, 1993). We report here the optimal recognition motifs for SH2 domains from GRB-2, Drk, Csk, Vav, fps/fes, SHC, Syk (carboxy-terminal SH2), 3BP2, and HCP (amino-terminal SH2 domain, also called PTP1C and SHPTP1). As predicted, SH2 domains from proteins that fall into group I on the basis of a Phe or Tyr at the PiD5 position (GRB-2, 3BP2, Csk, fps/fes, Syk C-terminal SH2) select phosphopeptides with the general motif phospho-Tyr-hydrophilic (residue)-hydrophilic (residue)-hydrophobic (residue). The SH2 domains of SHC and HCP (group III proteins with Ile, Leu, or Cys at the PD5 position) selected the general motif phospho-Tyr-hydrophobic-Xxx-hydrophobic, also as predicted. Vav, which has a Thr at the PD5 position, selected phospho-Tyr-Met-Glu-Pro as the optimal motif Each SH2 domain selected a unique optimal motif distinct from motifs previously determined for other SH2 domains. These motifs are used to predict potential sites in signaling proteins for interaction with specific SH2 domain-containing proteins. The Syk SH2 domain is predicted to bind to Tyr-hydrophilic-hydrophilic-LeuIIle motifs like those repeated at 10-residue intervals in T-and B-cell receptor-associated proteins. SHC is predicted to bind to a subgroup of these same motifs. A structural basis for the association of Csk with Src family members is also suggested from these studies.The activation of cellular protein tyrosine kinases by growth factors, lymphokines, and cytokines initiates a cascade of events critical for mitosis and other cellular responses.

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Cytolysis of target cells by natural killer (NK) cells and by some cytotoxic T cells occurs unless prevented by inhibitory receptors that recognize MHC class I on target cells. Human NK cells express a p58 inhibitory receptor specific for HLA-C. We report association of the tyrosine phosphatase HCP with the p58 receptor in NK cells. HCP association was dependent on tyrosine phosphorylation of p58. Phosphotyrosyl peptides corresponding to the p58 tail bound and activated HCP in vitro. Furthermore, introduction of an inactive mutant HCP into an NK cell line prevented the p58-mediated inhibition of target cell lysis. These data imply that the inhibitory function of p58 is dependent on its tyrosine phosphorylation and on recruitment and activation of HCP.

Mice homozygous for the recessive allelic mutation motheaten (me) or viable motheaten (mev) on chromosome 6 develop severe defects in hematopoiesis. In this paper we present the findings that the me and mev mutations are within the hematopoietic cell protein-tyrosine phosphatase (Hcph) gene. High resolution mapping localized me to an area tightly linked to Hcph on chromosome 6. Abnormalities of the Hcph protein product were demonstrated by Western blot analysis and by activity assays in both me/me and mev/mev mice. Molecular analysis of the Hcph cDNA identified abnormal transcripts in both mutants. DNA sequence analyses of cDNA and genomic clones revealed that both the me and mev mutations are point mutations that result in aberrant splicing of the Hcph transcript. These findings provide the first available animal models for a specific protein-tyrosine phosphatase deficiency, thus facilitating determination of the precise role of this signaling molecule in hematopoiesis.

Interleukin 3 (IL-3) regulates the proliferation and differentiation of hematopoietic cells. Although the 1L-3 receptor chains lack kinase catalytic domins, IL-3 induces tyrosine phosphorylation of cellular proteins. To investigate the potential role of the JAK family of protein-tyrosine kinases in EL-3 signal transduction, we have obtained fulllength cDNA clones for murine Jakl and Jak2 protein-tyrosine kcinas and prepared antiserum against the predicted proteins. Using antisera against Jak2, we demonstrate that 1L-3 stimulation results in the rapid and specific tyrosine phosphorylation of Jak2 and activates its in vitro kinase activity.

SHP-1 is an SH2-containing cytoplasmic tyrosine phosphatase that is widely distributed in cells of the hematopoietic system. SHP-1 plays an important role in the signal transduction of many cytokine receptors, including the receptor for erythropoietin, by associating via its SH2 domains to the receptors and dephosphorylating key substrates. Recent studies have suggested that SHP-1 regulates the function of Jak family tyrosine kinases, as shown by its constitutive association with the Tyk2 kinase and the hyperphosphorylation of Jak kinases in the motheaten cells that lack functional SHP-1. We have examined the interactions of SHP-1 with two tyrosine kinases activated during engagement of the erythropoietin receptor, the Janus family kinase Jak-2 and the c-fps/fes kinase. Immunoblotting studies with extracts from mouse hematopoietic cells demonstrated that Jak2, but not c-fes, was present in anti-SHP-1 immunoprecipitates, suggesting that SHP-1 selectively associates with Jak2 in vivo. Consistent with this, when SHP-1 was coexpressed with these kinases in Cos-7 cells, it associated with and dephosphorylated Jak2 but not c-fes. Transient cotransfection of truncated forms of SHP-1 with Jak2 demonstrated that the SHP-1-Jak2 interaction is direct and is mediated by a novel binding activity present in the N terminus of SHP-1, independently of SH2 domain-phosphotyrosine interaction. Such SHP-1-Jak2 interaction resulted in induction of the enzymatic activity of the phosphatase in in vitro protein tyrosine phosphatase assays. Interestingly, association of the SH2n domain of SHP-1 with the tyrosine phosphorylated erythropoietin receptor modestly potentiated but was not essential for SHP-1-mediated dephosphorylation of Jak2 and had no effect on c-fes phosphorylation. These data indicate that the main mechanism for regulation of Jak2 phosphorylation by SHP-1 involves a direct, SH2-independent interaction with Jak2 and suggest the existence of similar mechanisms for other members of the Jak family of kinases. They also suggest that such interactions may provide one of the mechanisms that control SHP-1 substrate specificity.Protein tyrosine phosphorylation is a common mechanism of signal transduction and is controlled by the balance of protein tyrosine kinases (PTKases) and protein tyrosine phosphatases (PTPases) (11). SHP-1 (1) is a PTPase predominantly expressed in cells of the hematopoietic system; it is also termed PTP1C, HCP, SHPTP1, and SHP (15,21,24,44). Loss of functional SHP-1 because of mutations in the mouse SHP-1 gene is associated with motheaten disease, characterized by increased phosphorylation in hematopoietic cells, hypersensitivity to extracellular stimuli, and heightened myelopoiesis (25,26,32). This indicates that SHP-1 is a critical negative regulator of tyrosine phosphorylation and signal transduction in hematopoietic cells.SHP-1 is a cytoplasmic protein with two src-homology 2 (SH2) domains at the amino region (SH2n and SH2c) and a PTPase catalytic domain at the carboxyl terminus (44). SHP-1 may therefore...

