Association of hematopoietic cell phosphatase with c-Kit after stimulation with c-Kit ligand.

Protein tyrosine phosphorylation and dephosphorylation have been implicated in the growth and functional responses of hematopoietic cells. Recent studies have identified a novel protein tyrosine phosphatase, termed hematopoietic cell phosphatase (HCP) or PTP1C, that is predominantly expressed in hematopoietic cells. HCP encodes a cytoplasmic phosphatase that contains two src homology 2 (SH2) domains. Since SH2 domains have been shown to target the association of signal-transducing molecules with activated growth factor receptors containing intrinsic protein kinase activity, we assessed the association of HCP with two hematopoietic growth factor receptors, c-Kit and c-Fms. The results demonstrate that HCP transiently associates with ligand-activated c-Kit but not c-Fms and that this association occurs through the SH2 domains. In both colony-stimulating factor 1- and stem cell factor-stimulated cells, there is a marginal increase in tyrosine phosphorylation of HCP. Lastly, HCP can dephosphorylate autophosphorylated c-Kit and c-Fms in in vitro reactions. The potential role of HCP in stem cell factor signal transduction is discussed.     

Tyrosine phosphorylation of p95Vav in myeloid cells is regulated by GM-CSF, IL-3 and steel factor and is constitutively increased by p210BCR/ABL.

Vav is a recently described proto-oncogene expressed only in hematopoietic cells which contains an SH2 and two SH3 domains and shares homology with the Dbl GDP-GTP exchange factor and BCR. p95Vav is phosphorylated on tyrosine residues in response to stimulation of the T cell antigen receptor, cross-linking of IgE or IgM receptors and stimulation of immature hematopoietic cells by Steel factor. Monoclonal antibodies to human Vav were generated and used to examine the events which regulate tyrosine phosphorylation of p95Vav in myeloid cells. In the factor-dependent MO7e cell line, p95Vav was rapidly phosphorylated on tyrosine residues in a dose- and time-dependent manner by GM-CSF, IL-3 and Steel factor. Introduction of the BCR/ABL oncogene into this cell line resulted in factor-independent proliferation and constitutive phosphorylation of p95Vav. Tyrosine phosphorylation of p95Vav was also substantially increased by treatment of cytokine-deprived cells with the tyrosine phosphatase inhibitor sodium vanadate. Since many of the cytokines known to induce tyrosine phosphorylation of p95Vav are also known to activate JAK family tyrosine kinases, we looked for an interaction of p95Vav with JAK kinases. p95Vav co-precipitated with JAK2 in MO7e cells stimulated with GM-CSF, but not in unstimulated cells. Also, JAK2 was found to be constitutively associated with p95Vav in vivo when expressed at high levels in insect cells using baculovirus vectors. A fusion protein consisting of glutathione-S-transferase and the SH2 domain of p95Vav (GST-Vav-SH2) precipitated JAK2, suggesting that this interaction is mediated by the SH2 domain of p95Vav.(ABSTRACT TRUNCATED AT 250 WORDS)     

A Phosphatidylcholine Phospholipase C Inhibitor, D609, Blocks Interleukin-3 (IL-3)-Inducedbcl-2Expression But Notc-mycExpression in Human IL-3-Dependent Cells

Activation of the interleukin-3 (IL-3) receptor is required for the induction of cell proliferation and suppression of apoptosis in primitive hematopoietic progenitor cells. A rapid activation of tyrosine kinases and a phosphatidylcholine-specific phospholipase C has been observed in these cells in response to IL-3. The signal transduction cascades regulating cell proliferation and the suppression of apoptosis are poorly understood. Using human IL-3-dependent TF-1 cells, we have found that the tyrosine kinase inhibitor genistein blocks both the IL-3 suppression of apoptosis and the expression of the cell survival genebcl-2.In addition, we have found that D609, a specific inhibitor of phosphatidylcholine-specific phospholipase C, also inhibits IL-3-induced expression of thebcl-2gene without affecting IL-3-induced tyrosine phosphorylation. D609 also drove these cells into apoptosis even in the presence of IL-3. Significantly, genistein inhibited the IL-3 induction of bothbcl-2andc-mycgene. The latter gene is related to the induction of cell proliferation. D609, however, blocked the induction only of the cell survival genebcl-2.Thus, phosphatidylcholine hydrolysis appears linked to the induction of genes related to cell survival. These data fit with the hypothesis that there is a bifurcation in the signaling pathways downstream of IL-3 receptor-induced tyrosine phosphorylation.     

