second Messengers G proteinsTRANSCRIPT
MCB Cell Signaling Lectures 1 & 2
Ken Blumer
Dept. of Cell Biology & Physiology
506 McDonnell Sciences
362-1668
Lecture 1
General Concepts of Signal TransductionCell CommunicationTypes of ReceptorsMolecular Signaling
Receptor BindingScatchard AnalysisCompetitive Binding
Second Messengers
G proteins
Modes of cell communication
Lodish, 20-1
Four classes of cell-surface receptors
Lodish, 20-3
Transmitting/transducing signals within cells:
3 basic modes (may be combined)
1. Allostery
2. Covalent modification
3. Proximity (= regulated recruitment)
P
Shape change, often induced by binding a protein or small moleculeSwitching can be very rapid
Modification itself changes molecule’s shapeMemory device; may be reversible (or not)
Regulated molecule may already be in “signaling mode;” induced proximity to a target promotes transmission of the signal
P P
Finding and analyzing receptors:Ligand binding assays
Saturation Binding studiesCan be performed in intact cells, membranes, or purified receptors1. Add various amounts of labeled ligand (drug, hormone, growth factor)2. To determine specific binding, add an excess of unlabeled ligand to compete for specific binding sites.QU: Why is there non-specific binding?3. Bind until at equilibrium4. Separate bound from unbound ligand5. Count labeled ligand
[Adapted from A. Ciechanover et al., 1983, Cell 32:267.]
Receptor: ligand binding must be specific, saturable, and of high affinity
Receptor abundance, affinity, cooperativity:Scatchard plots
Slope = - 1/Kd
X intercept = # rec
(Bound Lig)
(Bound Lig)(Free)
For an excellent discussion of principles of receptor binding, andpractical considerations, see http://www.graphpad.com; also posted on MCB website.
Cooperativity indicated by non-linearScatchard plots
(Bound Lig)
(Bound Lig)(Free)
Negative cooperativity: binding of ligand to first subunit decreases affinity of subsequent binding events.
Positive cooperativity: binding of ligand to first subunit increasesAffinity of subsequent binding events. Example: hemoglobin binding O2
What receptors do:Generate second messengers
• Cyclic nucleotides: cAMP, cGMP• Inositol phosphate (IP)• Diacylglycerol (DAG)• Calcium• Nitric oxide (NO)• Reactive oxygen species (ROS)
Molecular mediators of signal transduction. Cells carefully, and rapidly, regulate the intracellular concentrations. Second messengers can be used by multiple signaling networks (at the same time).
cAMP regulates protein kinase (PKA) activity
Alberts 15-31,32
Positive cooperativity--binding of increases affinity for second cAMP
PKA targets include Phosphorylase kinase and the transcription regulator, cAMP response element binding (CREB) protein
Lipid-derived second messengers:Diacylglycerol and inositol phosphates
Alberts, 15-35
IP3 evokes calcium release as third messenger
Lodish, 20-39
A key effector of Ca2+-CaM:CaM-kinase II
Alberts, 15-41
NO signaling
Lodish, 20-42
NO effects are local, since it has half-life of 5-10 seconds (paracrine).NO activates guanylate cyclase by binding heme ring (allosteric mechanism)
Gases can act as second messengers!
Discovery of NO signaling
Robert F Furchgott showed that acetylcholine-induced relaxation of blood vessels was dependent on the endothelium. His "sandwich" experiment set the stage for future scientific development. He used two different pieces of the aorta; one had the endothelial layer intact, in the other it had been removed.
Louis Ignarro reported that EDRF relaxed blood vessels. He also identified EDRF as a molecule by using spectral analysis of hemoglobin. When hemoglobin was exposed to EDRF, maximum absorbance moved to a new wave-length; and exposed to NO, exactly the same shift in absorbance occurred! EDRF was identical with NO.
Furchgott, Ignarro, Murad, Nobel Prize 1998
http://www.nobel.se/medicine/laureates/1998/illpres/index.html
G proteins:Switches linking receptors & 2nd messengers
• Discovery and Structure of Heterotrimeric G proteins
• Signaling pathways of G proteins• Receptors that activate G proteins• Small G proteins-discovery and structure• Activation and inactivation mechanisms• Alliance for Cell Signaling (AfCS)
Signal Transduction by G proteins
• Discovery and Structure of Heterotrimeric G proteins
• Signaling pathways of G proteins• Receptors that activate G proteins• Small G proteins-discovery and structure• Activation and inactivation mechanisms• Alliance for Cell Signaling (AfCS)
G protein signal transduction
Neves, Ram, Iyengar, Science 2002
Hydrolysis of GTP
• Arg & Gln stabilize the b and g phospates of GTP molecule in correct orientation for hydrolysis by H2O
• Hydrolysis leads to major conformation change in Gs a
• Mutations in the Gln or Arg (or ADP ribosylation by cholera toxin) blocks the ability to stabilize transition state, and therefore locks G protein in the “on” position.
