biochem 503 december 3, 2008 protein ser/thr phosphatases (ppp family) controlling 99% of cellular...
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Biochem 503 December 3, 2008Protein Ser/Thr Phosphatases (PPP family)
Controlling 99% of cellular phosphorylation
I. HistoryII. Type-1 Protein Phosphatase (PP1)III. Type-2A Protein phosphatase (PP2A)IV. Calcineurin (PP2B) Ca2+/CaM dependentV. PP2C or Mg2+-dependent PPase (MPP)
History of Ser/Thr Protein Phosphatases
1950’s 1. PR Enzyme Gerty Coriglycogen phosphorylase a to b conversion (phosphorylase phosphatase)
1960’s a lost decade? Ed Fischer students struggle
1970’s2. Glycogen synthase PPase - Tsuiki, Tamura, Kikuchi C’ville to Sendai connection…3 different enzymes separated later known as PP1, PP2A and PP2C (MPP)
3. The EtOH ppt method a grad student mistake becomes “standard” method - 35 kDa PP-ase (C subunit)
4. Isolation of inhibitor proteins (1976)..the C + I = CI idea of regulators
5. Same phosphatase for every substrate?…….histone, casein, myosin, tropomyosin, spectrin, ribosomes, enzymes……
“broad specificity” (i.e. non-specific) 35 kDa phosphatase
S. TamuraTohoku Univ.Sendai, Japan
E.Y.C. LeeU. Miami, now NYMC
History of Ser/Thr Protein Phosphatases
1980’s6. Separation of two phosphatases in 35 kDa fraction (Ingebritsen)
Classification (1983) Type 1 vs. type 2 A, B, C based on sensitivity to inhibitor proteins
7. Targeting hypothesis (Cohen, 1985) - glycogen-bound PP1 a dimer of [ PP1C plus glycogen-subunit]R subunits modulate activity and anchor C
8. PP1 binds to multiple different R subunits: GM and MYPT1myosin phosphatase PP1C plus MYPT1
9. PP2A binds 60 and 55 kDa subunits to form ABC trimercommon AC, different Bs (B55, B56, B72)NIH + Sendai + Hiroshima
Sir Phillip CohenDundee, Scotland
History
10. Cloning of C subunits of PP2A and PP1 unrelated to PTPs, but similar to purple acid PP-ase
11. Yeast-fungal genetics show functional conservation GLC7=PP112. Discovery of Okadaic Acid as selective PP1-PP2A inhibitor
1990’s13. Cloning of multiple PPP catalytic subunits PP4, PP5, PP6
Family Conservation & Diversity in Catalytic subunits (see PTW Cohen TIBS 22:245-251)
14. Many PP2A B subunits in Families- B, B’, B”15. Genomics - over 40 human genes, 24 in yeast
Marc MumbyUTSW
Tricia (PTW) Cohen(Lady Cohen)Dundee, Scotland
QuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture.Edgar de Cruz e SilvaDundee, NYC, Portugal
QuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture.
David VirshupUniv. Utah, now SingaporeNUS-Duke Med Sch.
QuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture.Anna DePaoli-RoachIndiana U Med.Sch.
B
The PPP Family of Protein Ser/Thr Phosphatases
Red = humanGreen = DrosophilaYellow = yeast
PP1
PP2APP4PP6
CaN (PP2B)
Toxin-sensitive PPPToxin-sensitive PPP
Natural Toxins from Diverse Sources Bind and Inhibit PPP Protein Phosphatases
DinoflagellatesProrocentrum lima
Blue-green Algae
Blister beetleColeoptera
Streptomyces (fostreus)
Protein Ser/Thr Phosphatases Are Dominant over Protein Kinases
most proteins are maintained in a de-phosphorylated state
Cell Signaling Technology 2002 catalogue (pg. 15): Western blot analysis of whole cell lysates of Jurkat cells,.untreated with 0.1M calyculin A for 20 minutes prior to lysis, using Phospho-Thr antibody.
