Activation of Src in human breast tumor cell lines: elevated levels of phosphotyrosine phosphatase activity that preferentially recognizes the Src carboxy terminal negative regulatory tyrosine 530.
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Elevated levels of Src kinase activity have been reported in a number of human cancers, including colon and breast cancer. We have analysed four human breast tumor cell lines that exhibit high levels of Src kinase activity, and have determined that these cell lines also exhibit a high level of a phosphotyrosine phosphatase activity that recognizes the Src carboxy-terminal P-Tyr530 negative regulatory site. Total Src kinase activity in these cell lines is elevated as much as 30-fold over activity in normal control cells and specific activity is elevated as much as 5.6-fold. When the breast tumor cells were grown in the presence of the tyrosine phosphatase inhibitor vanadate, Src kinase activity was reduced in all four breast tumor cell lines, suggesting that Src was being activated by a phosphatase which could recognize the Tyr530 negative regulatory site. In fractionated cell extracts from the breast tumor cells, we found elevated levels of a membrane associated tyrosine phosphatase activity that preferentially dephosphorylated a Src family carboxy-terminal phosphopeptide containing the regulatory tyrosine 530 site. Src was hypophosphorylated in vivo at tyrosine 530 in at least two of the tumor cell lines, further suggesting that Src was being activated by a phosphatase in these cells. In preliminary immunoprecipitation and antibody depletion experiments, we were unable to correlate the major portion of this phosphatase activity with several known phosphatases. (+info)
Shp-2 tyrosine phosphatase functions as a negative regulator of the interferon-stimulated Jak/STAT pathway.
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Shp-2 is an SH2 domain-containing protein tyrosine phosphatase. Although the mechanism remains to be defined, substantial experimental data suggest that Shp-2 is primarily a positive regulator in cell growth and development. We present evidence here that Shp-2, while acting to promote mitogenic signals, also functions as a negative effector in interferon (IFN)-induced growth-inhibitory and apoptotic pathways. Treatment of mouse fibroblast cells lacking a functional Shp-2 with IFN-alpha or IFN-gamma resulted in an augmented suppression of cell viability compared to that of wild-type cells. To dissect the molecular mechanism, we examined IFN-induced activation of signal transducers and activators of transcription (STATs) by electrophoretic mobility shift assay, using a specific DNA probe (hSIE). The amounts of STAT proteins bound to hSIE upon IFN-alpha or IFN-gamma stimulation were significantly increased in Shp-2(-/-) cells. Consistently, tyrosine phosphorylation levels of Stat1 upon IFN-gamma treatment and, to a lesser extent, upon IFN-alpha stimulation were markedly elevated in mutant cells. Furthermore, IFN-gamma induced a higher level of caspase 1 expression in Shp-2(-/-) cells than in wild-type cells. Reintroduction of wild-type Shp-2 protein reversed the hypersensitivity of Shp-2(-/-) fibroblasts to the cytotoxic effect of IFN-alpha and IFN-gamma. Excessive activation of STATs by IFNs was also diminished in mutant cells in which Shp-2 had been reintroduced. Together, these results establish that Shp-2 functions as a negative regulator of the Jak/STAT pathway. We propose that Shp-2 acts to promote cell growth and survival through two mechanisms, i.e., the stimulation of growth factor-initiated mitogenic pathways and the suppression of cytotoxic effect elicited by cytokines, such as IFNs. (+info)
gp49B1 inhibits IgE-initiated mast cell activation through both immunoreceptor tyrosine-based inhibitory motifs, recruitment of src homology 2 domain-containing phosphatase-1, and suppression of early and late calcium mobilization.
