Protein tyrosine phosphatases in signal transduction

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Protein tyrosine phosphatases in signal transduction Benjamin G Neel* and Nicholas K Tonkst Protein-tyrosyl phosphorylation, regulated by protein tyrosine kinases and protein tyrosine phosphatases (PTPs), is a key cellular control mechanism. Until recently, little was known about PTPs. However, the past two years have witnessed an explosion of information about PTP structure, regulation and function. Crystal structures of several PTPs have provided insights into enzymatic mechanisms and regulation and suggested the design of 'substrate-trapping' mutants. Candidate homophilic and heterophilic ligands for transmembrane PTPs have been identified, and roles for transmembrane PTPs in regulating cell-cell interactions have been suggested. Finally, progress has been made in understanding signaling by Src homology 2 domain containing PEPs and PTPs controlling yeast osmoregulatory pathways.

cell adhesion molecules, transmit signals via pathways involving tyrosyl phosphorylation of specific cellular proteins. These signal transduction pathways dictate whether a cell will grow and divide, change shape, move, differentiate, or die. T h e regulation of tyrosyl phosphorylation, controlled by protein tyrosine kinases (PTKs) and protein tyrosinc phosphatases (PTPs), is critical for homeostasis. Abnormal tyrosyl phosphorylation can result in neoplastic or non-neoplastic disease. Until recently, PTKs were considered to be the major enzymes regulating tyrosyl phosphorylation. Little was known about PTPs, which were believed to be few in number and to subserve primarily housekeeping functions. T h e discovery of a large, diverse family of PTPs in eukaryotes from yeast to man, as well as in some bacterial species, belies these notions.

Addresses

*Cancer Biology Program, Division of Hematology-Oncology, Department of Medicine, HIM 1047, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215, USA; e-maih [email protected] tDemerec Building, Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724-2208, USA; e-mail: [email protected] Current Opinion in Cell Biology 1997, 9:193-204

Electronic identifier: 0955-0674-009-00193 © Current Biology Ltd ISSN 0955-0674 Abbreviations

BCR B cell antigen receptor CA carbonic anhydrase CTLA-4 cytotoxic T lymphocyte antigen-4 Dos DSP

FcR Ig ITIM KIR LMP MAM

daughter of sevenless

dual-specificity phosphatase Fc receptor immunoglobulin immunereceptor tyrosine-based inhibitory motif killer-inhibitory receptor low molecular weight (acid) phosphatase rneprin-Xenopus A2-mu

MAP mitogen-activatedprotein MAPK MAP kinase me~me motheaten

MKP-1 PTK

MAP kinase phosphatase-1 protein tyrosine kinase PTP protein tyrosine phosphatase RPTP receptor-like PTP RTK receptor PTK SH2 Src homology 2 SHP PTP containing SH2 domains TCR T cell antigen receptor TM transmembrane ZAP-70 ~-associated protein-70

Introduction A wide variety of stimuli, including growth factors, cytokines, hormones, extracellular matrix components, and

At least three families of molecules, composing the tyrosine phosphatase superfamily (Fig. 1), have tyrosine phosphatase activity. There is little overall sequence similarity amongst members of the three groups, but they have similar tertiary structures and share the same general catalytic mechanism, characterized by the formation of a thiophosphate intermediate involving an essential catalytic cysteinyl residue (reviewed in [1,2°°]). 'Classical' PTPs, like PTKs, exist in transmembrane forms (receptor-like PTPs or RPTPs) and nontransmembrane (non-TM) forms. Approximately 75 PTPs have been identified to date; genome sequencing predicts the existence of as many as 500 human PTPs. These enzymes are characterized by at least one conserved catalytic domain o f - 2 4 0 residues (the P T P domain), containing the unique 'signature motif', [I/V]HCxAGxxR[S/T]G (single-letter code for amino acids,where x represents any amino acid). During P T P catalysis, the cysteinyl residue executes a nucleophilic attack upon the phosphate moiety of the substrate, leading to thio-phosphate intermediate formation. Most RPTPs contain two P T P domains. Usually only the most proximal (amino-terminal) of these two domains (domain 1) has significant enzymatic activity. The function of the more carboxy-terminal domain (domain 2) of RPTPs is unclear, but it may direct protein-protein interactions [3,4]. P T P catalytic domains are fused at their amino and/or carboxyl termini to noncatalytic regulatory sequences. Regulation is effected by post-translational modifications and/or protein-protein interactions that modulate activity directly or indirectly through control of subcellular location. Dual-specificity phosphatases (DSPs) and low molecular weight (acid) phosphatases (LMPs) can also dephosphorylate phosphotyrosyl proteins. Sequence similarity between these molecules and classical PTPs is largely confined to the signature motif (Fig. 1). DSPs, which include the cell cycle regulators cdc25A, B and C

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and the MAP (mitogen-activated protein) kinase phosphatases MKP-1 and PAC-1, generally dephosphorylate both phosphotyrosine and phosphothreonine in specific sequence contexts. LMPs are even more distantly related to PTPs, although structural studies show that they retain the essentials of P T P catalysis (reviewed in [2°°]). Much less is known about LMPs, although they also may function in cell cycle regulation [5].

as positive signal transducers, and some data suggest that the same P T P can have positive or negative effects in different signaling pathways. T h e implications of P T P crystal structures for P T P regulation and the design of 'substrate traps' will be discussed in this review, together with selected recent examples of studies in which insight has been gained into the physiological functions of specific P T P family members.

This review focuses on recent progress in studies of the classical P T P family. T h e past two years witnessed a plethora of P T P studies, using approaches from crystallography to genetics. These studies reveal that the structural diversity of the P T P family reflects a broad range of functions. Although, not surprisingly, some PTPs exert negative influences on P T K signaling pathways, other act

Structural studies of PTPs Crystal structures have now been solved at high resolution for prototypical non-TM PTPs [6,7,8 °°] and RPTPs [9°°]. It is ironic, and a tribute to the rapid progress in t-he PTP field, that the first P T P structures were solved befor~ the first P T K structure, a mere seven years after identification of the first PTP. T h e details of P T P structure, in addition

Figure 1

Domains I

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PTPs @ 1997 Current Opinion in Cell Biology

