PROTEIN PHOSPHATASES

Stephen R. J. Salton M.D. Ph.D.Fishberg Department of Neuroscience

Mount Sinai School of Medicine

Cellular phosphorylation pathways, triggered by NGF binding to TrkA in PNS and CNS neurons, activate downstream protein kinases and are controlled by protein phosphatases.

Salton Mt Sinai J Med. 2003 Mar;70(2):93-100.

Nobel Prize in Physiology or Medicine (1992) was awarded to Edmond H. Fischer and Edwin G. Krebs “for their discoveries concerning reversible protein phosphorylation as a biological regulatory mechanism”

From Nobelprize.org

--At least 1/3 of all proteins are reversibly phosphorylated by ~600 kinases.

--Only about 25 serine/threonine phosphatases have been identified, vs. ~400 serine/threonine kinases. Their diversity is determined by the variety of regulatory proteins that interact with these catalytic subunits

--Phospho-tyrosine represents 0.01-0.05% of the cellular phospho-amino acid content…. increases to 1-3% in cells transformed by oncogenic tumor viruses…. led to the recognition in the 1980s that growth factor receptors (e.g. EGF) and viral transforming proteins (e.g. src) were tyrosine kinases.

--Roughly equal numbers of tyrosine kinases and tyrosine phosphatases are encoded in each mammalian genome….about 100…. suggesting that each class of enzymes must have similarly high degrees of substrate specificity.

PROTEIN PHOSPHATASES

1. Protein Serine/Threonine Phosphatases

2. Protein Tyrosine Phosphatases

PPs are highly conserved through evolution

Proteins unit of evolution(millions of yrs for 1% change)

Histone H4 400Calmodulin 350Histone H3 330

PP-2A 100PP-1 88PP-2 66

Histone H-2A, H-2B 60

GDH 55Tubulin 40PKA 39Collagen 36Gai 32K+ channel 22

From H.C.-Li 2001

Dephosphorylation of phosphorylated serines and threonines is catalyzed by four types of serine/threonine phosphoprotein phosphatases—protein phosphatases 1, 2A, 2B, and 2C.

Except for protein phosphatase-2C (which is a monomer), PP1, PP2A and PP2B share a common catalytic domain of 280 residues (with divergent N- and C-termini) that is complexed with one or more of a large set of regulatory subunits--these help to control phosphatase activity and enable the enzyme to select specific targets.

PPs are metalloenzymes that dephosphorylate substrate in a single step using a metal-activated nucleophilic water molecule (in contrast, PTPs use a cysteinyl-phosphate enzyme intermediate)

Often, different enzyme types are distinguished by their substrates:-PP1 dephosphorylates many PKA-phosphorylated proteins (e.g. P-CREB) -PP2A has a broad specificity and dephosphorylates many serine/threonine kinase-phosphorylated proteins-PP2B (calcineurin) is activated by Ca2+ and calmodulin, and is especially abundant in the brain and in T-cells where increased Ca2+ triggered by antigen presentation stimulates NFAT1 dephosphorylation, which results in translocation to nucleus and stimulation of IL-2 expression and T-cell activation.

Protein Serine/Threonine Phosphatases

Phosphoprotein phosphatase (PPP) familyPP-1

PP-1i: RC where R=inhibitor 2 (I-2) (31 kDa) andC=catalytic (38 kDa)

PP-1g: GC where G=glycogen binding subunit (161 kDa)PP-1m: MC where M=myofibril binding subunit

PP-2A (spontaneously active)PP-2A0: AB’2C where A=structural subunit (65 kDa),B’=targeting subunit (53 kDa), C=catalytic subunit (36 kDa)

PP-2A is a family of holoenzymes containing a common core of a 36 kDa catalytic subunit and a 65 kDa A subunit associated with a variety of B subunits PP-2B (calcineurin) AB where A=catalytic subunit (61 kDa) and B=regulatory subunit (19 kDa); Ca2+ dependent

