Faculty Bibliography

SH-PTP2, the vertebrate homolog of Drosophila corkscrew, associates with several activated growth factor receptors, but its biological function is unknown. We assayed the effects of injection of wild-type and mutant SH-PTP2 RNAs on Xenopus embryogenesis. An internal phosphatase domain deletion (delta P) acts as a dominant negative mutant, causing severe posterior truncations. This phenotype is rescued by SH-PTP2, but not by the closely related SH-PTP1. In ectodermal explants, delta P blocks fibroblast growth factor (FGF)- and activin-mediated induction of mesoderm and FGF-induced mitogen-activated protein (MAP) kinase activation. Our results indicate that SH-PTP2 is required for early vertebrate development, acting as a positive component in FGF signaling downstream of the FGF receptor and upstream of MAP kinase.

The steady-state kinetic properties of SH-PTP1 (PTP1C, SHP, HCP), a Src homology 2 (SH2) domain-containing protein tyrosine phosphatase (PTPase), were assessed and compared with those of three truncation mutants, using p-nitrophenyl phosphate, phosphotyrosyl (pY) peptides, and reduced, carboxyamido-methylated, maleylated, and tyrosyl-phosphorylated lysozyme as substrates. At physiological pH (7.4), truncation of the two N-terminal SH2 domains [SH-PTP1(delta SH2)] or the last 35 amino acids of the C-terminus [SH-PTP1(delta C35)] activated the phosphatase activity by 30-fold and 20-34-fold relative to the wild-type enzyme, respectively. Truncation of the last 60 amino acids resulted in a mutant [SH-PTP1(delta C60)] with wild-type activity. SH-PTP1 and SH-PTP1(delta C60) displayed apparent saturation kinetics toward pNPP only at acidic pH (pH < or = 5.4); as pH increased above 5.5, their apparent KM values increased dramatically. In contrast, SH-PTP1(delta SH2) obeyed normal Michaelis-Menten kinetics at all pH values tested (pH 5.1-7.4) with a constant KM (10-14 mM). Furthermore, two synthetic pY peptides corresponding to known and potential phosphorylation sites on the erythropoietin (EPOR pY429) and interleukin-3 (IL-3R pY628) receptors bound specifically to the N-terminal SH2 domain of SH-PTP1 (KD = 1.8-10 microM) and activated the catalytic activity of SH-PTP1 and SH-PTP1(delta C60) but not SH-PTP1(delta SH2), in a concentration-dependent manner. Maximal activation (25-30-fold) of SH-PTP1 was achieved at 70 microM EPOR pY429, and the maximally activated enzyme approached the activity of SH-PTP1(delta SH2). Addition of EPOR pY429 peptide, which corresponds to the recently identified in vivo binding site for SH-PTP1, at 40 microM also completely restored the saturation kinetic behavior of SH-PTP1 (at pH 7.4) toward pNPP, with catalytic parameters (KM = 12.8 mM, kcat = 3.2 s-1) similar to those of SH-PTP1(delta SH2). These data suggest that the SH2 domains of SH-PTP1 serve to autoinhibit the phosphatase activity of the PTPase domain. A model is proposed in which the SH2 domains interact with the PTPase domain in a pY-independent fashion and drive the PTPase domain into an inactive conformation.

Protein-tyrosine-phosphatase SHPTP2 (Syp/PTP-1D/PTP2C) is the homologue of the Drosophila corkscrew (csw) gene product, which transmits positive signals from receptor tyrosine kinases. Likewise, SHPTP2 has been implicated in positive signaling from platelet-derived growth factor receptor beta (PDGFR). Upon PDGF stimulation, SHPTP2 binds to the PDGFR and becomes tyrosine-phosphorylated. We have identified tyrosine-542 (pY542TNI) as the major in vivo site of SHPTP2 tyrosine phosphorylation. The pY542TNI sequence conforms to the consensus binding site for the SH2 domain of Grb2, which, by association with Sos1, couples some growth factor receptors to Ras. Following PDGF stimulation, Grb2 binds tyrosine-phosphorylated SHPTP2. Moreover, a mutant PDGFR lacking its SHPTP2 binding site displays markedly reduced Grb2 binding. These data indicate that phosphorylation of SHPTP2 couples Grb2 to PDGFR in vivo, providing a mechanism for Ras activation by PDGFR and for positive signaling via SHPTP2 and Csw.