The SH2 domain containing tyrosine phosphatase-1 down-regulates activation of Lyn and Lyn-induced tyrosine phosphorylation of the CD19 receptor in B cells

SHP-1 is a cytosolic tyrosine phosphatase implicated in down-regulation of B cell antigen receptor signaling. SHP-1 effects on the antigen receptor reflect its capacity to dephosphorylate this receptor as well as several inhibitory comodulators. In view of our observation that antigen receptor-induced CD19 tyrosine phosphorylation is constitutively increased in B cells from SHP-l-deficient motheaten mice, we investigated the possibility that CD19, a positive modulator of antigen receptor signaling, represents another substrate for SHP-1. However, analysis of CD19 coimmunoprecipitable tyrosine phosphatase activity in CD19 immunoprecipitates from SHP-1-deficient and wild-type B cells revealed that SHP-1 accounts for only a minor portion of CD19-associated tyrosine phosphatase activity. As CD19 tyrosine phosphorylation is modulated by the Lyn protein-tyrosine kinase, Lyn activity was evaluated in wild-type and motheaten B cells. The results revealed both Lyn as well as CD19-associated Lyn kinase activity to be constitutively and inducibly increased in SHP-1-deficient compared with wild-type B cells. The data also demonstrated SHP-1 to be associated with Lyn in stimulated but not in resting B cells and indicated this interaction to be mediated via Lyn binding to the SHP-1 N-terminal SH2 domain. These findings, together with cyanogen bromide cleavage data revealing that SHP-1 dephosphorylates the Lyn autophosphorylation site, identify Lyn deactivation/dephosphorylation as a likely mechanism whereby SHP-1 exerts its influence on CD19 tyrosine phosphorylation and, by extension, its inhibitory effect on B cell antigen receptor signaling.     

Adenosine acts through A2 receptors to inhibit IL-2-induced tyrosine phosphorylation of STAT5 in T lymphocytes: role of cyclic adenosine 3',5'-monophosphate and phosphatases

Adenosine is a purine nucleoside with immunosuppressive activity that acts through cell surface receptors (A(1), A(2a), A(2b), A(3)) on responsive cells such as T lymphocytes. IL-2 is a major T cell growth and survival factor that is responsible for inducing Jak1, Jak3, and STAT5 phosphorylation, as well as causing STAT5 to translocate to the nucleus and bind regulatory elements in the genome. In this study, we show that adenosine suppressed IL-2-dependent proliferation of CTLL-2 T cells by inhibiting STAT5a/b tyrosine phosphorylation that is associated with IL-2R signaling without affecting IL-2-induced phosphorylation of Jak1 or Jak3. The inhibitory effect of adenosine on IL-2-induced STAT5a/b tyrosine phosphorylation was reversed by the protein tyrosine phosphatase inhibitors sodium orthovanadate and bpV(phen). Adenosine dramatically increased Src homology region 2 domain-containing phosphatase-2 (SHP-2) tyrosine phosphorylation and its association with STAT5 in IL-2-stimulated CTLL-2 T cells, implicating SHP-2 in adenosine-induced STAT5a/b dephosphorylation. The inhibitory effect of adenosine on IL-2-induced STAT5a/b tyrosine phosphorylation was reproduced by A(2) receptor agonists and was blocked by selective A(2a) and A(2b) receptor antagonists, indicating that adenosine was mediating its effect through A(2) receptors. Inhibition of STAT5a/b phosphorylation was reproduced with cell-permeable 8-bromo-cAMP or forskolin-induced activation of adenylyl cyclase, and blocked by the cAMP/protein kinase A inhibitor Rp-cAMP. Forskolin and 8-bromo-cAMP also induced SHP-2 tyrosine phosphorylation. Collectively, these findings suggest that adenosine acts through A(2) receptors and associated cAMP/protein kinase A-dependent signaling pathways to activate SHP-2 and cause STAT5 dephosphorylation that results in reduced IL-2R signaling in T cells.     