• Examples include adenomas of pituitary and thyroid glands (GH secreting tumors, acromegaly), and McCune-Albright syndrome.
Iiri, et al. NEJM (1999)
Signal Transduction by G proteins
• Discovery and Structure of Heterotrimeric G proteins
• Signaling pathways of G proteins• Receptors that activate G proteins• Small G proteins-discovery and structure• Activation and inactivation mechanisms• Alliance for Cell Signaling (AfCS)
G protein-coupled receptors (GPCRs)• Many ligands
• Robust switches• Multiple effectors• Conserved 7 TM
architecture• More than 50% of
drugs target GPCRs
Bockaert & Pin, EMBO J (1999)2012 Nobel Prize
Lefkowitz Kobilka
GPCR desensitization mechanisms
Arrestins act as scaffolds for ERK and JNK signaling pathways
Lefkowitz reviews
Signal Transduction by G proteins
• Discovery and Structure of Heterotrimeric G proteins
• Signaling pathways of G proteins• Receptors that activate G proteins• Small G proteins-discovery and structure• Activation and inactivation mechanisms• Alliance for Cell Signaling (AfCS)
Reverse genetics: express one or two mutant versions of the protein of interest
Depends on understanding how the machines work
1. Inhibit activity of the protein with a “dominant-negative” interfering mutant of that protein
2. Increase activity of the protein with a “dominant-positive” or “constitutively active” interfering mutant of the protein
The mutant titrates (binds up) a limiting component to block the normal protein’s signal
The mutant exerts the same effect as the normal protein would, if it were activated in the cell
Reverse genetics: small GTPases as examplesDepends on understanding how the machines work
“Dominant-negative” mutation “Dominant-positive”
mutation
The mutant titrates (binds up) a limiting component to block the normal protein’s signal
The mutant exerts the same effect as the normal protein would, if it were activated
GAP
GTP
Pi
GDPGEF
GEFGDP
Binds GEF but cannot replace GDP by GTP; so GEF not available for activating normal protein
Cannot hydrolyze GTP, so remains always active
Signal Transduction by G proteins
• Discovery and Structure of Heterotrimeric G proteins
• Signaling pathways of G proteins• Receptors that activate G proteins• Small G proteins-discovery and structure• Activation and inactivation mechanisms
Small G protein “turn on” mechanisms
First mammalian GEF, Dbl, isolated in 1985 as an oncogene in NIH 3T3 focus forming assay. It had an 180 amino acid domain with homology to yeast CDC24. This domain, named DH (Dbl homology) is necessary for GEF activity.
In 1991, Dbl shown to catalyze nucleotide exchange on Cdc42.
Schmidt & Hall, Genes & Dev. (2002)Dbl= Diffuse B-cell lymphoma
Many RhoGAPsRhoGAPs outnumber the small G proteins Rho/Rac/Cdc42 by nearly 5-fold.Why so much redundancy?Luo group did RNAi against 17 of the 20 RhoGAPs in fly.
Six caused lethality when expressed ubiquitously. Tissue specific expression of RNAi revealed unique phenotypes.
P190RhoGAP implicated in axon withdrawal. Increasing amounts of RNAi caused more axon withdrawal (panels C-G).
Why so many RhoGAPs?Billuart, et al. Cell (2001)
The GTPase switch
Schmidt & Hall, Genes & Dev. (2002)
Growth Factors and Receptor Tyrosine Kinases
• RTK’s--How do they work?• EGFR signaling and ras• MAP kinase cascades• PI3K, PKB, PLCg• PTPs (Protein Tyrosine Phosphatases)
How RTKs (& TK-linked Rs) work
1. Ligand promotes formation of RTK dimers, by different mechanisms:
Ligand itself is a dimer (PDGF)
One ligand binds both monomers (GH)
2. Dimerization allows trans-phosphorylation of catalytic domains, which induces activation of catalytic (Y-kinase) activity
3. Activated TK domains phosphorylate each other and proteins nearby, sometimes on multiple tyrosines
4. Y~P residues recruit other signaling proteins, generate multiple signals
EGF receptor as a model1st RTK to be characterized
v-erbB oncogene = truncated EGFR
How do we know that the EGFR auto- phosphorylates in trans?