+ PPP inhibitor
Catalytic Subunit of Protein Phosphatase-1 (PP1)
Okadaic Acid Binds at the Active Site of PPP Protein Phosphatases
Fe O O
H O P O-Ser-substrate
Zn O H-His
Mechanism of Phospho-Ester Hydrolysis by PPP Phosphatases:in-line attack of metal-activated hydroxide,with triginal bipyramid intermediate and inversion of stereochemistryprotonation of the alcoholic leaving group by active site His
A. Catalytic subunit1. Bimetal center Fe::Zn, Mechanism of direct hydrolysis
Type-1 Protein Phosphatase (PP1)
1. Bi-metallic active site with Fe and Zn
2. 3D structure - beta sheet and alpha helix clusters
3. Isoforms differences mostly in C terminal, allow specific antibodies alpha NPGGRPITPPRN--SAKAKK gamma --ATRPVTPPRGMITKQAKK delta NSG-RPVTPPRTANPPK-KR
4. Phosphorylation in RPITPPR motif first found in yeast sds22cyclin-dep kinases (CDK) phosphorylate to inactivate; reversed by auto-dephosphorylation
5. Toxins - microcystin, okadaic acid, calyculin A bind at active site (3D structures)
B. Regulatory/targeting subunits for PP1C1. The pioneer…..glycogen-binding GM
2. The RVxF Motif as primary recognition site3. Myosin phosphatase MYPT1 as regulator & scaffold4. Dozens more, >200 total…..phospho regulation
C. Inhibitor phospho-proteins1. Inh1 and DARPP-322. Inh2, Inh43. CPI-174. Linking kinases to PP1 inhibition-
inhibitors target selective R-C complexes
D. Substrates1. abundant phosphatase,
handles large capacity, abundant substrates…myosin, lamins, glycogen enzymes, histones
Protein Ser(P)/Thr(P) Phosphatase - PP1many different regulatory-targeting subunits
with common catalytic subunit
GL GM
PTG
GADD34
RVSF
KVKF
VXF
C
C
C
CMYPT1
myosin - cytoskeleton
protein synthesis
glycogen metabolism
Neurabin
dendritic spines
PP1
C
Dozens of R/K-VxF subunits
VXF
P
PKC
CPI-17
Myosin Phosphatase: PP1PP1--MYPT1MYPT1
Myosin Phosphatase inhibition by phosphorylation-dependent inhibitor protein CPI-17.
MYPT1
First example of an inhibitor specific for a PP1 Holoenzyme
Myosin Phosphatase Inhibitor CPI-17single residue phosphorylation (not T to D mutation)
condenses conformation and increases potency >2000-fold
Selective Inhibition of Myosin Phosphatase by CPI-17
same C subunit isoform bound to different R subunits
0
50
100
Pho
spha
tase
Act
ivity
(%
)
[P-CPI-17] (-log M)
MYPT1•PP1
Glycogen-bound PP1
10 9 8 7 6 5
Myosin LC Phosphatase: A complex of PP1C with MYPT1 Subunit
C subunit bound to specific R subunit
Type-2A Protein phosphatase (PP2A)
A. Catalytic subunit
1. Bimetal center Fe::Zn and catalytic Mechanism same as PP1
2. 3D structure…known in complex with A and in ABC
3. Isoforms 10:1 ratio, essential for development
DYFLCOOH motif at C terminus conserved phosphorylation - PTKs, eg. Src, JAKmethylation - PMT and PME, alters subunit
association
5. Toxins - MCLR, OA bind at active site. Differences between PP1 and PP2A in12-13
loop
Protein Ser(P)/Thr(P) Phosphatase - PP2A
ABC
C
HEAT Helicalrepeats
Pph21Pph22
ATpd3
B
ABC = { [A] Scaffold + [C] Catalytic} + [B] Regulatory
Binding of C to A decreases Vmax and alters KM
A subunit
Protein Ser(P)/Thr(P) Phosphatase - PP2Acovalent modifications of the catalytic subunit
ABC
C
ATpd3
BRTPDYFLCOOH
Tyr kinasesSrc, JAK, RTK
Tyr307
RTPDYFLCOOMe
PMT
PME
S-AdoMet
RTPDYFLCOOXRTPDYFLCOOX
PTK
self
inactive
OO-P-O
B
B. Scaffold and Regulatory subunits- the ABC’s of PP2A.
1. The Scaffold "A" or PR65 (HEAT repeats) the AC dimer A and A 85:15, but A specific tumor suppressor2. Many Bs….are they for dedicated functions?