Classical PTPs

1

The PEP superfamily. At least three families of molecules possess protein tyrosine phosphatase activity and shared elements of three-dimensional architecture. 'Classical PTPs' can be divided into transmembrane RPTP and nonreceptor, non-TM PTP subfamilies. These molecules consist of one or two conserved catalytic (PTP) domains (of approximately 240 amino acids; large open boxes), flanked by a wide variety of amino- and/or carboxy-terminal noncatalytic sequences (differently patterned or shaped areas) that function in regulation and/or targeting. Dual-specificity phosphatases (DSPs) and low molecular weight (acid) phosphatases (LMPs) are more distantly related to the classical PTPs, retaining only key residues within the PTP 'signature motif' (small open boxes) and a few other conserved residues. For DSPs and LMPs, the small open boxes represent the region comprising the PTP signature motif, the only region shared by all members of the PTP superfamily. The cross-hatched (in DSPs) and speckled (in LMPs) areas surrounding the signature motif region indicate the general lack of primary sequence conservation between PTPs, DSPs and LMPs outside of the signature motif. DSPs, as implied by their name, tend to dephosphorylate phosphotyrosine and phosphothreonine within highly restricted sequence contexts. LMPs are even more distantly related to the classical PTPs than are DSPs, although their three-dimensional structure and catalytic mechanism are similar to those of the classical PTPs and DSPs. The different PTPs are named beneath each structure. PEST, Pro-Glu-Ser-Thr motif.

Protein tyrosine phosphatases in signal transduction Neel and Tonks

to a comparison of classical PTPs with DSPs, LMPs and serine/threonine phosphatases, have been reviewed recently [2"o]. We will focus only on the implications of structural studies for P T P regulation and for the design of 'substrate-trapping' mutants. Structural studies of non-TM PTPs Structures have been solved for the catalytic domains of PTP-1B (i.e. PTP-1B that lacks its carboxy-terminal regulatory domain) [6,8"'] and for a Yersinia P T P [7]. These molecules share the same overall architecture, consisting of a central twisted, mixed 13 sheet flanked by cc helices. T h e signature motif residues are found within a single loop, nestled at the base of a cleft on the surface of the protein. T h e essential cysteinyl residue is in position for nucleophilic attack on an incoming phosphotyrosyl residue. T h e remaining residues of the signature motif function to increase the nucleophilicity of the catalytic cysteine and to bind to and position the incoming phosphate residue. The signature motif arginyl residue is particularly important for the latter function; mutation of this residue results in a marked decrease in both Km and Vmax [10"]. T h e depth of the cleft is set by an invariant tyrosyl residue (Y46 in PTP-1B). Thus, only phospho-tyrosine (not phospho-threonine or phospho-serine) is long enough to access the catalytic cysteine.

Comparison of the structure of PTP-1B with the phosphate analog tungstate bound at the active site with the structure of inactive PTP-1B (containing Cys---~Ser mutations) with a phosphopeptide bound at the active site yields insight into the dynamics of P T P catalysis ([8"']; reviewed in [2"']). For thiophosphate intermediate formation to proceed efficiently, the phenolic oxygen of the tyrosyl leaving group must be protonated. Elegant mutagenesis studies by Dixon and colleagues [I 1] predicted that an aspartyl residue (equivalent to D181 in PTP-1B) would donate this proton, serving as a general acid. In the tungstate-bound open structure, D181 is engaged in a salt bridge and is displaced from the active site. Binding of a phosphotyrosine-containing substrate is accompanied by a profound conformational change. In a dramatic example of induced fit, the cleft closes around the tyrosyl sidechain of the substrate and D181 is positioned appropriately to serve as a general acid (Fig. 2). These structural features have led to the design of mutants for 'trapping' P T P substrates. Early studies suggested that mutants of the catalytic cysteinyl residue of the Yersinia P T P (Cys---~Ser mutants) retained the ability to bind to phosphotyrosyl proteins [12]. Analogous mutants of MKP-1 were found to 'trap' the MAP kinases Erk-1 and Erk-2 [13], thus extending this strategy to DSPs. Recently, mutants (Asp--~Ala mutants) of the aspartyl residue that serves as the general acid (the analog of D181 in PTP-1B) were also found to act as substrate traps [10",14"o]. In a direct comparison of Cys---)Ser and

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Asp---~Ala mutants of the non-TM PTPs PTP-1B and PTP-PEST, Asp---~Ala mutants were found to be better substrate-trapping reagents. Replacement of D181 with an uncharged residue eliminates electrostatic repulsion between D181 and the phosphate moiety of the substrate. Thus, the hydrophobic interactions between the tyrosyl side chain and the enzyme that are generated upon loop closure are favored in Asp-+Ala mutants. Figure 2

© 1997 Current Opinion in Cell Biology

Position of Asp181 in the signature motif of PTP-1B. The position of Asp181 (the general acid) and a phosphate residue (PO 4) bound at the active site are shown. In an Asp-->Ala mutant (e.g. Asp181--~Ala), the replacement of the negatively charged aspartate residue with an uncharged alanine decreases electrostatic repulsion following loop closure. The tyrosyl residue attached to the phosphate as well as the rest of the target protein are not shown. Dotted lines represent hydrogen bonds. For details, see text.

Cys---~Ser mutants of several PTPs also have been used as putative 'dominant-negative' mutants in several studies. However, the same property that makes Cys--+Ser (and Asp--+Ala) mutants valuable reagents for the identification of P T P substrates renders their use as dominant-negatives problematic. As such mutants retain the ability to bind their substrates, by sterically interfering with their target molecule, they can, in principle and in practice [13,15"'], act biologically like the wild-type P T P (while at the same time acting as biochemical dominant-negatives, leading to an increase in tyrosyl phosphorylation of their substrates). P T P domain deletion mutants or mutants of the essential arginyl residue of the P T P signature motif (see above) are more appropriate choices as dominant-negatives. Implication of PTP(x structure for RPTP regulation

T h e topology of the PTP(x catalytic domain is highly similar to that of the non-TM PTPs, with one key

196 Cellregulation

difference: PTPet crystallizes as a dimer in which the amino-terminal helix-turn-helix motif of each monomer inserts, like a hairpin, into the catalytic domain of the other, making specific contacts with residues at the mouth of the catalytic cleft [9"]. In dimeric form, PTPet should be enzymatically inactive, the presence of the hairpin blocking substrate access to the active site and preventing generation of the closed structure (see above).

Figure3 f

.

.

.

.

.

RTK ligand

.

.

.

.

RPTP ligand

J

1

f

1

H

RTK

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i i ~.