The cytoplasmic insulin receptor substrate-1 (IRS-1), which is multiply phosphorylated in vivo on tyrosine residues, is a known binding protein for the tandem src homology 2 (SH2) domain-containing protein tyrosine phosphatase, SH-PTP2. Eleven phosphotyrosyl (pY) peptides from IRS-1 were screened for allosteric activation of SH-PTP2 phosphatase activity toward phosphorylated, reduced, carboxyamidomethylated, and maleylated-lysozyme. Peptides IRS-1pY895, IRS-1pY1172, and IRS-1pY1222 showed up to 50-fold acceleration of dephosphorylation. Analyses of Arg to Lys mutants in either or both SH2 domains indicate that both the N-terminal (N-SH2) and C-terminal (C-SH2) domains function in allosteric activation. Direct determination by surface plasmon resonance of the dissociation constants between pY peptides and glutathione S-transferase fusions to N-SH2 and C-SH2 domains reveals a 240-fold preference of the N-SH2 domain (compared with the C-SH2 domain) for IRS-1pY1172. The N-SH2 domain prefers IRS-1pY1172 > IRS-1pY895 > IRS-1pY1222, whereas C-SH2 domain prefers IRS-1pY1222 > IRS-1pY895 > IRS-1pY1172. These data suggest that each SH2 domain can bind to a distinct pY sequence of multiply phosphorylated protein substrates such as IRS-1, while activating hydrolysis at a third pY sequence bound in the SH-PTP2 active site. In addition, proteolysis and truncation studies reveal an autoregulatory function for the C-terminal region of SH-PTP2. Limited tryptic cleavage within the C-terminus results in 27-fold activation of protein tyrosine phosphatase activity. The activated tryptic fragment cannot be further activated by pY peptide binding to the SH2 domains indicating that autoregulatory functions of the SH2 domains are dependent on the C-terminal region. These data suggest that multiple levels for control of SH-PTP2 enzymatic activity may exist in vitro and in vivo.

Signaling by tyrosine kinases involves direct associations between proteins with Src homology 2 (SH2) domains and sites of tyrosine phosphorylation. Specificity in signaling pathways results in part from inherent selectivity in interactions between particular SH2 domains and phosphopeptide sequences. The cytoplasmic phosphotyrosine phosphatase SH-PTP2 (Syp, PTP 1D, PTP-2C) contains two SH2 domains (N and C) which mediate its association with and activation by the platelet-derived growth factor (PDGF) and epidermal growth factor receptors and IRS-1. We have developed a competitive phosphopeptide binding assay to analyze specificity of the SH-PTP2 N-SH2 domain for phosphorylation sites of these phosphoproteins. The sequence surrounding Tyr1009 bound with greatest affinity (ID50 = 14 microM) of eight PDGF receptor-derived phosphopeptides tested. No peptides corresponding to known epidermal growth factor receptor phosphorylation sites bound with high affinity. However, an alternative sequence surrounding Tyr954 bound tightly (ID50 = 21 microM). Of the 13 IRS-1-related peptides analyzed, sequences surrounding Tyr546, Tyr895, and Tyr1172 bound with highest affinity (ID50 = 11, 4, and 1 microM, respectively). Alternative phosphopeptides generally bound with much weaker affinity (ID50 > 150 microM). These findings are consistent with recent mutational analyses of the PDGF receptor and predict site-specific interactions between SH-PTP2 and each of these phosphoproteins. Comparisons between peptide sequences suggest that the N-terminal SH2 domain of SH-PTP2 binds with highest affinity to phosphotyrosine (pY) followed by a beta-branched residue (Val, Ile, Thr) at pY+1 and a hydrophobic residue (Val, Leu, Ile) at pY+3 positions. Peptide truncation studies also indicate that residues outside of the pY-1 to pY+4 motif are required for high affinity interactions.

We have developed a family of mammalian coexpression vectors that permit identification of living or fixed cells overexpressing a gene of interest by surrogate detection of a coexpressed marker protein. Using these 'pMARK' vectors, a fluorescence-based, single cell proliferation assay was developed and used to study the effect of retinoic acid receptor beta (RAR-beta) on cell cycling. We demonstrate that transient overexpression of RAR-beta in the presence, but not absence, of all-trans retinoic acid results in a dramatic suppression of cell proliferation. We further show that this effect requires the DNA binding (C) domain of RAR-beta. It has been previously shown that RAR-beta expression is markedly altered in a variety of neoplasms and cell lines. Our data support the hypothesis that loss of RAR-beta may contribute to tumor progression by removing normal restraints on proliferation. The pMARK vectors should be useful for studying other genes that putatively suppress or enhance proliferation.