PP-1, PP-2A and PP-2B share catalytic domain of 280 residues but have divergent N- and C-termini and different regulatory subunits

PPM family: PP-2C (45 kDa) monomer; Mg2+ dependent; structure dissimilar

From H.-C. Li (2001)

Figure 20-35. Regulation of glycogen breakdown and synthesis by cAMP in liver and muscle cells. Active enzymes are highlighted in darker shades; inactive forms, in lighter shades. (a) An increase in cytosolic cAMP activates a cAMP-dependent protein kinase (cAPK) that triggers a protein kinase cascade involving glycogen phosphorylase kinase (GPK) and glycogen phosphorylase (GP), leading to breakdown of glycogen. The active cAPK also phosphorylates and thus inactivates glycogen synthase (GS), inhibiting glycogen synthesis. Phosphorylation of an inhibitor of phosphoprotein phosphatase (PP) by cAPK (see Figure 20-36) prevents PP from dephosphorylating the activated enzymes in the kinase cascade or inactive GS. (b) A decrease in cAMP inactivates the cAPK, leading to release of the active form of phosphoprotein phosphatase. This enzyme then removes phosphate residues from GPK and GP, thereby inhibiting glycogen degradation. The phosphatase also removes phosphate from inactive GS, thereby activating this enzyme and stimulating glycogen synthesis.

From Lodish et al, Molecular Cell Biology, p886, Figure 20-35, (2000).

Figure 20-36. Regulation of phosphoprotein phosphatase activity by cAMP is mediated by an inhibitor protein. At high levels of cAMP, a cAMP-dependent protein kinase (cAPK) phosphorylates an inhibitor protein (IP), which then binds to phosphoprotein phosphatase (PP), forming a complex that lacks phosphatase activity. When the cAMP level decreases, constitutive phosphatases dephosphorylate the inhibitor, releasing phosphoprotein phosphatase in its active form.

From Lodish et al, Molecular Cell Biology, p887, Figure 20-36, (2000).

PP-1 and PP-2A inhibited by okadaic acid (shellfish toxin) and microcystin (cyclic peptides produced by cyanobacteria whichare potent hepatotoxins), also PP-4, PP-5 and PP-6 are inhibited, while PP-2B is inhibited by higher (M) concentrations

PP-2B is a target of cyclosporin A and FK506 (immunosuppressants)

Cyclosporin A is a lipid soluble fungal undecapeptide (Mr=1,203)Widely used in transplantation for graft rejectionFunctions as blocker of T cell activation/proliferationCsA binds cyclophilin and this complex binds B subunit of calcineurin

in presence of calcium/calmodulin to inhibit PP activityFK506 is a bacterial (Streptomyces) product, a macrocyclic lactone

structurally unrelated to cyclophilin that complexes withFKB binding protein to inhibit calcineurin PP activity

Protein phosphatase 2A describes a panoply of phosphatases. The common heterotrimeric form of PP2A containing the catalytic subunit, the structural A/PR65 subunit and a regulatory/targeting B subunit (at least 15 distinct B subunits are known) is shown. In addition, various cellular and viral proteins that interact with PP2A components are indicated.

Virshup, DM (2000) Current Opinion in Cell Biol 12:180-185

CATALYTIC REGULATORYVARIABLE

Domain Organization of PP2B Calcineurin A

Calcineurin B-subunit bind. helix Calmodulin bind. dom. Autoinhibitory dom.

Adapted from Aramburu et al. (2000) Current Topics Cell. Reg. 36:237

Protein phosphatase 1 in complex with microcystin LR (MCLR). MCLR interacts with the hydrophobic groove (via the Adda side chain), the metal sites (via a carboxylate group and a carbonyl oxygen of the toxin), and to Cys 273 (via the Mdha side chain). This structure, combined with the PP1-tungstate complex structure, reveals that microcystin inhibits the activity of PP1 by directly blocking substrate binding to the catalytic site.From Barford et al. (1998) Annu. Rev. Biomol. Struct. 27:133-64

Interface of 3 -sheets at top of -sandwich creates a shallow catalytic site.