Yeast Cdc55 and Rts1 - distinct phenotypes, not complemented (conclusion?)
now 3 B families cloned - more than 15 genesB B55 B’ B56 B’’ B72(130)
3. Tumor antigens, sT, mT, E4orf replace B subunits to re-direct activity.
4. More and more Bs?, p107, p48, etc. proposed as alternates
Protein Ser(P)/Thr(P) Phosphatase - PP2Amultiple dedicated enzymes on one platform
C4
TAP42
B55CDC55
kinase inactivationG-like propeller structure
B’56RDS1
anti-apoptosis integrin signalingpolarity-Wnt signaling
P
B’’72 P retinoids
anti - apoptosis
ABC
CPPH21PPH22
ATpd3
B
PP2A can be > 80 different “enzymes”
sm-t tumor virustransformation
The B56 Family of PP2A Regulatory Subunits
D-weiderborst
Paxillin bindinganti-metastatic
Wnt-B56-Dishevelled
The 3D Structure of PP2A Trimer AB56CNature, Nov. 2006
Side view Top view
C. Inhibitor proteins for PP2A1. Inh1(PP2A) Damuni et al2. Inh2(PP2A) SET protein - important in solid tumors
ceramide “receptor” for PP2A activation
D. Alternative partners1. A binds HSF2 and PP52. C binds alpha4; in yeast part of TOR signaling
E. Substrates1. Signaling Kinases (MEK, PKB, PI3K, p70S6K, etc.)2. anti-apoptosis (PKB, Bad, Bcl)3. integrin signaling - Paxillin B56 deletions & increased invasion
Calcineurin (PP2B)(PPP3)Ca2+/CaM activated Phosphatase
A. Catalytic subunit1. C has Fe::Zn active site like PP1, PP2A2. Suppressor domain, CaM dependent
B. Regulatory subunits1. B subunit (Ca2+)2. additional CaM
3. FKBP + FK-506 or cyclosporin ($B product)C. Inhibitor Proteins
1. induced in response to signaling have reported activity as activator-inhibitor.
D. Substrates1. NFAT (co-transport into nucleus)2. Elk-1 (TCF transcription factor)3. DARPP-32 (NMDA receptor)
Calcineurin (CnA +CaB) Inhibited byCyclosporin (CsA) and its Binding protein (Cyp)
$B drug forimmunosuppressionfollowing organ transplants
PP2C = Mg2+-dependent PPase (MPP)
A. Catalytic subunit1. unrelated to PPP but bimetallic Mg:Mg active center 2. isoforms , etc.3. many new family members in genome
B. Regulatory subunits - none?C. Inhibitor Proteins - none?D. Substrates
1. CDKs2. the kinase activation loop3. PI3K4. Glycogen synthase
Maybe activated by small molecule second messenger? Lipids?
Protein Phosphatase 2C Mg2+-dependent Phosphatase (MPP)
Protein Phosphorylationrapid and reversible biochemical reactions
A molecular on/off switching mechanism.
TARGET
ATP ADP
KINASES
PHOSPHATASES
Pi H2O
TARGET P
Protein Tyr(P) Phosphatases
Yeast 5C. elegans 95Drosophila 22Human 56
Protein Phosphorylation: Kinases & Phosphatases
TARGET P-Tyr TARGET TARGET P-Ser/Thr-TARGET
Protein Tyr Kinases Protein Ser/Thr Kinases
Protein Ser(P)/Thr(P) Phosphatases
Yeast 13C. elegans 51Drosophila 19Human 15
KINASE Superfamily ~ 500 enzymes
Cys-SH Fe::Zn
ATP ATP
Mg MgPPP MPP
Protein Phosphorylation: Kinases & Phosphatases
KINASE Superfamily ~ 500 enzymes
Cys-SH
Fe::Zn
ATP ATP
Mg Mg MPP
ATP
ATPATPATP
ATP
ATP
ATP
Cys-SH
Cys-SH
ATP
ATP
Fe::Zn
Fe::Zn
Phosphatase families and ensembles ~ 500 enzymes
ATP
Protein Kinases and
Protein Phosphatases
ancient enzymes essential to cell signaling
and cellular regulation
New targets for Pharmaceuticals