PTP-1B is a major nontransmembrane phosphotyrosine phosphatase in human cell lines and tissues, but its physiological function(s) and mechanism(s) of regulation are largely unknown. We have found that in human diploid fibroblasts a novel PTP-1B mRNA isoform is produced upon stimulation of quiescent cells with a variety of growth factors. Generation of this isoform requires protein synthesis, suggesting that the product(s) of an immediate early response gene(s) is required. RNase protection and reverse transcription polymerase chain reaction analysis demonstrate that the isoform arises as a consequence of alternative pre-mRNA splicing, leading to retention of the last intron. The novel isoform is found on polyribosomes, indicating that it is actively translated, and is variably expressed in human tissues. Sequence analysis indicates that the isoform encodes a PTP-1B protein with an altered C terminus. To our knowledge, this is the first example of growth factor-regulated alternative splicing.

The non-transmembrane phosphotyrosine phosphatase 1B (PTP-1B) is an abundant enzyme, normally localized to the cytosolic face of the endoplasmic reticulum via a C-terminal targeting sequence. We have found that agonist-induced platelet activation results in proteolytic cleavage of PTP-1B at a site upstream from this targeting sequence, causing subcellular relocation of its catalytic domain from membranes to the cytosol. PTP-1B cleavage is catalyzed by the calcium-dependent neutral protease calpain and is a general feature of platelet agonist-induced aggregation. Moreover, PTP-1B cleavage correlates with the transition from reversible to irreversible platelet aggregation in platelet-rich plasma. Engagement of gpIIb-IIIa is necessary for inducing PTP-1B cleavage, suggesting that integrins regulate tyrosine phosphatases as well as tyrosine kinases. PTP-1B cleavage is accompanied by a 2-fold stimulation of its enzymatic activity, as measured by immune complex phosphatase assay, and correlates with discrete changes in the pattern of tyrosyl phosphorylation. Cleavage and subcellular relocation of PTP-1B represents a novel mechanism for altering tyrosyl phosphorylation that may have important physiological implications in cell types other than platelets.

The importance of tyrosyl phosphorylation in regulating growth factor-receptor-mediated signal transduction is firmly established, but the roles of protein tyrosine phosphatases (PTPases) in these pathways are unclear. PTPases that contain src-homology 2 (SH2) domains, which mediate interactions with specific phosphotyrosyl proteins, probably play an important role in early signaling events following growth factor stimulation. In this review, the two mammalian SH2-containing PTPases, SH-PTP1 and SH-PTP2, are described and their possible roles in signal transduction discussed. In addition, the implications of recent genetic studies in the mouse and Drosophila, which shed light on the actions of these PTPases in physiology and pathology, will be addressed.

A human protein tyrosine phosphatase containing two src homology 2 (SH2) domains (SH-PTP2) was expressed in Escherichia coli under T7 promoter control and purified to near homogeneity. The purified protein, with molecular mass of 68 kDa on SDS-polyacrylamide gel electrophoresis, was identified as SH-PTP2 by its protein tyrosine phosphatase activity and N-terminal amino acid sequence analysis. Its protein tyrosine phosphatase activity was sensitive to pH and salt concentration. Whereas its optimum pH for the low molecular weight substrate para-nitrophenyl phosphate is 5.6, the pH optima for peptide substrates were shifted toward neutral. With the artificial protein substrate reduced, carboxyamidomethylated, and maleylated lysozyme, it displays 2000-fold lower Km (1.7 microM) and 2.4-fold higher kcat (0.11 s-1) than with para-nitrophenyl phosphate. Among the phosphopeptides from autophosphorylation sites of receptors for epidermal growth factor and platelet-derived growth factor, SH-PTP2 displayed high activity toward phosphopeptides corresponding to pY992 of the epidermal growth factor receptor and pY1009 and pY1021 of the platelet-derived growth factor receptor. In further enzymatic studies with phosphopeptides corresponding to pY1009, SH-PTP2 showed nonlinear Line-weaver-Burk double-reciprocal plots, suggesting that the phosphopeptide corresponding to pY1009 may have a substrate and allosteric effect.