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RPTP activity off

Little direct experimental evidence exists for R P T P dimer formation in vivo, but some indirect evidence is supportive. Other RPTPs have conserved residues at the amino termini of their domain Is, suggesting that they too may fold into a similar helix-turn-helix structure [9°°]. Interestingly, there is little conservation of the specific hairpin residues predicted to make contacts with the catalytic domains [9"°]. Furthermore, there is no analogous structure upstream from domain 2 of RPTPs or in the catalytic domains of single domain RPTPs or non-TM PTPs [9"']; this may suggest ways by which to design specific inhibitors of RPTPs. Most importantly, inactivation of RPTPs by dimerization may explain earlier observations regarding the behavior of EGF (epidermal growth factor) receptor-CD45 chimeras. T-cell lines lacking the tyrosine phosphatase CD45 cannot respond to T cell antigen receptor (TCR)-induced activation signals; reconstituting CD45 expression restores this capability. Desai et al. [16] found that EGF-receptor-CD45 chimeras can also rescue CD45 deficiency, but only in the absence of E G E Extrapolating from the P T P a crystal structure, EGF-induced dimerization of the chimera, with resultant P T P inhibition, may account for the results of Desai et al. Teleologically, regulation of RPTPs by dimerization presents an attractive model for controlling tyrosyl phosphorylation in response to cell-cell contact (Fig. 3). Upon cell-cell interaction, surface-bound ligands for RPTPs and receptor PTKs (RTKs) would have converse effects on their cognate receptors, thus promoting synergistic control of tyrosyl phosphorylation. Further studies are required to test whether the effects of mutating specific residues in the P T P ~ helix-turn-helix are consistent with the dimerization model, as well as to demonstrate the existence of ligand-induced dimers/muhimers in vivo; such studies should be aided by the recent identification of candidate ligands for RPTPs (see below).

J

© 1997CurrentO p i ~ in CellBiology Model for simultaneous activation of RTKs and inhibition of RPTPs by surface-bound ligands. RTKs and RPTPs found on one cell (bottom) may be engaged simultaneously by ligands expressed on the surface of an apposing cell (top). Upon cell-cell interaction, each of the respective ligands would promote clustering (oligomerization) of its cognate receptor. Whereas oligomerization leads to RTK activation, the crystal structure of PTP~. suggests that RPTP dimers would be enzymatically inhibited. Reciprocal regulation of RTKs and RPTPs provides a potential mechanism for synergistic control of tyrosyl phosphorylation.

RPTPs and cell adhesion Many R P T P ectodomains contain structural features (immunoglobulin domains, fibronectin type III repeats, etc.) that suggest a role in cell--cell or cell-matrix adhesion. Several recent studies, including genetic analyses of Drosophila RPTPs and the identification of homotypic or heterotypic ligands/binding proteins for some mammalian RPTPs, have demonstrated a role for RPTPs in cell adhesion signaling pathways.

RPTPsand the regulationof neuronaladhesion T h e P T P ~ structure suggests that dimerization/ muhimerization may be a general mechanism for R P T P regulation (Fig. 3). R P T P ligands, which should promote P T P oligomerization, would thus promote R P T P inactivation. This contrasts with the activating role of oligomerization for receptor PTKs.

Four of the five RPTP genes identified in Drosophila encode adhesion molecule like PTPs expressed selectively in the nervous system (reviewed in [17]). Recently, mutant embryos lacking expression of some of these RPTPs were generated [18"',19°']. Disruption of DPTP69D results in pupal lethality [18°°]. In mutant embryos, motor neuron growth cones show defects in their ability to recognize muscle targets, or follow pathways that bypass these

Protein tyrosine phosphatases in signal transduction Neel and Tonks

targets altogether [18°°]. Mutants lacking DPTP99A show no detectable phenotype, but this P T P probably also has a role in neuronal targeting, as the D69D/99A double mutant is more defective than the D P T P 6 9 D mutant [18°']. Loss-of-function mutations in DLAR illustrate a role in controlling the ability of different, but overlapping, subsets of neurons to navigate through various migration choice points and to innervate the appropriate muscle targets [19"°]. Overexpression of adhesion molecules such as fasciclin III in Drosophila motor neurons produces a similar phenotype [20], suggesting that RPTPs may regulate the same signaling pathways as these adhesion molecules or modulate common downstream targets. These observations support a critical role for RPTPs in regulating the adhesive events that control axonal pathfinding. Tyrosine phosphorylation dependent pathways involving homologs of fibroblast growth factor receptor (FGFR) or Eph family PTKs are potential targets for these PTPs. Studies aimed at identifying candidate ligands for PTPI3/~ revealed a role for mammalian RPTPs in the control of neuronal adhesion. PTPI3/~, which is expressed in glia, is characterized by an extracellular segment containing an amino-terminal carbonic anhydrase (CA)-like domain (Fig. 1). This domain lacks key CA catalytic residues, and instead functions as a binding pocket for a specific heterophilic ligand, contactin, a glycophosphatidylinositolanchored cell recognition molecule expressed on neurons [21"°]. T h e CA domain of PTPI3/~ can promote neuron binding and induce neurite outgrowth and differentiation; in such assays, surface contactin expression is required for neuronal response. Contactin also binds to a distinct ligand, restrictin, with opposite physiological effects (i.e. cell repulsion). During neuronal development, contactin-PTPl3/~ and contactin-restrictin interactions may mediate uni- or bidirectional signaling pathways between glia and neurons. Although contactin binds to PTPI3/~, and PTPI3/~ affects contactin function, contactin has not been shown to affect either PTPI3/~ enzymatic activity or downstream pathways regulated by this RPTP, although presumably it does. T h e effect of the PTPI3/~ ectodomain on contactin argues for caution in interpreting the phenotypes of the Drosophila R P T P mutants; some or even all of the observed defects could result from loss of the ability of RPTPs to function as ligands, independent of their capacity to dephosphorylate target substrates.