Hematopoietic cell phosphatase (HCP) is a tyrosine phosphatase with two Src homology 2 (SH2) domains that is predominately expressed in hematopoietic cells, including cells whose growth is regulated by interleukin-3 (IL-3). The potential effects of HCP on IL-3-induced protein tyrosine phosphorylation and growth regulation were examined to assess the role of HCP in hematopoiesis. Our studies demonstrate that, following ligand binding, HCP specifically associates with the 13 chain of the IL-3 receptor through the amino-terminal SH2 domain of HCP, both in vivo and in vitro, and can dephosphorylate the receptor chain in vitro. The effects of increasing or decreasing HCP levels in IL-3-dependent cells were assessed with dexamethasone-inducible constructs containing an HCP cDNA in sense and antisense orientations. Increased HCP levels were found to reduce the levels of IL-3-induced tyrosine phosphorylation of the receptor and to dramatically suppress cell growth. Conversely, decreasing the levels of HCP increased IL-3-induced tyrosine phosphorylation of the receptor and marginally increased growth rate. These results support a role for HCP in the regulation of hematopoietic cell growth and begin to provide a mechanistic explanation for the dramatic effects that the genetic loss of HCP, which occurs in motheaten (me) and viabk motheaten (me') mice, has on hematopoiesis.The viability, proliferation, differentiation, and functional activation of hematopoietic cells are regulated by hematopoietic growth factors through interaction with their cognate receptors (2,18,20,32,36). Although a few hematopoietic growth factor receptors (c-Fms and c-Kit) are receptor tyrosine kinases, the majority of the hematopoietic growth factor receptors belong to a large cytokine receptor superfamily that does not have intrinsic kinase activity. Nevertheless, ligand binding to receptors of the cytokine receptor superfamily rapidly induces tyrosine phosphorylation of cellular substrates as well as one or more of the receptor chains. A critical role for tyrosine phosphorylation in growth regulation has been suggested by the observations that inhibitors of tyrosine kinases inhibit cell growth (56), that inhibitors of tyrosine phosphatases can stimulate cell proliferation in the absence of ligand (47,54,57), and that, among mutant receptors, there is a direct correlation between the ability to induce tyrosine phosphorylation and the ability to support ligand-induced cell growth (34,35,45

Using in vitro protein tyrosine phosphatase (PTPase) assays, we found that sodium stibogluconate, a drug used in treatment of leishmaniasis, is a potent inhibitor of PTPases Src homology PTPase1 (SHP-1), SHP-2, and PTP1B but not the dual-specificity phosphatase mitogen-activated protein kinase phosphatase 1. Sodium stibogluconate inhibited 99% of SHP-1 activity at 10 μg/ml, a therapeutic concentration of the drug for leishmaniasis. Similar degrees of inhibition of SHP-2 and PTP1B required 100 μg/ml sodium stibogluconate, demonstrating differential sensitivities of PTPases to the inhibitor. The drug appeared to target the SHP-1 domain because it showed similar in vitro inhibition of SHP-1 and a mutant protein containing the SHP-1 PTPase domain alone. Moreover, it forms a stable complex with the PTPase: in vitro inhibition of SHP-1 by the drug was not removed by a washing process effective in relieving the inhibition of SHP-1 by the reversible inhibitor suramin. The inhibition of cellular PTPases by the drug was suggested by its rapid induction of tyrosine phosphorylation of cellular proteins in Baf3 cells and its augmentation of IL-3-induced Janus family kinase 2/Stat5 tyrosine phosphorylation and proliferation of Baf3 cells. The augmentation of the opposite effects of GM-CSF and IFN-α on TF-1 cell growth by the drug indicated its broad activities in the signaling of various cytokines. These data represent the first evidence that sodium stibogluconate inhibits PTPases and augments cytokine responses. Our results provide novel insights into the pharmacological effects of the drug and suggest potential new therapeutic applications.