Experiment: test WT and short EGFRs, each with or without a kin- mutation
Honneger et al. (in vitro) PNAS 1989; (in vivo) MCB 1999
wt +
Does this result rule out phosphorylation in cis as well?
If not, how can you find out?
PS: What do trans and cis mean?
kin- + +
short kin+ + +short kin- +
How can we know that the EGFR does not autophosphorylate in cis?
Need an EGFR that cannot homodimerize
EGFR family is huge, with many RTK members and many EGF-like ligands
Such receptors often form obligatory heterodimers with a similar but different partnerIf A can dimerize only with A’, then we can inactivate the kinase domain of A’ and ask whether A phosphorylates itself
Answer: NO
QED
Growth Factors and Receptor Tyrosine Kinases
• RTK’s--How do they work?• EGFR signaling and ras• MAP Kinase Cascades• PI3K, PKB, PLCg• PTPs (Protein Tyrosine Phosphatases)
.P
.P .PPP
.P
P PP
Signals generated by the EGFR
The activated dimer phosphorylates itself
T-loop only Multiple sites
Individual Y~P residues recruit specific proteins, generate different signals
SOS, a Ras GEFDocks via intermediate adapters to activate Ras
Ras activates multiple targets (MAPK)
PLC-gDocking of Y-kinases allows Tyr-phos’n of PLC-g, which activates it
PI3-kinaseAdapters again
Docking allosterically activates PI3K
Each signal, in turn, activates a different set of pathways, which cooperate to produce the overall response
Adapters connect A with B, B with C . . . to create complex, localized assemblies of signaling proteins
Each adapter has at least 2 interaction domains, and may have other functions as well
Types of adapter interactions
Y~P sequence motifs allow regulatable adapter functionsSH2 Tyrosine phosphatesPTB Tyrosine phosphates
AlsoSH3 Polyproline-containing sequences
PDZ Specific 4-residue sequences at C-terminiPleckstrin homol. (PH) Phosphoinositides
Many others
A CBP
Adapter 1
Adapter 2
EGF
EGFR
EGFR~P
Grb2
SOS
Ras
Raf
Mek
ERKs
C-Jun
Extracellular GF
RTK
Phospho-RTK
Adapter
Ras-GEF
Small GTPase
Ser kinase
Tyr/thr kinase
Ser kinase
Transcription factor
MechanismProximity
Allostery
Covalent modification
EGF activates the MAPK pathway in multiple steps, with multiple mechanisms
.PPP
.P
PP
SH2SH3
SH3
Grb2SOS
EGFR Activation of Ras: Proximity & Allostery
The PlayersRTK = EGFR
“Rat Sarcoma”Small GTPase, attached to PM by prenyl group
“GF receptor binding 2”Adapter, found in screen for binders to EGFR~P
“Son of Sevenless”GEF, converts Ras-GDP to Ras-GTPFound in Drosophila, homol. To S.c. Cdc25
RasGDP
. .
SH2SH3
SH3
Grb2 SOS
EGFR Activation of Ras: Proximity & Allostery
SOS is “ready to go”: already (mostly) associated with Grb2 in cytoplasm, in the resting state
Even before EGF arrives . . .
RasGDP
EGFR Activation of Ras: Proximity & Allostery
Then . . . Covalent modification
RasGDP
.PPP
.P
PP
EGF-bound dimers trigger phosphorylation, in trans SH2
SH3
SH3
Grb2 SOS
SH2SH3
SH3
Grb2
.PPP
.P
PP
SOS
RasGDP
Grb2’s SH2 domain binds Y~P on EGFR, bringing SOS to the plasma membrane
EGFR Activation of Ras: Proximity & Allostery
Then . . . Proximity
SH2SH3
SH3
Grb2
.PPP
.P
PP
SOS
EGFR Activation of Ras: Proximity & Allostery
RasGDP
GDP
SOS now binds Ras-GDP, causing GDP to dissociate, and . . .
Then . . . Allostery
SH2SH3
SH3
Grb2
.PPP
.P
PP
SOS
EGFR Activation of Ras: Proximity & Allostery
Ras
GTP
GTP enters empty pocket on Ras, which dissociates from SOS and converts into its active conformation
Then . . . Allostery continues
GTP
Raf
SH2SH3
SH3
Grb2
.PPP
.P
PP
SOS
EGFR Activation of Ras: Proximity & Allostery
Ras
GTP
Ras-GTP brings Raf to the PM for activation, and the MAPK cascade is initiated
Finally . . . Proximity again!