RPTPs and regulation of adherens junctions T h e ectodomains of PTP~t and P T P ~ contain an immunoglobulin (Ig) domain, three fibronectin type III repeats, and an MAM domain. MAM domains are newly appreciated motifs named for their appearance in several different molecules (meprin-Xenopus A5-mu) that may be involved in cell-cell contact. Several studies have shown that PTPla [22] and PTPK: [23] participate in homophilic binding interactions (reviewed in [24]). PTPI-t and PTP~:

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do not interact with each other, indicating specificity in these interactions. Mutagenesis studies suggest that both the MAM and the Ig domains participate in directing homophilic interactions. T h e Ig domain alone, when coated onto latex beads (covaspheres), can direct adhesive interactions, and deletion of the Ig domain prevents homophilic interactions in heterologous expression systems [25]. T h e MAM domain probably confers interaction specificity. A chimera in which the P T P ~ MAM domain is fused to the rest of PTPI.t gains new binding properties: it can bind to neither PTPrt nor P T P ~ but only to a molecule of itself [26]. These data suggest that the MAM domain guides the PTPp. or PTP~c molecule into the appropriate position in which to form strong contacts between apposing Ig domains. Although, when expressed at high levels, PTP~t and PTPK: can mediate homophilic cell adhesion, these PTPs probably do not promote cell adhesion per se, but rather transduce signals generated by cell adhesion, perhaps by regulating cadherin-catenin function. Both PTP~t [25] and PTP~c [27], together with other less well defined PTPs [28], have been shown to associate with cadherin-catenin complexes in various tissues and cell lines. Cadherins are implicated in tissue development and morphogenesis and represent the adhesive component in adherens junctions (reviewed in [29]). Association between the intracellular portion of cadherins and the actin cytoskeleton, mediated by catenins, is important for adhesion. Disruption of this multiprotein complex abrogates adhesion and may result in invasion and/or metastasis. Several cytoplasmic and receptor PTKs have been shown to phosphorylate components of the cadherin-catenin complex with concomitant disruption of adhesion. PTPs such as PTP~t and P T P ~ probably regulate the tyrosine phosphorylation, and thus the adhesive properties, of cadherin-catenin complexes. This may contribute to the mechanism by which cell-cell contact is stabilized and growth inhibited in, for example, confluent cell cultures.

R e g u l a t i o n of growth factor, cytokine, and o l i g o m e r i c receptor signaling by S H P s Non-TM PTPs containing Src homology 2 (SH2) domains (SHPs) have been identified in mammals, Xenopus and Drosophila. Two SHPs exist in vertebrates. SHP-1 is expressed at highest levels in hematopoietic cells. T h e motheaten (me~me) mouse, with a phenotype caused by absence of SHP-1, provides a murine model of SHP-1 deficiency. SHP-2 and its likely Drosophila homolog Corkscrew are expressed ubiquitously. Whether an additional SHP exists in Drosophila or whether Corkscrew also serves SHP-1 functions remains unclear. T h e presence of SH2 domains in PTPs suggested that these molecules might interact with known P T K signaling pathways. Recent evidence, combining biochemical

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and genetic approaches, validates this notion. Pathways regulated by the two SHPs have been specified, and several potential targets identified. Remarkably, despite their shared domain structure and considerable (55%) overall sequence identity, the SHPs appear to have distinct biological roles. Early studies indicated that SHP-1 is predominantly a negative regulator of PTK signaling, whereas SHP-2 and Corkscrew play a positive (i.e. signal-enhancing) role. More recent work, although not conclusive, raises the possibility that, depending on the specific signaling pathway, either SHP may have positive or negative effects. Regulation of hematopoietic cell signaling by SHP-1

T h e me~me mouse displays a panoply of hematopoietic abnormalities, affecting virtually every lineage. It is now clear that SHP-1 negatively regulates multiple hematopoietic signaling pathways, including those that are downstream of cytokine receptors, of immune recognition receptors, such as antigen and Fc receptors, and of RTKs.

SHP-1 regulates cytokine receptor signaling by controlling Janus family PTKs Initial studies, in which SHP-1 levels were lowered using inducible antisense RNA expression, suggested that SHP1 controlled IL3 receptor (IL3R) 13-chain phosphorylation [30]. Cells expressing lower levels of SHP-1 were also mildly hypersensitive to IL3 in proliferation assays [30]. Subsequent work using erythropoietin (EPO) receptor (EPOR) mutants indicated that an inability to recruit SHP-1 to the activated EPOR resulted in prolonged tyrosyl phosphorylation of the receptor-associated PTK Jak2 [31°°]. Likewise, IFNoc-stimulated macrophages from me~me mice exhibit markedly increased tyrosyl phosphorylation of Jakl, suggesting that one general function of SHP-1 may be to inactivate cytokine receptor associated Janus family PTKs [32]. Thus, hyperphosphorylation of the IL3R 13 chain in the presence of lowered SHP-1 could be an indirect consequence of increased receptorassociated PTK activity, rather than reflecting direct SHP1-mediated dephosphorylation of the 13 chain. However, not all Jaks (even those activated by the same cytokine receptor) are inactivated by SHP-1, as IFNcz-induced Tyk2 activation is comparable in normal and me~memacrophages [32]. IL3-directed mast cell proliferation is unaffected in me~me mice, although hematopoietic progenitor cells from such mice are hypersensitive to IL3 [33°°]. It is not clear whether SHP-2 or another PTP substitutes for SHP-1 in these pathways. Together, these studies are consistent with the hypothesis that SHP-1 regulates at least some cytokine receptor associated Jaks, but direct dephosphorylation of Jaks by SHP-1 has not been demonstrated. Use of novel substrate-trapping mutants of the SHPs should help to identify the proximal SHP-1 targets in these pathways. It also is unclear why SHP-1 appears to be recruited to cytokine receptors (e.g. the EPOR) substantially before

dephosphorylation of Jaks is detected; a second event may be required to activate SHP-1.