Human protein phosphatase 2C. The catalytic domain consists of a central -sandwich surrounded by -helices. The Mn2+ ions, spheres, are coordinated by Asp and Glu residues from the central -sandwich structure. From Barford et al. (1998) Annu. Rev. Biomol. Struct. 27:133-64

PROTEIN TYROSINE PHOSPHATASES

Phospho-tyrosine 0.01-0.05% cellular phosphamino acids

Increases to 1-3% upon transformation by oncogenic viruses**RSV src gene and EGF receptor shown in 80s to be tyrosine kinases

1988 PTPase 1B was purified from placenta and aa sequence determined{Tonks, Diltz and Fischer, JBC 263:6722 and 263:6731 (1988)}** not similar to serine/threonine phosphatases at active site

** region of similarity to major lymphocyte cell surfaceglycoprotein CD45 (LCA)-LCA heavily glycosylated-differentially spliced extracellular domain-single transmembrane domain-cytosolic domain containing 2 repeats of a 200 aa domain with

identity to PTP1B-antibody studies suggested that CD45 was involved in early

lymphocyte activation-LCA and LAR (leuk Ag-related) had PTPase activity in vitro and a

200 aa conserved catalytic domain

Andersen et al. (2001) Mol Cell Biol 21:7117-36

Receptor and non-receptor PTPs

*receptor PTPs generally have 2 catalytic domainsmembrane proximal activemembrane distal regulatory

*non-receptor PTPs generally have 1 catalytic domain

*receptor PTPs have extracellular adhesion domains includingIgG repeats, Fibronectin FNIII repeats, CA domain

*intracellular PTPs have signaling modules such as SH2 domainsand PEST sequences

*intracellular and receptor PTPs can interact with other signalingdomainse.g. PTP-PEST (C-terminal proline rich domain) with SH3domain of p130cas (substrate) (Garton et al., 1997)

RPTPs are regulated by alternative splicing

A. Extracellular domains of RPTPs*altered N- and O-linked glycosylation*isoform expression is developmentally regulated (e.g. CD45 during

lymphoid development)

B. Intracellular domains of RPTPs*removal of catalytic domains of RPTP by alternative splicing to

generate the secreted proteoglycan phosphacan*108 bp alternatively spliced insertion in first phosphatase domain

of LRP- modulate activity? * RPTP alternatively spliced 21bp mini-exon in juxtamembrane

domain near ‘wedge’ region-modulate activity? *alternative splicing of PTP1B C-terminus alters targeting to ER

(Frangioni et al., Cell 68, 1992)*dPTTP61F drosophila PTP gene encodes 2 non-receptor PTPs-

alternative splicing of C-terminal sequences determinestargeting to nucleus or to cytoplasmic membranes (McLaughlinand Dixon, JBC 268, 1993)

Do RPTPs function as cell adhesion molecules?

RPTP expressed in non-adherent SF9 cells leads to cell aggregationGebbink MF et al. (1993) J. Biol. Chem. 268:16101

Diversity and tissue specificity of expression suggest adhesion proteinshave functional roles

Selective expression on different subsets of cells in the embryonic CNS,including on different axons and pioneer neuronsDLAR, DPTP10D, DPTP99A (Tian et al. & Yang et al., Cell 67, 1991)

Conserved residues within the core PTP catalytic domain:

VHCSAGV GR(S/T)G (invariant)

Conserved regions within the 200 aa PTP catalytic domain allowed PCR-cloning of additional family members

Nucleophilic cysteine attacks phosphate---critical to mechanismcysteinyl-phosphate intermediate hydrolyzed and Pi released

Mutagenesis of cysteine revealed that in general only membraneproximal domain of RPTPs is active (distal is regulatory)

Nucleophilic cysteine attacks phosphate---critical to mechanismcysteinyl-phosphate intermediate hydrolyzed and Pi released

From Barford et al. (1998) Annu. Rev. Biomol. Struct. 27:133-64

*Structural analysis of PTP1B indicated that the catalytic domainsits at the bottom of a cleft

**Depth of this cleft accounts for the specificity of recognition andcatalysis to the extended phosphotyrosine residue(vs. phosphoserine and phosphothreonine). Recall that the crystal structures of PPs show a much more shallow active site cleft.

