Direct Association with and Dephosphorylation of Jak2 Kinase by the SH2-Domain-Containing Protein Tyrosine Phosphatase SHP-1

Abstract

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...

Reduced Tyk2/SHP-1 interaction and lack of SHP-1 mutation in a kindred of familial hemophagocytic lymphohistiocytosis

“…To determine whether such associations are defective in FHLH, we immunoprecipitated SHP-1 from PBMC of the patient and the parents, and probed the immunocomplexes with antibodies against the Jak kinases. Consistent with our previous reports, 36,41 Tyk2 was detected in the SHP-1 immunocomplexes from the father (Figure 4a, lane 2). However, the levels of Tyk2 in the SHP-1 immunocomplexes from the patient and the mother were markedly reduced, representing approximately 5-10% of the father's.…”

“…The mutant has Jak-binding activity but contains point mutations inactivating the SHP-1 SH2 domains to prevent its association with phosphotyrosine receptors (Figure 5c). 36 We detected Tyk2 protein from both parents in the anti-SHP-1/N immunocomplexes in the transfected cells (Figure 5d, lanes 3 and 4). The amount of Tyk2 from the parent M1, whose PBMC showed reduced Tyk2/SHP-1 association, was smaller than that of the F1 Tyk2 (compare lane 4 to lane 3 of Figure 5d, upper panel).…”

“…Since dephosphorylation and inactivation of Tyk2 by SHP-1 may depend on intermolecular interactions, 36 the reduced Tyk2 association with SHP-1 may lead to increased Tyk2 activity and enhanced cellular responses that depend on the Tyk2 kinase for signal transduction. Given the functional involvement of Tyk2 in the signal transduction of multiple hematopoietic growth factors/cytokines, a hyperactive Tyk2 is not inconsistent with the hyperproliferation of hematopoietic cells that is characteristic of FHLH.…”

“…36 The wild-type human Tyk2 cDNA was a gift from Dr George Stark, The Cleveland Clinic Foundation, Cleveland, OH, USA. The cDNA clones were inserted into the eukaryotic cell expression vector pRK5 or pXM as indicated.…”

“…24 Our recent study demonstrates that SHP-1 directly binds to the Jak family kinases via a region in its N-terminus independently of SH2/phosphotyrosine interactions. 36 As this interaction leads to SHP-1 activation and the dephosphorylation of Jak kinases, it may be a critical factor required for specific and efficient dephosphorylation of SHP-1 substrates. Because the Jak kinases play critical roles in signal transduction of many hematopoietic growth factors and cytokines, 35,37 defects in SHP-1/Jak interactions may affect SHP-1 dephosphorylation of these substrates and cause hematopoietic disorders.…”