Effective Dephosphorylation of Src Substrates by SHP-1

Abstract

The protein-tyrosine phosphatase SHP-1 is a negative regulator of multiple signal transduction pathways. We observed that SHP-1 effectively antagonized Srcdependent phosphorylations in HEK293 cells. This occurred by dephosphorylation of Src substrates, because Src activity was unaffected in the presence of SHP-1. One reason for efficient dephosphorylation was activation of SHP-1 by Src. Recombinant SHP-1 had elevated activity subsequent to phosphorylation by Src in vitro, and SHP-1 variants with mutated phosphorylation sites in the C terminus, SHP-1 Y538F, and SHP-1 Y538F,Y566F were less active toward Src-generated phosphoproteins in intact cells. A second reason for efficient dephosphorylation is the substrate selectivity of SHP-1. Pull-down experiments with different GST-SHP-1 fusion proteins revealed efficient interaction of Src-generated phosphoproteins with the SHP-1 catalytic domain rather than with the SH2 domains. Phosphopeptides that correspond to good Src substrates were efficiently dephosphorylated by SHP-1 in vitro. Phosphorylated "optimal Src substrate" AEEEIpYGEFEA (where pY is phosphotyrosine) and a phosphopeptide corresponding to a recently identified Src phosphorylation site in p120 catenin, DDLDpY 296 GMMSD, were excellent SHP-1 substrates. Docking of these phosphopeptides into the catalytic domain of SHP-1 by molecular modeling was consistent with the biochemical data and explains the efficient interaction. Acidic residues N-terminal of the phosphotyrosine seem to be of major importance for efficient substrate interaction. Residues C-terminal of the phosphotyrosine probably contribute to the substrate selectivity of SHP-1. We propose that activation of SHP-1 by Src and complementary substrate specificities of SHP-1 and Src may lead to very transient Src signals in the presence of SHP-1. The SH21 domain PTP SHP-1 regulates multiple signal transduction events by dephosphorylation (1-3). These comprise signaling of cytokine receptors such as the erythropoietin receptor (4), and the interleukin-3 receptor (5), and of receptor tyrosine kinases such as c-Kit (6 -8), the colony-stimulating factor-1 receptor (9, 10), and the epithelial kinase Ros (11). SHP-1 modulates also the function of immunoreceptors (12), and cytoplasmic tyrosine kinases such as Lck (13,14). In these and many other cases, SHP-1 regulates signaling in a negative manner. In other pathways, SHP-1 may also exert a positive function. Thus, a role of SHP-1 for differentiation of glia cells (15), and for Ras-dependent activation of mitogen-activated protein kinase (16) have been reported. Also, SHP-1 has the capacity to activate Src kinase by dephosphorylation of the inhibitory phosphotyrosine in the Src C terminus and may thus stimulate Src-dependent phosphorylations in certain cell types (17). SHP-1 can translocate into the nucleus, however, its nuclear substrates are still elusive (18 -21). Among the recently identified substrates of SHP-1 is p120 ctn (22), a cytoplasmic protein that possesses armadillo-like repeats, participates in ...

Temporal quantitation of mutant Kit tyrosine kinase signaling attenuated by a novel thiophene kinase inhibitor OSI-930

“…Genetic studies with Kit null and tyrosine phosphatase Shp-1 null mice have implicated Shp-1 as a negative regulator of Kit function in vivo (25,26); in vitro studies indicate that ubiquitinmediated Shp-1 degradation may contribute to transformation by Kit mutation (27). The phosphorylation of Shp-1 has been shown to be important for maximal dephosphorylation of substrates, and consistent with this model mutation of Shp-1 Y 538 and Y 566 were shown to impair its function (28). The PEST domain tyrosine phosphatase BDP-1 (29, 30) shared a similar temporal phosphorylation profile following Kit inhibition.…”

STAT3- and DNA methyltransferase 1-mediated epigenetic silencing of SHP-1 tyrosine phosphatase tumor suppressor gene in malignant T lymphocytes

“…Importantly, we show that STAT3 used this inhibitory mechanism to target SHP-1 tyrosine phosphatase, a well recognized tumor suppressor (9). Because in normal cells SHP-1 down-regulates signaling mediated by a spectrum of cytokines, growth factors, chemokines, antigens and other molecules (9)(10)(11), loss of SHP-1 renders the malignant cells hypersensitive to a whole array of extra-and intracellular stimuli. Noteworthy, activation of STAT3 by tyrosine 705 phosphorylation, and the simultaneous expression of DNMT1 and HDAC1 is insufficient to mediate the fully effective SHP-1 gene silencing.…”

PU.1 Activates Transcription of SHP-1 Gene in Hematopoietic Cells

“…SHP-1 acts in the immune and other hematopoietic cells by inhibiting signaling through receptors for cytokines, growth factors, and chemokines as well as receptors directly involved in the immune responses and programmed cell death (5). SHP-1 down-regulates cell activation by binding and de-phosphorylating the receptors, receptor associated tyrosine kinases, and the down-stream signaling molecules such as Vav1 (6) and src kinase substrates (7). SHP-1 acts as tumor suppressor and loss of its expression has been identified in the whole spectrum of lymphoid and myeloid cell malignancies (8 -11).…”