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...

We recently reported that phosphatidylinositol (PI) 3-kinase becomes associated with the activated erythropoietin receptor (EpR), most likely through the Src homology 2 (SH2) domains within the p85 subunit of PI-3 kinase and one or more phosphorylated tyrosines within the EpR. We have now investigated this interaction in more detail and have found, based on both blotting studies with glutathione S-transferase-p85-SH2 fusion proteins and binding of these fusion proteins to SDS-denatured EpRs, that this binding is direct. Moreover, both in vitro competition studies, involving phosphorylated peptides corresponding to the amino acid sequences flanking the eight tyrosines within the intracellular domain of the EpR, and in vivo studies with mutant EpRs bearing tyrosine to phenylalanine substitutions, indicate that phosphorylation of Tyr 503 within the EpR is essential for the binding of PI 3-kinase. The presence of PI 3-kinase activity in EpR immunoprecipitates from DA-3 cells infected with wild-type but not Y503F EpRs confirms this finding. Our results demonstrate that the SH2 domains of p85 can bind, in addition to their well established Tyr-Met/Val-X-Met consensus binding sequence, a Tyr-Val-Ala-Cys motif that is present in the EpR. A comparison of erythropoietin-induced tyrosine phosphorylations and proliferation of wild-type and Y503F EpR-infected DA-3 cells revealed no differences. However, the PI-3 kinase inhibitor, wortmannin, markedly inhibited the erythropoietin-induced proliferation of both cell types, suggesting that PI 3-kinase is activated in Y503F EpR expressing cells. This was confirmed by carrying out PI 3-kinase assays with anti-phosphotyrosine immunoprecipitates from erythropoietin-stimulated Y503F EpR-infected DA-3 cells and suggested that PI 3-kinase has a role in regulating erythropoietin-induced proliferation, but at a site distinct from the EpR.Erythropoietin is the principal in vivo stimulator of mammalian erythropoiesis (1) and exerts its action by binding to receptors on the surface of relatively mature erythroid progenitors (2-4). These erythropoietin receptors (EpRs) 1 belong to a family of hematopoietin receptors whose members are characterized by the presence of conserved cysteines and Trp-Ser-XTrp-Ser motifs in their extracellular domains and the absence of any known catalytic activity in their intracellular regions (5, 6). Nonetheless, although the EpR lacks tyrosine kinase activity, it, along with a number of other cellular proteins, becomes transiently phosphorylated on tyrosine residues within minutes of binding erythropoietin (7-13). Recent studies suggest that this rapid phosphorylation is carried out, at least in part, by the cytoplasmic tyrosine kinase, Jak 2 (14, 15). Following this activation of the EpR, a number of intracellular proteins apparently become physically associated with it (8, 16 -19). These proteins include Grb2 (8, 18), Shc (8,18), and the enzyme, phosphatidylinositol (PI) 3-kinase (16,17,19). All three of these proteins contain at least one Src homology 2 (SH2)...

The tyrosine kinase Tyk-2 is physically associated with the Type I interferon (IFN) receptor complex and is rapidly activated during IFN alpha stimulation. We report that Tyk-2 forms stable complexes with the SH2-containing hematopoietic cell phosphatase (HCP) in several hematopoietic cell lines in vivo, and that the IFN alpha-induced tyrosine-phosphorylated form of Tyk-2 is a substrate for the phosphatase activity of HCP in in vitro assays. Furthermore, treatment of cells with the phosphatase inhibitor sodium orthovanadate induces tyrosine phosphorylation of Tyk-2 and an associated 115-kDa protein. Altogether, these data suggest that HCP regulates tyrosine phosphorylation of the Tyk-2 kinase, and thus its function may be important in the transmission of signals generated at the Type I IFN receptor level.

In kidney epithelial cells, an angiotensin II (Ang II) type 2 receptor subtype (AT 2 ) is linked to a membrane-associated phospholipase A 2 (PLA 2 ) and the mitogenactivated protein kinase (MAPK) superfamily. However, the intervening steps in this linkage have not been determined. The aim of this study was to determine whether arachidonic acid mediates Ang II's effect on p21ras and if so, to ascertain the signaling mechanism(s). We observed that Ang II activated p21ras and that mepacrine, a phospholipase A 2 inhibitor, blocked this effect. This activation was also inhibited by PD123319, an AT 2 receptor antagonist but not by losartan, an AT 1 receptor antagonist. Furthermore, Ang II caused rapid tyrosine phosphorylation of Shc and its association with Grb2. Arachidonic acid and linoleic acid mimicked Ang II-induced tyrosine phosphorylation of Shc and activation of p21ras. Moreover, Ang II and arachidonic acid induced an association between p21ras and Shc. We demonstrate that arachidonic acid mediates linkage of a G protein-coupled receptor to p21ras via Shc tyrosine phosphorylation and association with Grb2͞Sos. These observations have important implications for other G protein-coupled receptors linked to a variety of phospholipases.