Structure of protein tyrosine phosphatase 1B. The PTP loop (dark shading) and WPD loop are indicated, as is Cys 215 and Arg 221 of the PTP loop and the position of the C-atom of Tyr 46 of the phosphotyrosine recognition loop. From Barford et al. (1998)

Annu. Rev. Biomol. Struct. 27:133-64.

The PTP signature motif, containing the catalytic cysteinyl residue, forms a continuous loop located at the base of the catalytic cleft. In classical PTPs, a tyrosyl residue (Tyr46 in PTP1B) forms one side of the cleft, determining the depth of the pocket and contributing to the absolute specificity of classical PTPs for tyrosyl phosphoproteins. Only the side chain of a pTyr residue in a target substrate is of sufficient length to be accessible to the nucleophilic cysteinyl residue when inserted into the catalytic site; pSer and pThr residues are too short to be dephosphorylated. Another important feature is the dramatic change in conformation that occurs upon substrate binding. In the absence of substrate, the active site adopts an ‘open’ conformation in which the general acid Asp is pointed away from the active site, precluding it from serving as a proton donor. Upon substrate binding, the active site closes around the side chain of the pTyr residue. In this ‘closed’ conformation the loop containing the general acid residue (the so-called ‘WPD loop’) has moved, repositioning the Asp for its catalytic function. This also juxtaposes the Asp residue to the negatively charged phosphate group, which limits the stability of the enzyme–substrate complex. Tonks and Neel, Current Opin Cell Biol 13:182 (2001).

Receptor PTPs catalytic activity is regulated by dimerization

Monomeric RTKs exhibit weak basal activity. Ligand binding ofRTKs leads to dimerization, trans autophosphorylation,and activation

Monomeric RPTPs exhibit enhanced catalytic activity. Ligandbinding of RPTPs leads to dimerization of membrane-proximal PTP domains. ‘Inhibitory wedge’ sequencesfrom each phosphatase domain interact with the othercatalytic domain, preventing substrate binding.

RPTP mutants in the ‘wedge’ are not inhibited bydimerization.

Inhibitory Wedge Experiments :Majeti et al., Dimerization-induced inhibition of receptor protein tyrosine phosphatase function through an inhibitory wedge. Science 279:88-91 (1998)

A chimeric EGFR-CD45 molecule restores TCR-mediated signal transduction in a CD45-deficient T cell line; furthermore, treatment of these cells with EGF blocks TCR-mediated signaling, which suggests that CD45 is negatively regulated by ligand-induced dimerization. A possible explanation for this negative regulation comes from the crystal structure of the membrane-proximal phosphatase domain of the RPTP which revealed a putative inhibitory wedge in symmetrical dimers. Two acidic residues found in this wedge are strongly conserved among the membrane-proximal phosphatase domains of RPTP

Effects of EGF on TCR-mediated ZAP-70 and MAPK phosphorylation in CD45-deficient T cells expressing EGFR-CD45 wild-type (A and B, lanes 1 through 5) or EGFR-CD45/E624R (A and B, lanes 6 through 10). Cells were stimulated as indicated: no stimulation (lanes 1 and 6); 2 min with antibody to the TCR (lanes 2 and 7); 3 min with EGF (lanes 3 and 8); 2 min with both antibody to the TCR and EGF (lanes 4 and 9); and 1 min pretreatment with EGF, then 2 min with antibody to the TCR (lanes 5 and 10).

Jiang G et al., Dimerization inhibits the activity of receptor-likeprotein-tyrosine phosphatase-alpha. Nature (1999) 401:606-10.