Regulation of lymphoid ceil signaling by SHP-1 SHP-1 also regulates lymphocyte signaling. Activation of signaling through the B cell antigen receptor (BCR) is abrogated upon co-cross-linking of the inhibitory Fc receptor (FcR) Fc)'RIIB; this serves to attenuate antibody production in the presence of circulating immune complexes (reviewed in [34]). Mutation of a specific Fc)'RIIB tyrosyl residue eliminates its ability to block B-cell activation [35]. A phosphotyrosyl peptide comprising the sequence surrounding this tyrosyl residue (i.e. the tyrosyl residue in Fc),RIIB) binds SHP-1, a 70kDa protein subsequently identified as SHP-2, and a 150kDa protein [36°°], which appears to be the inositol 5' monophosphatase SHIP [37°°]. As me~me mice were found to be refractory to Fc)'RIIB-mediated inhibition, a model was proposed in which SHP-1 is a critical signaling molecule used by FcyRIIB to terminate BCR signals [36°°]. Interestingly, Fc)'RIIB engagement does not result in major changes in tyrosyl phosphorylation, suggesting that Fc)'RIIB-bound SHP-1 has a limited number of targets [36°°]. One study suggests that phospholipase C (PLC) y is a target [38]. However, as FcyRII-indveed inhibition predominantly blocks BCR-induced calcium influx, not calcium release from intracellular stores [39], it is unclear whether and/or how PLC), dephosphorylation might be of regulatory importance. Moreover, SHP-1 is not generally required for Fc'[RIIB-mediated inhibition. Fc),RIIB also blocks activation signals generated through FccR, but FcyRIIB-mediated inhibition is normal in mast cells from me~me mice [37°°]. Instead, the inositol monophosphatase SHIP is proposed to play the key role in FcyRIIB signaling in mast cells [37°°]. It is not clear whether and/or why Fc),RIIB uses distinct signaling pathways in mast cells and B cells. Both SHIP and an SHP could be necessary for inhibition. Perhaps SHP-2 can substitute for SHP-1 in mast cells but not B cells. Alternatively, the actions of SHP-1 and SHIP could converge to produce similar effects, with different cell types using predominantly one or the other. A final possibility is that the lack of an FcyRlI effect in me~me B cells does not reflect a primary role for SHP-1 in this pathway, but instead is a secondary consequence of profoundly abnormal B-cell development in these mice. A motif similar to that found in Fc)'RIIB, now termed the ITIM (immune receptor tyrosine-based inhibitory motif) [40"], is also found in other receptors, notably killer-inhibitory receptors (KIRs) of natural killer (NK) cells and the B-cell coreceptor CD22. MHC class I antigens bind to KIRs, which then prevent activating receptors on NK cells from directing lysis of host cells. SHP-1 is recruited to KIRs [40°°,41-43] and phosphotyrosyl peptides from the KIR ITIM activate SHP-1 in vitro [40°°]. Furthermore, overexpression of a presumptive dominant-negative (Cys--->Ser) mutant of

Protein tyrosine phosphatases in signal transduction Neel and Tonks

SHP-1 blocks KIR-mediated inhibition [40°°]. However, as $HP-1, SHP-2 and SHIP can all bind to a similar motif (although with differing affinities; see [43]), it is difficult to be certain from these experiments that SHP-1 alone mediates KIR signals. SHP-1 binds tightly to CD22 [44°°,45--47], and it has been proposed that this interaction helps set the BCR activation threshold [44°°]. This model is consistent with elegant genetic studies of mice expressing transgenic BCRs in the me~me background [48°°]. Recently, CD22-deficient mice were generated [49°°,50°°]. T h e phenotype of these mice, whose B-cell lineage shows some similarity to that found in me~me mice (although the phenotype is quantitatively much less severe), is also consistent with a negative regulatory role for CD22. However, the signaling defects in CD22-/- mice must be interpreted with caution. As is the case for me~me mice, in the presence of altered B-cell development it is difficult to be certain whether observed defects reflect primary signaling roles for CD22 or secondary consequences of developmental abnormalities. Moreover, it has not been demonstrated directly that CD22 I T I M s are required for CD22-mediated inhibition, nor have the precise targets of SHP-1 in B-cell signaling been identified. SHP-1 also negatively regulates signaling from the TCR. Thymocytes and peripheral T cells from me~me mice hyperprolifetate in response to T C R stimulation and display hyperphosphorylation of several phosphotyrosyl proteins [51,52°']. It is not yet clear how SHP-1 antagonizes T C R signaling. A direct interaction between ZAP-70 (k-associated protein-70) and SHP-1 has been reported in T-cell lines and in heterologous expression systems, and it has been suggested that $HP-1 inactivates ZAP-70 [53°°]. However, Lorenz et al. [541 report that the PTKs Lck and Fyn (which are upstream of ZAP-70) are hyperactive in me~me thymocytes. Interaction between CD5 and the epsilon chain of the T C R has also been reported [52°°]. T h e latter is of particular interest, as thymocytes from CD5-deficient mice display T C R hypersensitivity comparable to that found in me~me mice [55]. However, there is no clear ITIM-like sequence within the cytoplasmic domain of CDS, so the nature of the SHP-1-CD5 interaction remains unclear. Further studies of the role of SHP-1 in T-cell signaling are needed to resolve these issues.

Regulation of the receptor protein tyrosine kinase c-Kit by SHP- 1 Two recent studies used genetic analysis to explore the role of SHP-1 in regulating the receptor P T K c-Kit in vivo [33"°,56°°]. SHP-1 binds to tyrosyl-phosphorylated c-Kit and itself becomes tyrosyl-phosphorylated in response to Kit ligand (KL) stimulation [57]. Mice bearing kinasedefective, but not kinase-dead, mutants of c-Kit (Wv/+ mice) were crossed with me/+ mice to generate all possible allelic combinations of SHP-1 and c-Kit. These studies

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reveal strong intergenic complementation between these two loci. Marked diminution of c-Kit signaling (in the Wv/IVv genotype) resulted in substantial improvement of the me~me phenotype. These results suggest that SHP-I negatively regulates c-Kit in hematopoietic progenitors and that excess signaling through the c-Kit pathway is an important contributor to the me/me phenotype. However, SHP-1 does not negatively regulate c-Kit in all cell types [33°',56°°]. There is no effect of loss of SHP-1 on coat color; SHP-1 is probably not even expressed in the neural crest cells that determine this phenotype. SHP-1 also has a relatively small effect on c-Kit signaling in mast cells. Loss of SHP-1 improves the mast cell deficiency in Wv/Wv mice, at least in some body areas. This may be explained by the increased survival of mast cells from Wv/lVv : me~me mice compared with those from Wv/Wv:+/+ mice [56°°], although proliferation in response to KL is unaffected by SHP-1 genotype [33°°]. T h e primary target(s) of SHP-1 in mast cells is also not clear. Lorenz et al. [33 °°] reported increased c-Kit tyrosyl phosphorylation in the absence of SHP-1, whereas Paulson et al. [56 °°] found no consistent difference. T h e latter workers also observed an increase in KL-directed Shc tytosyl phosphorylation and MAP kinase activation in the absence of SHP-1. Although further work is required to resolve these discrepancies, these studies provide compelling genetic evidence for the importance of SHP-1 in c-Kit regulation. T h e tissue specificity of this regulation emphasizes the complexity of the interplay between even a single RTK and P T P in the context of the whole organism. Identification of presumptive substrates for Corkscrew and SHP-2