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We define by molecular, pharmacologic, and physiologic approaches the proximal mechanism by which the immunoglobulin superfamily member gp49B1 inhibits mast cell activation mediated by the high affinity Fc receptor for IgE (FcepsilonRI). In rat basophilic leukemia-2H3 cells expressing transfected mouse gp49B1, mutation of tyrosine to phenylalanine in either of the two immunoreceptor tyrosine-based inhibitory motifs of the gp49B1 cytoplasmic domain partially suppressed gp49B1-mediated inhibition of exocytosis, whereas mutation of both abolished inhibitory capacity. Sodium pervanadate elicited tyrosine phosphorylation of native gp49B1 and association of the tyrosine phosphatases src homology 2 domain-containing phosphatase-1 (SHP-1) and SHP-2 in mouse bone marrow-derived mast cells (mBMMCs). SHP-1 associated transiently with gp49B1 within 1 min after coligation of gp49B1 with cross-linked FcepsilonRI in mBMMCs. SHP-1-deficient mBMMCs exhibited a partial loss of gp49B1-mediated inhibition of FcepsilonRI-induced exocytosis at concentrations of IgE providing optimal exocytosis, revealing a central, but not exclusive, SHP-1 requirement in the counter-regulatory pathway. Coligation of gp49B1 with cross-linked FcepsilonRI on mBMMCs inhibited early release of calcium from intracellular stores and subsequent influx of extracellular calcium, consistent with SHP-1 participation. Because exocytosis is complete within 2 min in mBMMCs, our studies establish a role for SHP-1 in the initial counter-regulatory cellular responses whereby gp49B1 immunoreceptor tyrosine-based inhibition motifs rapidly transmit inhibition of FcepsilonRI-mediated exocytosis. (+info)
The tyrosines in the bidentate motif of the env-sea oncoprotein are essential for cell transformation and are binding sites for Grb2 and the tyrosine phosphatase SHP-2.
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The transforming gene product of the S13 avian erythroblastosis virus, the env-sea protein, is a member of the hepatocyte growth factor receptor family of tyrosine kinases comprising Met, Ron, and Sea. Like all three members of this family, the env-sea protein has a so-called bidentate motif (Y557INMAVTY564VNL) composed of two tandemly arranged tyrosines in the carboxyl terminus. To investigate whether the tyrosine residues in this motif are essential for the env-sea-mediated transformation, we generated tyrosine to phenylalanine mutations. Substitutions of both tyrosine residues resulted in complete loss of the transforming activity. In contrast, single mutations at either tyrosine did not inhibit transformation of Rat1 cells, and mutation of tyrosine 564 actually increased transformation of Rat 1 cells. To define signaling pathways activated by the env-sea protein, we looked for protein-protein interactions mediated by these tyrosine residues. We show that the bidentate motif is responsible for interaction with the adapter protein Grb2, phosphatidylinositol 3-kinase, and the tyrosine phosphatase SHP-2. Furthermore, we show that microinjected Src homology 2 domains from either Grb2 or SHP-2 blocked the transforming activity of the env-sea protein. Together, these results suggest that the tyrosines within the bidentate motif are essential for the env-sea transformation. (+info)
Gab-family adapter proteins act downstream of cytokine and growth factor receptors and T- and B-cell antigen receptors.
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We previously found that the adapter protein Gab1 (110 kD) is tyrosine-phosphorylated and forms a complex with SHP-2 and PI-3 kinase upon stimulation through either the interleukin-3 receptor (IL-3R) or gp130, the common receptor subunit of IL-6-family cytokines. In this report, we identified another adapter molecule (100 kD) interacting with SHP-2 and PI-3 kinase in response to various stimuli. The molecule displays striking homology to Gab1 at the amino acid level; thus, we named it Gab2. It contains a PH domain, proline-rich sequences, and tyrosine residues that bind to SH2 domains when they are phosphorylated. Gab1 is phosphorylated on tyrosine upon stimulation through the thrombopoietin receptor (TPOR), stem cell factor receptor (SCFR), and T-cell and B-cell antigen receptors (TCR and BCR, respectively), in addition to IL-3R and gp130. Tyrosine phosphorylation of Gab2 was induced by stimulation through gp130, IL-2R, IL-3R, TPOR, SCFR, and TCR. Gab1 and Gab2 were shown to be substrates for SHP-2 in vitro. Overexpression of Gab2 enhanced the gp130 or Src-related kinases-mediated ERK2 activation as that of Gab1 did. These data indicate that Gab-family molecules act as adapters for transmitting various signals. (+info)
Expression of dominant-negative src-homology domain 2-containing protein tyrosine phosphatase-1 results in increased Syk tyrosine kinase activity and B cell activation.