Dimerization inhibits the activity of a full-length RPTP in vivo. We generated stable disulphide-bonded full-length RPTP homodimers by expressing mutants with single cysteines at different positions in the ectodomain juxtamembrane region. Expression of wild-type RPTP and Phe135Cys and Thr141Cys mutants in RPTP -null mouse embryo cells increased dephosphorylation and activity of Tyr 529 in the protein tyrosine kinase c-Src; in contrast, expression of a Pro137Cys mutant did not. Mutation of Pro 210/211 to leucine in the inhibitory wedge of the Pro137Cys mutant restored its ability to activate c-Src, indicating that dimerization may inhibit full-length RPTP activity.

Any way to get Figure 2 from this paper?

From Weiss, A and Schlessinger, J (1998) Cell 94:277

Domain 1 of receptor protein tyrosine phosphatase . An example of a receptor-like PTP. In two independent crystal forms, the protein forms a homodimer such that the catalytic site (PTP loop) of each molecule is blocked by a wedge within a helix-turn-helix segment. This inhibits the enzyme by preventing substrate binding. The reason is that Asp 227 of one subunit interacts with the pTyr recognition loop of the opposite subunit and the WPD loop containing the catalytic Asp residue is restrained in the open, inactive conformation. From Barford D. et al. (1998).

I. RPTPs-How do you identify ligands?II. RPTPs and PTPs-How do you identify substrates?

I. Ligands:Binding partner is easier to define than ligand which should bind and also

decrease catalytic activity (then you need to know substrate)

II. Substrates:PTPs are usually active and when (over)expressed, quite toxic.

Determine substrate biochemically by:”Substrate trapping mutation”-mutate active site so catalysis is not

possible and PTP functions as affinity reagent. Cys to Ser(Yersinia PTP and MKP-1, dual specificity PTPs) and Asp to Ala(T cell PTP and PTP1B trapped EGF receptor).

Knockout PTP and look for selectively increased tyr phosphorylation(e.g. PTP1B ko indicated role in IR signaling not EGFR!!)

Structural basis for PTP1B specificity for the insulin receptor. Surface representation of PTP1B in complex with a bisphosphopeptide derived from the activation loop of the insulin receptor (IR). Critical residues in PTP1B are in red and those in the IR activation loop in blue and the latter are numbered according to their positions in the IR. From Tonks and Neel (2001) Cur Opin Cell Biol 13:182.

Fig. 1 Insulin-signaling pathway. The metabolic arm of the insulin transduction pathway is shown schematically. Larger round circles represent insulin binding to its receptor; smaller circles depict glucose being taken up by the membrane-embedded GLUT4 transporter. Other key protein molecules have been identified: PTP1B, protein tyrosine phosphatase 1B; PI3K, phosphatidylinositol 3-kinase; PDK1, phosphoinositide-dependent kinase-1; GSK3, glycogen synthase kinase 3; PKC/, protein kinase C/; GS, glycogen synthase; IRS, insulin receptor substrate. From Asante-Appiah and Kennedy Am J Physiol Endocrinol Metab 284:E663 (2003).

How PTP1B Affects Leptin Signaling

(A) Depiction of the putative mechanism by which PTP1B causes resistance to leptin signaling believed to occur in diet-induced obesity. Although leptin binds normally to its cell surface receptor (1) and phosphorylates Jak2 (2), the receptor complex comes into proximity with PTP1B on the ER (3). There, PTP1B dephosphorylates Jak2, blocking the phosphorylation of the receptor and Stat3 (4). Unphosphorylated Stat3 is therefore unable to exert transcriptional control over its target genes that encode enzymes of lipid homeostasis. Consequently, ACC expression is abnormally high and CPT-1 is low (5); malonyl CoA levels are elevated and they inhibit CPT-1, thereby reducing oxidation of fatty acids (6). This is believed to lead to steatosis, lipotoxicity, and lipoapoptosis of nonadipocytes.