Corkscrew is a required positive component of the Torso pathway, which directs embryonic head and tail development, as well as of several other RTK pathways in Drosophila [58,59°°,60°']. Recent work by two groups [61°°,62°°], using biochemical and genetic approaches, established that Corkscrew also regulates Sevenless signaling, most likely by dephosphorylating the product of the daughter of sevenless (dos) gene. These studies suggest that dephosphorylation of Dos by Corkscrew is required to generate a positive signal needed for Sevenless signaling. However, although Dos may be direct substrate of Corkscrew, the data do not exclude the possibility that Corkscrew actually targets a P T K that controls Dos phosphorylation. T h e precise pathway(s) in which Corkscrew and Dos participate remains unclear. T h e structure of Dos, which contains an amino-terminal PH (plecksttin homology) domain and multiple potential tyrosine-phosphorylation sites, is reminiscent of the structures of the mammalian proteins Gabl, IRS-1 and IRS-2 [62°°]. Intriguingly, these all bind SHP-2, and appear to serve as scaffolding proteins downstream of mammalian PTKs, but the precise pathways that they regulate are not fully understood. Given their structure, one possibility is that they collect

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Cell regulation

secondary signaling molecules, which are then released by dephosphorylation so that they may participate in downstream signaling. Which pathway(s) further downstream (of Dos) is regulated by Corkscrew and SHP-2 remains unclear. In the Sevenless pathway, Corkscrew must be either upstream and downstream of Raf or operate in a parallel pathway [60°°]. Multiple studies, in mammals [63-66] and Xenopus [67"], have placed SHP-2 upstream of MAP kinase, and

one study places it upstream of Ras activation [64]. Perhaps Corkscrew and SHP-2 do have multiple points of action in RTK signaling. Alternatively, SHP-2/Corkscrew might not act directly on RTK signaling pathways per se, but could instead function in a pathway that is permissive for RTK signaling, for example in response to attachment to the extracellular matrix (ECM). A distinctly different candidate substrate for mammalian SHP-2 was reported recently. Earlier work, by several

Figure 4 S. cerevisiae

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S. pombe Osmotic stress Oxidative stress Heat shock Ultraviolet light

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° 13

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lgg7 Current Opinion in Cetl Biology

Control of osmoregulatory pathways in S. cerevisiae and S. pombe by PTPs. A general pathway is shown at the left in bold, and more specific pathways, with named protein components, are shown to the right of this general pathway. Similar pathways regulate the response to stress in both types of yeast. Membrane sensors (histidine kinases) control the activation of downstream MAP kinase cascades. Many mammalian MAP kinase pathways are inactivated by DSPs. These two yeast pathways are inactivated by tyrosine phosphatases (PTP2, Pypl and Pyp2) (and probably by serine phosphatases also). Note that, in S. pombe, the same MAP kinase cascade has been shown to be involved in both cell cycle regulation (by controlling cell size at division) and control of sporulation (by controlling meiosis). Similar, as yet unidentified PTPs may be important for regulating some MAP cascades in higher eukaryotes. Dashed arrows indicate that the number of intervening steps is unclear; question marks indicate that the identity of a given component is unknown. MAPKK, MAPK kinase; MAPKKK, MAPKK kinase.

Protein tyrosine phosphatases in signal transduction Neel and Tonks

groups, showed that expression of a Cys---~Ser mutant of SHP-2 resulted in the increased tyrosyl phosphorylation of a l l 5 k D a protein which also was found to bind to SHP-2 [63,68,69,70"]. In a heroic effort, Fujioka et al. [71"'] purified this molecule, which they termed SHPS-1, and cloned its encoding gene. Unlike Dos, SHPS-1 is a transmembrane protein, with extracellular Ig domains and an intracellular domain containing multiple potential tyrosyl-phosphorylation sites, including several that conform to the consensus for binding SHP-2. Again, as is the case for Dos, the possibility exists that SHP-2 actually regulates a P T K that phosphorylates SHPS-1 (which then merely binds to SHP-2), rather than regulating SHPS-1 itself. Nevertheless, the structure of SHPS-1 implicates SHP-2 in ECM and/or cell adhesion signaling.

Negative regulation of T-cell signaling by SHP-2 SHP-2 associates with, and may be responsible for inhibitory signals delivered through, CTLA-4 (cytotoxic T lymphocyte antigen-4), a negative regulator of T-cell activation [72°']. CTLA-4-knockout mice display lymphoproliferation, with markedly increased numbers of activated lymphocytes. SHP-2 coimmunoprecipitates with CTLA-4, and a phosphotyrosyl peptide derived from the CTLA-4 sequence (pYz01VKM, where p represents phosphorylation) binds SHP-2 in vitro, reportedly via SHP-2's SH2 domains. T cells from lymph nodes of CTLA-4-knockout mice reveal marked increases in Fyn, Lck, and ZAP-70 activity, suggesting that recruitment of SHP-2 to CTLA-4 inactivates T C R signaling as binding of SHP-1 to CD22 is proposed to inactivate BCR signals. Although these findings are provocative, no direct evidence for tyrosine phosphorylation of CTLA-4 has been presented; thus, the nature of the proposed interaction between CTLA-4 and SHP-2 is unclear. Furthermore, it is not clear that the putative SHP-2-binding site is required for CTLA-4 function; notably, this site differs from other known high-affinity binding sites for SHP-2, all of which have isoleucine, valine, or leucine at the +3 position. Thus, it is possible that interaction of SHP-2 with CTLA-4 requires an intermediate protein. Most importantly, it is not clear whether the observed hyperactivation of T cell signaling components is the cause of the lymphoproliferation in CTLA-4 mice or the indirect consequence of the increased number of activated T cells in these mice.

201

systems indicates that classical PTPs also play key roles in regulating at least some MAPKs (Fig. 4). T h e elegant work of Saito and his colleagues [74] defined a classical two-component osmosensing system in Saccharomyces cerevisiae that is analogous to those in bacterial chemotaxis pathways. T h e histidine kinase Sin-l, via the response regulator Sskl, activates a kinase cascade which includes the MAPK family member Hogl, which is most similar to Schizosaccharomyces pombe Styl/Spcl and mammalian p38. Genetic and biochemical studies indicate that Hogl is inactivated by PTP2 [74,75]. An analogous MAPK-dependent pathway linking changes in environmental stimuli to the onset of mitosis has been characterized in S. pombe. In this pathway, Spcl/Styl is inactivated by specific tyrosyl dephosphorylation by Pypl and Pyp2 [15"',76"°-78°']. Spcl/Styl is also implicated in regulation of a distinct pathway in sexual development in response to environmental stress [77°',78°°]. Spcl/Styl activation evokes the stress-induced phosphorylation of the transcription factor Aft-l, which in turn is required for induction of meiotic genes. One such gene encodes Pyp2, which, in a feedback inhibitory loop, dephosphorylates Spcl. Although it seems likely that PTPs are also involved in regulation of some MAPK pathways in higher eukaryotes, no specific P T P has yet been implicated in MAPK regulation. Interestingly, however, PTP-1B is phosphorylated on the same carboxy-terminal seryl residues in response to a variety of environmental stresses and at mitosis [79], suggesting a link between stress-induced and mitotic pathways similar to that found in S. pombe.