GTP
MAPKCascade
Raf
Growth Factors and Receptor Tyrosine Kinases
• RTK’s--How do they work?• EGFR signaling and ras• MAP Kinase Cascades• PI3K, PKB, PLCg• PTPs (Protein Tyrosine Phosphatases)
.
The best understood MAPK cascadeMAPK = Mitogen-activated protein kinase
Raf-1A-rafB-raf
MEK1MEK2
ERK1ERK2
C-Jun
Altered gene expression
Phos’n of T-loop Ser residues
.PP
.PP Phos’n of Ser/Thr
Phos’n of T-loop Thr and Tyr
MAPKKK
MAPKK
MAPK
Res
pons
e 1.0
0.5
00 1 5
Stimulus (multiples of EC50)
MAPKKK
MAPKK
MAPK
Frogoocyte
Progesterone
G2-M transition
Switch-like behavior*
*JE Ferrell, Tr Bioch Sci 22:288, 1997
Responses are not always graded
Amplified sensitivity: reduces noise @ low stimulus; reversible
Bistable responses: off or on, often via positive feedback & used for irreversible responses (e.g., cell cycle)
Other examples?
Instead . . .
All or nothing response in Xenopus oocytesProgesterone, or fertilization, induces germinal vesicle breakdown of Xenopus oocytes--a process mediated by the MAPK cascade.
Question: At a concentration of progesterone that half-maximally activates MAPK (0.01 uM, panel A), are all the oocytes activated halfway (panel B), or are half of the oocytes activated fully (panel C)?
Since Xenopus oocytes are HUGE, one can look at MAPK on a cell by cell basis.
Answer: All or nothing.Ferrell, et al., Science (1998)
Dhanasekaran (2007) Oncogene
Scaffold proteins involved in ERK-signaling pathways
Growth Factors and Receptor Tyrosine Kinases
• RTK’s--How do they work?• EGFR signaling and ras• MAP Kinase Cascades• PI3K, PKB, PLCg• PTPs (Protein Tyrosine Phosphatases
SH2
SH2 p85
.P
P
.P
Pp110
EGFR Activation of PI3K combines Proximity & Allostery
How do we know proximity is not enough?
SH2
SH2 p85 p110
PIP2 PIP3
Recruitment from cytoplasm to PM, via SH2 domains
Activated by EGFR/p85
Can also be activated by Rac or Ras!
1. p85 mutants that activate without binding to RTKs2. Tethering to membrane does not activate
P
PP
P
PIP3 targets include many GEFs, many tyrosine kinases, and others, including . . .
PKB (aka Akt) = ser/thr kinase that promotes cell survival
PIP3(= membrane lipid)
PH K
PKB . . . is inactive in cytoplasm
. . . contains a PH (pleckstrin homology) domain & a kinase domain
P
PP
P PH K
Multi-step activation of PKB: proximity
PIP3
PH K
PH domain recognizes 3’- phosphate of PIP3, bringing kinase domain to the PMProximity to PM
alone does not activate the kinase
P
PP
P PH K P
PP
P PH KP
PPIP3PDK1*
Multi-step activation of PKB: covalent modification
Inactive PKB Active (phos’d) PKB
*PDK1 is also recruited to the membrane via a PIP3-binding PH domain
Overall, two proximity steps plus (at least) one phosphorylation step
.P
P
.P
P
EGFR Activation of PLCg combines THREE inputs
PIP2
1. PROXIMITY: Recruitment from cytoplasm to PM, via SH2 domains
P
PP
P
SH2
SH2PH
Cata- lytic
P
P
PLCg (Inactive, in cytoplasm)
PIP3
.P
P
.P
P
EGFR Activation of PLCg combines THREE inputs
PIP2 DAG
2. COVALENT: Activated by EGFR phosph’n
3. PROXIMITY: Binds to PIP3 via PH domain
P
PP
P
SH2
SH2PH
Cata- lyticP
P
P
InsP3
Growth Factors and Receptor Tyrosine Kinases
• RTK’s--How do they work?• EGFR signaling and ras• MAP Kinase Cascades• PI3K, PKB, PLCg• PTPs (Protein Tyrosine Phosphatases)
PTEN opposes PI3K by removing PI3-phosphatePTEN discovered as a tumor suppressor gene.
Mutated in brain, breast and prostate cancers.
Has homology to dual specificity phosphates, but shows little activity toward phosphoproteins.
Was discovered to remove phosphates from PIPs; thereby providing likely mechanism for tumor suppression.
Cantley & Neel, PNAS (1999)
Gleevec--proof that you can target kinases for drug therapy