Phorbol 12-myristate 13-acetate inhibits granulocyte-macrophage colony stimulating factor-induced protein tyrosine phosphorylation in a human factor-dependent hematopoietic cell line.

The human myeloid cell line MO7 requires either granulocyte-macrophage colony stimulating factor (GM-CSF) or interleukin 3 (IL-3) for proliferation. We have previously shown that both GM-CSF and IL-3 transiently induce tyrosine phosphorylation of a number of proteins, including two cytosolic proteins, p93 and p70, which are maximally phosphorylated 5-15 min after addition of growth factor to factor-deprived cells. GM-CSF-induced proliferation of MO7 cells was found to be inhibited by two activators of protein kinase C, phorbol 12-myristate 13-acetate (PMA) and bryostatin-1. PMA did not affect surface expression or affinity of the GM-CSF receptor but significantly inhibited GM-CSF- or IL-3-induced tyrosine phosphorylation of p93 and p70. In contrast, PMA augmented GM-CSF-induced tyrosine phosphorylation of another protein, p42. Pretreatment of cells with sodium orthovanadate to inhibit protein tyrosine phosphatases (PTPase) partially reversed the inhibitory effects of PMA. These results suggest that one aspect of GM-CSF and IL-3 signal transduction, protein tyrosine phosphorylation, can be inhibited by a mechanism which does not involve receptor down-regulation, and may involve either receptor down-regulation, and may involve either inhibition of a receptor-activated tyrosine kinase, activation of a protein tyrosine phosphatase, or both. This mechanism could be important in exerting control of proliferation of some types of hematopoietic cells.     

EGF induces differentiation of an IL-3-dependent cell line expressing the EGF receptor.

Interleukin 3 (IL-3) is a T cell-derived lymphokine that supports the growth and development of hematopoietic cells. Tyrosine phosphorylation has been suggested to play an important role in IL-3-dependent cell proliferation. To test whether a growth factor receptor carrying a tyrosine kinase can be functional in IL-3 dependent cells, we used a retroviral vector to introduce the human EGF receptor into a murine IL-3-dependent pre-mast cell line, IC2. The EGF receptors expressed on the infected clones bind EGF with both high and low affinities. EGF stimulates the infected cells for a short term growth response. In the presence of IL-3 and EGF, infected clones differentiate into more mature mast cells characterized by increases in intracellular granulation and histamine content. This differentiation is reversible when EGF is removed. EGF induces tyrosine phosphorylation of several cellular proteins and the expression of oncogenes c-fos and c-myc, in a manner analogous to IL-3 stimulation. These results indicate that the EGF receptor is functional in the pre-mast IC2 cells; EGF can support short-term proliferation and activates the signals that induce cell differentiation. Thus, EGF receptor-expressing IC2 cells provide a unique cellular system for in vitro study of mast cell differentiation.     

Immunoreceptor tyrosine-based inhibitory motif of the IL-4 receptor associates with SH2-containing phosphatases and regulates IL-4-induced proliferation.