(B) Depiction of mechanism of leptin sensitivity in PTP1B knockout mice. Steps (1) and (2) are presumably the same as in (A). However, PTP1B is absent and Stat3, phosphorylated by Jak2 (3), enters the nucleus to alter the transcription of its target genes (4). ACC and FAS are downregulated and CPT-1 and ACO are upregulated. The reduction of ACC expression, coupled with inactivation of the enzyme by AMP-activated kinase, a crucial control system not depicted here, reduces malonyl CoA activity and thereby disinhibits CPT-1 (5). Fatty acid synthesis is reduced, FA-CoA oxidation increases, and normal intracellular liporegulation is restored. ACC, acetyl CoA carboxylase; FAS, fatty acid synthase; CPT-1, carnitine palmitoyl transferase 1; ACO, acyl CoA oxidase; TG, triacylglycerol. (Figure by K. McCorkle.)From Cook and Unger (2001) Dev. Cell 2:385-7

Substrate Specificity and Function

I. Dual specificity phosphatases

Catalytic domain ~240 amino acid residues[I/V]HCXAGXXR[S/T]GDephosphorylates Tyr & Thr

i. Cell signaling. MKP-1 is encoded by an immediate early gene that is activated by growth factor stimulation. MKP-1 recognizes Erk1 and Erk2 in vitro and in vivo with a high degree of substrate specificity (Sun et al., Cell 75, 1993).

ii. Cell cycle. CDK1 (CDC2 or p34cdc2) is inactive in G1 due tophosphorylation on Thr14, Tyr15 and Thr161(nucleotide bindingpocket GXXGX14X15G-phosphorylation interferes with ATP binding). Critical threshold concentration of CDC2 at G2M transition results in increased dephosphorylation of Thr14, Tyr15 by p80cdc25, a dual specificity phosphatase, and CDC2 activation.

MKP signature sequence is HCXXXXXR

-Nucleophilic attack of cysteine thiolate anion on MAPK P-Tyr-Aspartate in acid loop donates proton-Arginine coordinates phosphate group of P-Tyr or P-Thr-Histidine decreases pKa of cysteine so it exists as anion

From Farooq, A and Zhou, MM Cellular Signalling 16:769 (2004)

Fig. 1. MAPK signaling and down-regulation by MKPs. Stimuli such as stress, cytokines, hormones and growth factors activates MKKs. MKKs in turn phosphorylate tyrosine and threonine residues within the motif –pTXpY– located in the activation loop of MAPKs—such dual phosphorylation results in dimerization and subsequent activation of MAPKs. MAPKs may then interact with and phosphorylate cytoplasmic proteins, or alternatively translocate to the nucleus, where MAPKs may interact with specific transcription factors (TFs) leading to gene transcriptional activation of specific proteins including MKPs. MKPs in turn provide a negative feedback regulatory mechanism by inactivating MAPKs via dual dephosphorylation of –pTXpY– in the cytoplasm and the nucleus.

From Farooq, A and Zhou, MM Cellular Signalling 16:769 (2004)

Fig. 2. Catalytic mechanism of dephosphorylation of MAPKs by MKPs. (i) Nucleophilic attack of the thiolate anion of the active site Cys of an MKP on the phosphate of pY of an MAPK results in the formation of a transient phospho-enzyme intermediate with concomitant release of MAPK-Y aided by the donation of a proton from the active site Asp acting as a general acid. (ii) The active site Asp, acting as a general base, accepts a proton from a water molecule and the resulting hydroxyl group attacks the phosphate atom within the phospho-enzyme intermediate to eliminate phosphate and regenerate a thiolate anion at the active site Cys of the MKP. (iii) The regenerated thiolate anion of the MKP binds phosphorylated MAPK and the catalytic cycle is repeated.

From Farooq, A and Zhou, MM Cellular Signalling 16:769 (2004)

Fig. 7. A model for the substrate-induced catalytic activation of MKPs by MAPKs. In the absence of its substrate, the DSP domain of MKP exists in an inactive state. Binding of the MKB domain of MKP to dual-phosphorylated activated MAPK alters interactions between the DSP and the MKB domains of MKP. This conformational effect, along with the interaction of the DSP domain to MAPK, allosterically triggers the active site residues, cysteine and arginine within the signature sequence –HCXXXXXR– and an aspartate in the general acid loop, to reconfigure to a conformation optimal for dephosphorylation of MAPK.

