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Biochem 503 December 3, 2008 Protein Ser/Thr Phosphatases (PPP family) Controlling 99% of cellular phosphor

Biochem 503 December 3, 2008 Protein Ser/Thr Phosphatases (PPP family) Controlling 99% of cellular phosphorylation I. History II. Type-1 Protein Phosphatase (PP1) III. Type-2A Protein phosphatase (PP2A) IV. Calcineurin (PP2B) Ca 2+ /CaM dependent

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Biochem 503 December 3, 2008 Protein Ser/Thr Phosphatases (PPP family) Controlling 99% of cellular phosphor

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  1. Biochem 503 December 3, 2008 Protein Ser/Thr Phosphatases (PPP family) Controlling 99% of cellular phosphorylation I. History II. Type-1 Protein Phosphatase (PP1) III. Type-2A Protein phosphatase (PP2A) IV. Calcineurin (PP2B) Ca2+/CaM dependent V. PP2C or Mg2+-dependent PPase (MPP)

  2. Historyof Ser/Thr Protein Phosphatases 1950’s 1. PR Enzyme Gerty Cori glycogen phosphorylase a to b conversion (phosphorylase phosphatase) 1960’s a lost decade? Ed Fischer students struggle 1970’s 2. 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. Tamura Tohoku Univ. Sendai, Japan E.Y.C. Lee U. Miami, now NYMC

  3. Historyof Ser/Thr Protein Phosphatases 1980’s 6. 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 MYPT1 myosin phosphatase PP1C plus MYPT1 9. PP2A binds 60 and 55 kDa subunits to form ABC trimer common AC, different Bs (B55, B56, B72) NIH + Sendai + Hiroshima Sir Phillip Cohen Dundee, Scotland

  4. History 10. Cloning of C subunits of PP2A and PP1 unrelated to PTPs, but similar to purple acid PP-ase • Yeast-fungal genetics show functional conservation GLC7=PP1 • Discovery of Okadaic Acid as selective PP1-PP2A inhibitor 1990’s 13. Cloning of multiple PPP catalytic subunits PP4, PP5, PP6 Family Conservation & Diversity in Catalytic subunits • (see PTW Cohen TIBS22:245-251) • 14. Many PP2A B subunits in Families- B, B’, B” • 15. Genomics - over 40 human genes, 24 in yeast Marc Mumby UTSW Edgar de Cruz e Silva Dundee, NYC, Portugal Tricia (PTW) Cohen (Lady Cohen) Dundee, Scotland David Virshup Univ. Utah, now Singapore NUS-Duke Med Sch. B Anna DePaoli-Roach Indiana U Med.Sch.

  5. The PPP Family of Protein Ser/Thr Phosphatases Toxin-sensitive PPP PP1 Red = human Green = Drosophila Yellow = yeast PP2A PP4 PP6 CaN (PP2B)

  6. Natural Toxins from Diverse Sources Bind and Inhibit PPP Protein Phosphatases Blister beetleColeoptera Blue-green Algae Dinoflagellates Prorocentrum lima Streptomyces (fostreus)

  7. Protein Ser/Thr Phosphatases Are Dominantover Protein Kinasesmost proteins are maintained in a de-phosphorylated state + PPP inhibitor Cell Signaling Technology 2002 catalogue (pg. 15): Western blot analysis of whole cell lysates of Jurkat cells,.untreated with 0.1M calyculin A for 20 minutes prior to lysis, using Phospho-Thr antibody.

  8. Catalytic Subunit of Protein Phosphatase-1 (PP1) Okadaic Acid Binds at the Active Site of PPP Protein Phosphatases

  9. Mechanism of Phospho-Ester Hydrolysis by PPP Phosphatases: in-line attack of metal-activated hydroxide, with triginal bipyramid intermediate and inversion of stereochemistry protonation of the alcoholic leaving group by active site His Fe O O H O P O-Ser-substrate Zn O H-His

  10. A. Catalytic subunit 1. Bimetal center Fe::Zn, Mechanism of direct hydrolysis

  11. 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 sds22 • cyclin-dep kinases (CDK) phosphorylate to inactivate; • reversed by auto-dephosphorylation • 5. Toxins - microcystin, okadaic acid, calyculin A bind at active site • (3D structures)

  12. B. Regulatory/targeting subunits for PP1C • 1. The pioneer…..glycogen-binding GM • 2. The RVxF Motif as primary recognition site • 3. Myosin phosphatase MYPT1 as regulator & scaffold • 4. Dozens more, >200 total…..phospho regulation • C. Inhibitor phospho-proteins • 1. Inh1 and DARPP-32 • 2. Inh2, Inh4 • 3. CPI-17 • 4. Linking kinases to PP1 inhibition- • inhibitors target selective R-C complexes • D. Substrates • 1. abundant phosphatase, • handles large capacity, abundant substrates… • myosin, lamins, glycogen enzymes, histones

  13. Protein Ser(P)/Thr(P) Phosphatase - PP1many different regulatory-targeting subunitswith common catalytic subunit GL GM PTG Cd Cg1 PP1 a, d, g1 RVSF VXF Neurabin C glycogen metabolism GADD34 C dendritic spines VXF Cd MYPT1 protein synthesis KVKF myosin - cytoskeleton Dozens of R/K-VxF subunits