Conclusions It now seems clear that PTPs are at least as important as PTKs in regulating protein-tyrosyl phosphorylation. As disorders of P T K structure and regulation cause a variety of diseases in animals and man, it seems likely that P T P disorders will be of similar pathogenetic importance. T h e availability of structural data, together with detailed enzymological studies, has led to a sophisticated understanding of P T P catalysis, the design of powerful tools for defining P T P targets and function, and potential strategies for developing specific P T P inhibitors. T h e next few years should bring a wealth of new information about the biological roles of individual PTPs. If past is prolog, then combining genetic and biochemical approaches offers the greatest hope of rapid progress.

PTPs and dephosphorylation of MA P kinases MAPK (MAP kinase) family members are essential components in several signaling pathways. For activation, MAPKs require phosphorylation, by a dual-specificity kinase, of both the tyrosine and the threonine residues in the amino acid sequence TXY that is found in their activation loops. A significant body of literature implicates DSPs, such as MKP-1, in the dephosphorylation of MAPKs in vivo (reviewed in [73]). Recent work in yeast

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18. -•

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Together with [19"], this paper presents genetic evidence for the involve. ment of RPTPs in neuron guidance in Drosophila. 19. •.

Krueger NX, Van Vactor D, Wan HI, Gelbart WM, Goodman CS, Saito H: The transmembrane tyrosine phosphatase DLAR controls motor axon guidance in Drosophila. Cell 1996, 84:611-622. Together with [18°'], this paper presents genetic evidence for the involvement of RPTPs in neuron guidance in Drosophila. 20.

Lin DM, Goodman CS: Ectopic and increased expression of fasiculin III alters motoneuron growth cone guidance. Neuron 1994, 13:507-523.

21. • ,,

Peles E, Nativ M, Campbell PL, Sajurai T, Martinez R, Lev S, Clary DO, Schilling J, Barnea G, Plowman GD et aL: The carbonic anhydrase domain of receptor tyrosine phosphatase ~ is a functional ligand for the axonal recognition molecule contactin. Cell 1995, 82:251-260. This paper identifies the ectodomaln of RPTP~/~ as a ligand for the neuronal recognition molecule contactin and the ectodomaln of contactin as a putative ligand for RPTP~/~. 22.

Gebbink MFBG, Zondag GCM, Wubbolts RW, Beijersbergen RL, Van Etten I, Moolenaar WH: Cell-cell adhesion mediated by a receptor-like protein tyrosine phosphatase. J Biol Chem 1993, 268:16101-16104.

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31. ••

Klingmuller U, Lorenz U, Cantley LC, Neel BG, Lodish HF: Specific recruitment of SH-PTP1 to the erythropoietin receptor causes inactivation of JAK2 and termination of proliferative signals. Cell 1995, 80:729-738. Together with [32], this paper provides the first evidence that SHP-1 negatively regulates cytokine receptor signaling by dephosphorylating and inactivating cytokine receptor associated Janus family PTKs. 32.

David M, Chen HE, Ling L, Goelz S, Lamer AC, Neel BG: Differential regulation of the crJ~ interferon-stimulated Jak/Stat pathway by the SH2-domain containing tyrosine phosphatase SHPTP1. Mo/ Cell Bio/1995, 15:7050-7058.

33. •.

Lorenz U, Bergemann AD, Steinberg HN, Flanagan JG, Li X, Galli SJ, Neel BG: Genetic analysis reveals cell type-specific regulation of receptor tyrosine kinase c-Kit by the protein tyrosine phosphatase SHP1. J Exp Med 1995, 184:1111-1126. Together with [56"°], this paper uses a genetic approach to evaluate the biological significance of interactions between c-Kit and SHP-1. These studies conclude that SHP-1 does, indeed, negatively regulate c-Kit in vivo, but, unexpectedly, in a tissue-specific manner. 34.

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This paper provides evidence for the involvement of SHP-1 in the negative regulation of BCR activation pathways via the inhibitory Fc receptor, FcyRIIB. 37. •.

Ono M, Bolland S, Tempst P, Ravetch JV: Role of the inositol phosphatase SHIP in negative regulation of the immune system by the receptor Fc'fRIIB. Nature 1996, 383:263-266. This paper shows that SHP-1 is not required for signaling through Fc"yRIIBin mast cells, and instead suggests the involvement of the inositol monophosphatase SHIP. 38.

39.

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52. •o

Pani G, Fischer K-D, Rascan IM, Siminovitch KA: Signaling capacity of the T cell antigen receptor is negatively regulated by the PTPIC tyrosine phosphatase. J Exp Med 1996, 184:839-852. The authors of this paper provide evidence that SHP-1 is a negative regulator of activation of thymocytes and peripheral T cells, and that SHP-1 possibly acts through CD6. 53. •.

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40. •=

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Doody GM, Justement LB, Delibrias CC, Matthews PJ, Lin J, ThomasML, Fearon DT: A role in B cell activation for CD22 and the protein tyrosine phosphatase SHP. Science 1995, 269:242-244. This paper demonstrates that SHP-1 associates with CD22 upon B-cell activation, and suggests that CD22 acts as a negative regulator of BCR signaling.

59. ..

PerkinsLA, Johnson MR, Melnick MB, Perrimon N: The nonreceptor protein tyrosine phosphatase Corkscrew functions in multiple receptor tyrosine kinase pathways in Drosophila. Dev Biol 1997, in press. This paper provides evidence for the involvement of Corkscrew in multiple RTK pathways in Drosophila (in addition to its involvement in the Torso pathway, which was first described by these authors).

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60. •-

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43.