From Bartek 2004 Nat Rev Cell Biology 5:792-804 dual specificity phosphatases and cell cycle

Figure 2 | Checkpoint-induced degradation of the CDC25A phosphatase.   a | During unperturbed S phase, the ATR (ataxia-telangiectasia and RAD3 related)-activated checkpoint kinase CHK1 phosphorylates many serines of CDC25A, thereby facilitating its recognition by the SCF -TrCP ubiquitin ligase84, 85. The resulting ubiquitylation and proteolysis of CDC25A limits its accumulation to a physiological threshold that is necessary and sufficient for coordinated S-phase progression78. b | After DNA double-strand break (DSB) generation and ATM (ataxia-telangiectasia mutated) activation, the rate of phosphate incorporation into CDC25A increases through the combined action of CHK1 and CHK2. This leads to a stronger interaction with the SCF -TrCP ubiquitin ligase and the acceleration of CDC25A protein turnover. The resulting reduction of CDC25A levels is instrumental in slowing the progression through S phase76, 78. See also Fig. 3 for an overview of the CDC25A phospho-acceptor sites that are involved in the regulation of its turnover. E2, ubiquitin-conjugating enzyme; P, phosphorylated serine residues; Ub, ubiquitin.

From Bartek 2004 Nat Rev Cell Biology 5:792-804 dual specificity phosphatases and cell cycle

Figure 3 | Phosphorylation-mediated control of CDC25A protein turnover.   The phosphatase activity of CDC25A closely correlates with its total protein abundance throughout the cell cycle and under diverse stress conditions. During interphase, CDC25A is kept labile through a 'maintenance mode' that is controlled by the checkpoint kinase-1 (CHK1)-mediated phosphorylation of five serine residues78, 79, 84, 85, 108, 109 (in orange), together with the phosphorylation of serine residues within the PHOSPHODEGRON MOTIF (in blue; the dominant S82 is indicated in bold) by an as-yet-unknown kinase84, 85. This mode can be rapidly accelerated into a 'checkpoint mode' after DNA damage, through enhanced phosphorylations of the same five residues — however, now jointly mediated by CHK1 and CHK2 — at least in response to ionizing radiation (IR)78. At the G2–M transition, the degradation mode is cancelled through the CDK1-mediated phosphorylation of S18 and S116, and CDC25A is stable until exit from mitosis77. It is important to note the numbering of the phosphorylation sites. The original numbering (indicated in brackets) was based on the first sequence of CDC25A as reported by Galaktionov and Beach110. This sequence included an error in the sequence that encoded amino acids 6–11, which resulted in the loss of one amino acid.

Autoinhibition of Cytoplasmic PTPs

PTKs like Src are maintained in an inactive state by SH3- and SH2-mediated intramolecular interactions.

PTP Shp2 is maintained in an autoinhibited state by intramolecularinteractions between the N-SH2 domain and the catalyticcleft of the PTP domain. Binding of specific p-tyr sequencesto SH2 domain will release autoinhibition and activate Shp2.

From Tonks and Neel (2001) Cur Opin Cell Biol 13:182-195

SH-PTPs: 2 SH2 domains and a PTPase domain. Deletion of N-terminal SH2 domain activates PTPase suggesting intramolecular inhibition which crystal structure supports. Difficult to predict whether function as positive or negative signaling molecules.

SH-PTP1 is mutated in moth eaten mouse (effect on splicing leading to no protein or aberrant splice within PTPase domain). Results in severe combined immunodeficiency (impaired T-cell responses, decreased NK function, decreased B cell precursors, increased plasma cells and macrophages). Thought to negatively regulate hematopoietic signaling and JAK-STAT pathway.