Immunoreceptor tyrosine-based inhibitory motifs (ITIM) have been implicated in the negative modulation of immunoreceptor signaling pathways. The IL-4R alpha-chain (IL-4Ralpha) contains a putative ITIM in the carboxyl terminal. To determine the role of ITIM in the IL-4 signaling pathway, we ablated the ITIM of IL-4Ralpha by deletion and site-directed mutagenesis and stably expressed the wild-type (WT) and mutant hIL-4Ralpha in 32D/insulin receptor substrate-2 (IRS-2) cells. Strikingly, 32D/IRS-2 cells expressing mutant human (h)IL-4Ralpha were hyperproliferative in response to IL-4 compared with cells expressing WT hIL-4Ralpha. Enhanced tyrosine phosphorylation of Stat6, but not IRS-2, induced by hIL-4 was observed in cells expressing mutant Y713F. Using peptides corresponding to the ITIM of hIL-4Ralpha, we demonstrate that tyrosine-phosphorylated peptides, but not their nonphosphorylated counterparts, coprecipitate SH2-containing tyrosine phosphatase-1, SH2-containing tyrosine phosphatase-2, and SH2-containing inositol 5'-phosphatase. The in vivo association of SH2-containing inositol 5'-phosphatase with IL-4Ralpha was verified by coimmunoprecipitation with anti-IL-4Ralpha Abs. These results demonstrate a functional role for ITIM in the regulation of IL-4-induced proliferation.     

Direct association with and dephosphorylation of Jak2 kinase by the SH2-domain-containing protein tyrosine phosphatase SHP-1.

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.     

SHP-1 regulates STAT6 phosphorylation and IL-4-mediated function in a cell type-specific manner

SHP-1 has been shown to play positive and negative regulatory roles in IL-4-induced STAT6 phosphorylation and in IL-4-mediated functions. To determine whether SHP-1 can regulate STAT6 phosphorylation and IL-4-mediated functions in a cell type-specific manner in the immune system, we examined the IL-4 receptor (IL-4R) expression, STAT6 phosphorylation, and IL-4-mediated functions in CD4+ and CD8+ T cells of viable motheaten (me(v)/me(v)) and littermate control (+/-) mice. CD4+ T cells as well as CD8+ T cells from the lymph node of me(v)/me(v) and +/- mice expressed comparable levels of IL-4R. In CD4+ T cells, the loss of SHP-1 activity did not affect IL-4-induced STAT6 phosphorylation or IL-4-mediated function. In contrast, SHP-1-deficient CD8+ T cells from me(v)/me(v) mice failed to develop into IL-4-producing type-2 cytotoxic T cells (Tc2) in the presence of IL-4 despite that they showed comparable levels of STAT6 phosphorylation to that of +/- CD8+ T cells. Loss of SHP-1 activity also abolished IL-4-mediated inhibition of c-kit expression in bone marrow-derived mast cell (BMMC). Thus, our data suggest that SHP-1 may regulate IL-4-induced STAT6 phosphorylation and IL-4-mediated functions in a cell type-specific manner.     

SDF-1 synergistically enhances IL-3-induced activation of the Raf-1/MEK/Erk signaling pathway through activation of Rac and its effector Pak kinases to promote hematopoiesis and chemotaxis

Stromal cell-derived factor 1 (SDF-1) cooperates with cytokines to promote hematopoiesis. Here we demonstrate that SDF-1 activates Erk synergistically with interleukin-3 (IL-3) in hematopoietic cells. Small GTPases Ras and Rac were prominently activated by IL-3 and SDF-1, respectively. In accordance with this, Raf-1 was significantly activated by IL-3 but not by SDF-1. SDF-1 strongly induced phosphorylation of Raf-1 on S338, the target site for the Rac effector Paks, and enhanced the IL-3-induced activation of Raf-1 and MEK. Furthermore, the synergistic activation of Erk was inhibited by expression of a dominant-negative mutant of Pak1 or that of Rac and was enhanced by an activated mutant of Pak1. SDF-1 and IL-3 also showed synergistic effects on expansion of hematopoietic cells and on induction of chemotaxis, which were both inhibited by the MEK inhibitor PD98059. These results suggest that SDF-1 synergistically enhances IL-3-induced Erk activation by up-regulating Raf-1 activity through the Rac effector Pak kinases to promote hematopoiesis.     

IL-4 activates a distinct signal transduction cascade from IL-3 in factor-dependent myeloid cells.