44. •o

48. °°

Cyster JG, Goodnow CC: Protein tyrosine phosphatase 1C negatively regulates antigen receptor signaling in B lymphocytes and determines thresholds for negative selection. Immunity 1995, 2:1-20. An elegant genetic analysis of the role of SHP-1 in setting the BCR activation threshold. The authors explored the effect of superimposing loss of SHP-1 on clonal selection and deletion in transgenic mice expressing specific B-cell receptors. They conclude that loss of SHP-1 decreases the BCR activation threshold. 49. °°

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Sato S, Miller AS, Inaoki M, Bock CB, Jansen PJ, Tang LK, Tedder TF: CD22 is both a postitive and negative regulator of lymphocyte antigen receptor signal transduction: altered signaling in CD22-deficient mice. Immunity 1996, 5:551-562. See annotation [49"']. 51.

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Allard JD, Chang HC, Herbst R, McNeill H, Simon MA: The SH2containing tyrosine phosphatase corkscrew is required during signaling by sevenless, Rasl and Raf. Deve/oprnent 1996, 122:1137-1146. This paper provides genetic evidence for the involvement of Corkscrew in Sevenless signal transduction and places Corkscrew either parallel to, or both upstream and downstream of, Ras in this pathway. 61. •.

Herbst R, Carroll PM, Allard JD, Schilling J, Raabe T, Simon MA: Daughter of Sevenless is a substrate of the phosphotyrosine phosphatase corkscrew and functions during Sevenless signaling. Ce//1996, 85:899-909. This paper, together with [62°'], provides biochemical and genetic evidence supporting the identification of Dos as a substrate for Corkscrew. 62. •.

Raabe T, Riesgo-Escovar J, Liu X, Bausenwein BS, Deak P, Maroy P, Hafen E: DOS, a novel pleckstrin homology domaincontaining protein required for signal transduction between sevenless and Rasl in Drosophila. Ceil 1996, 85:911-920. This paper reports the cloning of the dos gene, the initial characterization of the Dos protein, and the position of the Dos protein relative to Corkscrew in the Sevenless pathway. 63.

MilarskiKL, Saltiel AR: Expression of catalytically inactive Syp phosphatase in 3T3 cells blocks stimulation of mitogenactivated protein kinase by insulin. J Bio/Chern 1994, 269:21239-21243.

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Noguchi T, Matozaki 1", Horita K, Fujioka Y, Kasuga M: Role of SH-PTP2, a protein-tyrosine phosphatase with src homology 2 domains, in insulin-stimulated ras activation. Mo/Cell Bio/ 1994, 14:6674-6682.

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Yamauchi K, Milarski KL, Saltiel AR, Pessin JE: Protein-tyrosinephosphatase SHPTP2 is a required positive effector for insulin downstream signaling. Proc Nat/Acad Sci USA 1995, 92:664-668.

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Bennett AM, Hausdorff SF, O'Reilly AM, Freeman RM Jr, Neel BG: Multiple requirements for SHPTP2 in epidermal growth

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Tang TL, Freeman RM, O'Reilly AM, Neel BG, Sokol SY: The SH2-containing protein tyrosine phosphatase SH-PTP2 is required upstream of MAP kinase for early Xenopus development. Ce//1995, 80:473-483. Demonstrates that vertebrate SHP-2, like Corkscrew, is required for early embryonic development.

Provides evidence suggesting that SHP-2 may be a negative regulator of T-cell signaling, acting through the CTLA-4 pathway. ,73.

TonksNK: Protein tyrosine phosphatases end the control of cellular signaling responses. Adv Pharmaco/1996, 3 6 : 9 1 - 1 1 9 .

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Maeda T, Wurgler-Murphy SM, Saito H: A two-component system that regulates an osmosensing MAP kinase cascade in yeast. Nature 1994, 369:242-245.

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Maeda T, Tsai AY, Saito H: Mutations in a protein tyrosine phosphatase gene (PTP2) and a protein serine/threonine phosphatase gene (PTCl) cause a synthetic growth defect in Saccharomyces cerevisiae. Mo/ Cell Bio/1993, 13:5409-5417.

•

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YamauchiK, Pessin JE: Epidermal growth factor-induced association of the SHPTP2 protein tyrosine phosphatase with a 115-kDa phosphotyrosine protein. J Bio/Chem 1995, 270:14871-148,74.

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YamauchiK, Ribon V, Saltiel AR, Pessin JE: Identification of the major SHPTP2-binding protein that is tyrosine-phosphorylated in response to insulin. J Biol Chem 1995, 270:17,716-1 7722.

'70. •

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Provides a detailed biochemical characterization of the SHPS-1 protein, whose encoding gene was later cloned by these investigators (see [,71"']). ,71. •,

FujiokaY, Matozaki T, Noguchi T, Iwamatsu A, Yamao T, TakahashiN, Tsuda M, Takada T, Kasuga M: A novel membrane glycoprotein, SHPS-1, that binds the SH2-domain-containing protein tyrosine phosphatase SHP-2 in response to mitogens and cell adhesion. Mo/Cell Bio/1996, 16:6887-6899. Describes the purification of a novel SHP-2-binding protein/potential substrate, and also describes the molecular cloning of the gene encoding this protein. 72. •=

Marengere LEM, Waterhouse P, Duncan GS, Mittrucker H-W, Feng G-S, Mak TW: Regulation of T cell receptor signaling by tyrosine phosphatase SYP association with CTLA-4. Science 1996, 272:11,70-1173.

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Millar JB, Buck V, Wilkinson MG: Pypl and Pyp2 PTPases dephosphorylate an osmosensing MAP kinase controlling cell size at division in fission yeast. Genes Dev 1995, 9:211,77-2130. Together with [15°°], this paper implicates Pypl and Pyp2 as negative regulators of the Spcl/Styl pathway in S. pombe. This pathway functions in the response to cell stress and in mitotic control. 77. °-

Wilkinson MG, Samuels M, Takeda T, Toone WM, Shieh J-C, Toda T, Millar JBA, Jones N: The Aft transcription factor is a target for the Sty1 stress-activated MAP kinase pathway in fission yeast. Genes Dev 1996, 10:2289-2301. Together with [,78"], this paper provides additional details about the S. pombe Styl/Spcl pathway and its involvement in the regulation of multiple physiological functions. ,78.

Shiozaki K, Russell P: Conjugation, meiosis, and the osmotic

•°

stress response are regulated by Spcl kinase through Aft1 transcription factor in fission yeast. Genes Dev 1996, 10:22,76-2288. See annotation [,7,7"']. 79.

ShifrinVI, Davis RJ, Neel BG: Phosphorylation of proteintyrosine phosphatase PTP-1B on identical sites suggests activation of a common signaling pathway during mitosis and stress reponse in mammalian cells. J Bio/Chem 199"7, 272:295,7-2962.