From BG Neel et al., Trends in Biochemical Sciences 28:284-293 (2003) SH-PTPs

SH-PTP2 (csw or corkscrew; Syp) is downstream from activated receptor PTKs (such as torso and sevenless in flies or EGFR, PDGFR, and IRS-1).

Binds to activated receptor with N-terminal SH2 domain and signals with C-terminal SH2 or with PTP domain (to activate src-like kinases).

Positive signal transduced via Ras-Raf-MAPK and PI3K. Also can negatively regulate JAK-STAT and recently shown to function as a dual specificity PTPase (STAT1 and STAT5A).

Mutations in human gene for SHP-2 (PTPN11) causes Noonan Syndrome, perhaps as gain of PTPase function, and results in dysmorphic facial features, short stature and heart disease (Tartaglia et al., 2001, Nat Genetics 29:465).

II. PTP and c-src

Keeping src innactive requires extensive C-terminal phosphorylationby C-terminal src kinase (Csk). Overexpression of PTP in ratembryo fibroblasts leads to transformation and dephosphorylationof Tyr 527 on c-src (and src activation).

Schematic representation of c-Src activation at the onset of mitosis. (A) During most of the cell cycle, c-Src is largely phosphorylated at Tyr527 and therefore in an inactive tail-bite conformation, whereas 20% of RPTP is phosphorylated at Tyr789 and saturated with Grb2. (B) At the transition from G2 to M phase, c-Src is first phosphorylated in its NH2-terminus by p34cdc2, and RPTP is phosphorylated at several serines by an unknown kinase, perhaps PKC. Subsequently, Grb2 dissociates from RPTP. (C) c-Src binds through its SH2 domain to phospho-Tyr789 on RPTP. This binding event exposes phospho-Tyr527 for dephosphorylation by the D1 domain of RPTP, allowing c-Src to autophosphorylate at Tyr416, thereby becoming fully active. PTK, protein tyrosine kinase. From Mustelin and Hunter (2002) Sci STKE. Jan 15;2002(115):PE3.

PTP Inhibitor Design (e.g. vs. PTP1B for diabetes and Cdc25 for cancer)

pTyr alone insufficient for high affinity binding to PTPs--adjacent residues contribute to specificity

By analogy, kinase inhibitor specificity determined by binding to region outside ATP binding pocket--for PTPase, pTyr binding domain is smaller than kinase ATP pocket (pTyr takes up ~50% of binding pocket)

So small molecule inhibitors:

--Need to bind PTP catalytic domain and another adjacent region unique to a specific PTP simultaneously to confer specificity (based on structure PTP1B and inh. BPPM)

--Need to penetrate cell membranes

A strategy for creating selective and high-affinity PTP1B inhibitors. Based on the principle of additivity of free energy of binding, high-affinity ligands can be obtained by linking two functional groups that bind to the active site (pTyr binding site) and a peripheral site X. Specificity arises from the fact that site X is not conserved and from the fact that the tethered ligand has to bind both sites simultaneously. Zhang ZY (2002) Annu Rev Pharmacol Toxicol. 42:209-34

Crystal structures of vertebrate PTP domains show conserved fold and consistent C-backbone trace. PTP1B (magenta), RPTP (gray), RPTPµ (red), LAR (blue), SHP1 (green), and SHP2 (yellow) were aligned and superimposed using Quanta (Molecular Simulations Inc.). For clarity, residues 280 to 298 (C terminal) of PTP1B, 250 to 281 (N terminal) and 522 to 532 (C terminal) of SHP1, and 2 to 218 (N terminal) of SHP2 were omitted from the figure, as well as D2 of LAR.

Drug Design Specificity--Bad News (left) and Good News (right)

Bad News

Nonconserved amino acids in the proximity of the PTP active site are involved in the recognition of PTP substrates and nonpeptide PTP inhibitors. Shown is the visualization of four selectivity-determining regions on the molecular surface of PTP1B. Areas of conservation (blue, most conserved; red, least conserved) represent the C-regiovariation score values of 37 aligned human PTP catalytic domainsFrom Andersen et al., 2001, Mol Cell Biol 21:7117