Interleukin-4 (IL-4) was shown to induce a potent mitogenic response in the IL-3-dependent myeloid progenitor cell line, FDCP-2. Although IL-4 could not sustain long-term growth of FDCP-2 cells, it enhanced their growth in serum-free medium containing IL-3. IL-4 triggered prominent tyrosine phosphorylation of a substrate(s) migrating at 170 kDa and less striking phosphorylation of several other proteins, including the IL-4 receptor. By contrast, IL-3 induced distinct tyrosine phosphorylation of proteins migrating at 145, 97, 70, 55 and 52 kDa in the same cell line. IL-4 treatment of FDCP-2 cells caused a dramatically strong association of phosphatidylinositol 3-kinase (PI 3-kinase) both with the 170 kDa tyrosine phosphorylated substrate and with the IL-4 receptor itself. By contrast, IL-3 triggered only weak association of PI 3-kinase activity with the 97 kDa substrate. While IL-4 did not affect cellular raf, IL-3 stimulation did induce a shift in its mobility presumably due to serine/threonine phosphorylation. Taken together, our results indicate that IL-4 and IL-3 activate distinct phosphorylation cascades in the same cell background; this may reflect a difference in the biological function of these two cytokines.     

Expression of dominant-negative src-homology domain 2-containing protein tyrosine phosphatase-1 results in increased Syk tyrosine kinase activity and B cell activation

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.     

Tec kinase associates with c-kit and is tyrosine phosphorylated and activated following stem cell factor binding.

Stem cell factor (SCF) plays a crucial role in hematopoiesis through its interaction with the receptor tyrosine kinase c-kit. However, the signaling events that are activated by this interaction and involved in the control of growth or differentiation are not completely understood. We demonstrate here that Tec, a cytoplasmic, src-related kinase, physically associates with c-kit through a region that contains a proline-rich motif, amino terminal of the SH3 domain. Following SCF binding, Tec is tyrosine phosphorylated and its in vitro kinase activity is increased. Tyrosine phosphorylation of Tec is not detected in the response to other cytokines controlling hematopoiesis, including colony-stimulating factor-1 (CSF-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-3 (IL-3). Conversely, the cytoplasmic kinase JAK2 is activated by IL-3 but not by SCF stimulation. The activation of distinct cytoplasmic kinases may account for the synergy seen in the actions of SCF and IL-3 on hematopoietic stem cells.     

IFN-gamma suppresses STAT6 phosphorylation by inhibiting its recruitment to the IL-4 receptor

Polarized Th1 cells show a stable phenotype: they become insensitive to IL-4 stimulation and lose the potential to produce IL-4. Previously, we reported that IFN-gamma played a critical role in stabilizing Th1 phenotype. However, the mechanism by which IFN-gamma stabilizes Th1 phenotype is not clear. In this study, we compared STAT6 phosphorylation in wild-type (WT) and IFN-gamma receptor knockout (IFNGR(-/-)) Th1 cells. We found a striking diminution of STAT6 phosphorylation in differentiated WT Th1 cells, but not in differentiated IFNGR(-/-) Th1 cells. The impairment of STAT6 phosphorylation in differentiated WT Th1 cells was not due to a lack of IL-4R expression or phosphorylation. Jak1 and Jak3 expression and phosphorylation were comparable in both cell types. No differential expression of suppressor of cytokine signaling 1 (SOCS1), SOCS3, or SOCS5 was observed in the two cell types. In addition, Src homology 2-containing phosphatase mutation did not affect IL-4-induced STAT6 phosphorylation in differentiated Th1 cells derived from viable motheaten (me(v)/me(v)) mice. These results led us to focus on a novel mechanism. By using a pulldown assay, we observed that STAT6 in WT Th1 cells bound less effectively to the phosphorylated IL-4R/GST fusion protein than that in IFNGR(-/-) Th1 cells. Our results suggest that IFN-gamma may suppress phosphorylation of STAT6 by inhibiting its recruitment to the IL-4R.