  14. Myosin Phosphatase inhibition by phosphorylation-dependent inhibitor protein CPI-17. PKCd P MYPT1 CPI-17 Myosin Phosphatase: PP1-MYPT1 First example of an inhibitor specific for a PP1 Holoenzyme

  15. Myosin Phosphatase Inhibitor CPI-17single residue phosphorylation (not T to D mutation) condenses conformation and increases potency >2000-fold

  16. 100 50 0 Selective Inhibition of Myosin Phosphatase by CPI-17same C subunit isoform bound to different R subunits Glycogen-bound PP1d Phosphatase Activity (%) MYPT1•PP1d 10 9 8 7 6 5 [P-CPI-17] (-log M)

  17. Myosin LC Phosphatase: A complex of PP1C with MYPT1 Subunit C subunit bound to specific R subunit

  18. 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, JAK • methylation - PMT and PME, alters subunit association • 5. Toxins - MCLR, OA bind at active site. • Differences between PP1 and PP2A in b12-b13 loop

  19. Protein Ser(P)/Thr(P) Phosphatase - PP2A ABC ={ [A] Scaffold + [C] Catalytic} + [B] Regulatory ABC HEAT Helical repeats B A Tpd3 A subunit C Pph21 Pph22 Binding of C to A decreases Vmax and alters KM

  20. Protein Ser(P)/Thr(P) Phosphatase - PP2Acovalent modifications of the catalytic subunit Tyr307 Tyr kinases Src, JAK, RTK PMT ABC S-AdoMet B RTPDYFLCOOH RTPDYFLCOOMe B A Tpd3 PME C PTK O O-P-O RTPDYFLCOOX RTPDYFLCOOX inactive self

  21. 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 suppressor 2. Many Bs….are they for dedicated functions? Yeast Cdc55 and Rts1 - distinct phenotypes, not complemented(conclusion?) now 3 B families cloned - more than 15 genes B 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

  22. Protein Ser(P)/Thr(P) Phosphatase - PP2Amultiple dedicated enzymes on one platform B55 CDC55 kinase inactivation Gb-like propeller structure anti-apoptosis integrin signaling polarity-Wnt signaling B’56 RDS1 P B’’72 P retinoids sm-t tumor virus transformation a4 TAP42 C ABC B A Tpd3 C PPH21 PPH22 PP2A can be > 80 different “enzymes” anti - apoptosis

  23. The B56 Family of PP2A Regulatory Subunits anti-metastatic Paxillin binding Wnt-B56e-Dishevelled D-weiderborst

  24. The 3D Structure of PP2A Trimer AB56C Nature, Nov. 2006 Side view Top view

  25. C. Inhibitor proteins for PP2A • 1. Inh1(PP2A) Damuni et al • 2. Inh2(PP2A) SET protein - important in solid tumors • ceramide “receptor” for PP2A activation • D. Alternative partners • 1. A binds HSF2 and PP5 • 2. C binds alpha4; in yeast part of TOR signaling • E. Substrates • 1. Signaling Kinases (MEK, PKB, PI3K, p70S6K, etc.) • 2. anti-apoptosis (PKB, Bad, Bcl) • 3. integrin signaling - Paxillin B56 deletions & increased invasion

  26. Calcineurin (PP2B)(PPP3) Ca2+/CaM activated Phosphatase A. Catalytic subunit 1. C has Fe::Zn active site like PP1, PP2A 2. Suppressor domain, CaM dependent B. Regulatory subunits 1. 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. Substrates 1. NFAT (co-transport into nucleus) 2. Elk-1 (TCF transcription factor) 3. DARPP-32 (NMDA receptor)

  27. Calcineurin (CnA +CaB) Inhibited by Cyclosporin (CsA) and its Binding protein (Cyp) $B drug for immunosuppression following organ transplants

  28. PP2C = Mg2+-dependent PPase (MPP) A. Catalytic subunit 1. 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. CDKs 2. the kinase activation loop 3. PI3K 4. Glycogen synthase Maybe activated by small molecule second messenger? Lipids?

  29. Protein Phosphatase 2C Mg2+-dependent Phosphatase (MPP)

  30. KINASES ATP ADP Pi H2O PHOSPHATASES Protein Phosphorylationrapid and reversible biochemical reactions TARGET P TARGET A molecular on/off switching mechanism.

  31. Protein Phosphorylation: Kinases & Phosphatases Fe::Zn Protein Tyr Kinases Protein Ser/Thr Kinases KINASE Superfamily ~ 500 enzymes ATP ATP TARGET P-Tyr TARGET TARGET P-Ser/Thr-TARGET Protein Tyr(P) Phosphatases Yeast 5 C. elegans 95 Drosophila 22 Human 56 Protein Ser(P)/Thr(P) Phosphatases Yeast 13 C. elegans 51 Drosophila 19 Human 15 Mg Mg MPP PPP Cys-SH

  32. Protein Phosphorylation: Kinases & Phosphatases Fe::Zn Fe::Zn Fe::Zn ATP ATP ATP ATP ATP ATP ATP ATP ATP KINASE Superfamily ~ 500 enzymes ATP ATP ATP Cys-SH Cys-SH Cys-SH Mg Mg Phosphatase families and ensembles ~ 500 enzymes MPP

  33. Protein Kinases and Protein Phosphatases ancient enzymes essential to cell signaling and cellular regulation New targets for Pharmaceuticals

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