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The Src-homology domain 2 (SH2)-containing cytoplasmic tyrosine phosphatase, SHP-1 (SH2-containing protein tyrosine phosphatase-1), interacts with several B cell surface and intracellular signal transduction molecules through its SH2 domains. Mice with the motheaten and viable motheaten mutations are deficient in SHP-1 and lack most mature B cells. To define the role of SHP-1 in mature B cells, we expressed phosphatase-inactive SHP-1 (C453S) in a mature B cell lymphoma line. SHP-1 (C453S) retains the ability to bind to both substrates and appropriate tyrosine-phosphorylated proteins and therefore can compete with the endogenous wild-type enzyme. We found that B cells expressing SHP-1 (C453S) demonstrated enhanced and prolonged tyrosine phosphorylation of proteins with molecular masses of 110, 70, and 55-60 kDa after stimulation with anti-mouse IgG. The tyrosine kinase Syk was hyperphosphorylated and hyperactive in B cells expressing SHP-1 (C453S). SHP-1 and Syk were coimmunoprecipitated from wild-type K46 cells, K46 SHP-1 (C453S) cells, and splenic B cells, and SHP-1 dephosphorylated Syk. Cells expressing SHP-1 (C453S) showed increased Ca2+ mobilization, extracellular signal-regulated kinase activation, and homotypic adhesion after B cell Ag receptor engagement. Thus, SHP-1 regulates multiple early and late events in B lymphocyte activation. (+info)
Immune signalling: SHP-2 docks at multiple ports.
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The protein tyrosine phosphatase SHP-2 functions in many diverse signalling pathways. The recent identification of a SHP-2-binding protein as a homologue of the Grb2-associated adaptor protein Gab1 sheds light on the role of SHP-2 in immune signalling. (+info)
Flt3 signaling involves tyrosyl-phosphorylation of SHP-2 and SHIP and their association with Grb2 and Shc in Baf3/Flt3 cells.
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Flt3 ligand (FL) is an early-acting potent co-stimulatory cytokine that regulates proliferation and differentiation of a number of blood cell lineages. Its receptor Flt3/Flk2 belongs to class III receptor tyrosine kinases that also include the receptors for colony-stimulating factor 1, Steel factor, and platelet-derived growth factor. Using CSF-1 receptor/Flt3 chimeras, two groups have characterized some of the post-receptor signaling events and substrate specificity of murine Flt3 receptor. However, there are few studies on the signaling pathway through human Flt3. We examined human Flt3 signaling pathways in a murine IL-3-dependent hematopoietic cell line Baf3, which stably expresses full-length human Flt3 receptor. This subline proliferates in response to human FL. Like the chimeric murine Flt3, human Flt3 undergoes autophosphorylation, associates with Grb2, and leads to tyrosine phosphorylation of Shc on ligand binding. We found that SHP-2, but not SHP-1, is tyrosine-phosphorylated by FL stimulation. SHP-2 does not associate with Flt3, but binds directly to Grb2. SHIP is also tyrosine-phosphorylated and associates with Shc after FL simulation. We further examined the downstream signaling pathway. FL transiently activates MAP kinase. This activation could be blocked by PD98059, a specific MEK inhibitor. PD98059 also blocked cell proliferation in response to FL. These results demonstrate that SHP-2 and SHIP are important components in the human Flt3 signaling pathway and suggest that SHP-2 and SHIP, by forming complexes with adapter proteins Grb2 and Shc, may modulate MAP kinase activation, which may be necessary for the mitogenic signaling of Flt3. (+info)