Catalytic-dependent and -independent roles of SHP-2 tyrosine phosphatase in interleukin-3 signaling

SHP-2 tyrosine phosphatase is highly expressed in hematopoietic cells, however, the function of SHP-2 in hematopoietic cell signaling is not well understood. Here we focus on the role of SHP-2 phosphatase in the signal transduction of interleukin (IL)-3, a cytokine involved in hematopoietic cell survival, proliferation, and differentiation. We established immortalized SHP-2(-/-) hematopoietic cell pools and showed that IL-3-induced proliferative response was diminished in SHP-2(-/-) cells. Moreover, inhibition of the catalytic activity of SHP-2 in wild-type (WT) bone marrow hematopoietic progenitor cells and Ba/F3 cells by overexpression of catalytically inactive SHP-2 mutant suppressed their differentiative and proliferative responses to IL-3, demonstrating an important positive role for SHP-2 in IL-3 signal transduction. Further biochemical analyses revealed that IL-3-induced Jak/Stat, Erk, and PI3 kinase pathways in SHP-2(-/-) cells were impaired and reintroduction of WT SHP-2 into mutant cells partially restored IL-3 signaling. Interestingly, in catalytically inactive SHP-2-overexpressing Ba/F3 cells, although IL-3-induced activation of Jak2 and Erk kinases was reduced and shortened, PI3 kinase activation remained unaltered. Taken together, these results suggest that SHP-2 tyrosine phosphatase plays multiple roles in IL-3 signal transduction, functioning in both catalytic-dependent and -independent manners in the Jak/Stat, Erk, and PI3 kinase pathways.     

Differences in phosphorylation of the IL-2R associated JAK/STAT proteins between HTLV-I(+), IL-2-independent and IL-2-dependent cell lines and uncultured leukemic cells from patients with adult T-cell lymphoma/leukemia

To determine activation status of the IL-2R-associated (Jak/STAT) pathway in the HTLV-I infected cells, we examined tyrosine phosphorylation of Jak3, STAT3, and STAT5 in several HTLV-I(+) T-cell lines and in uncultured leukemic T cells isolated from patients with adult T-cell lymphoma/leukemia (ATLL). Constitutive basal phosphorylation of Jak3 and, usually, STAT3 and STAT5 was detected in all four IL-2-independent cell lines tested, but in none of the three IL-2-dependent cell lines. Similarly, there was no detectable basal phosphorylation of Jak3 and STAT5 in the leukemic cells from ATLL patients (0/8 and 0/3, respectively). However, stimulation with IL-2 resulted in Jak3 and STAT5 phosphorylation in both leukemic ATLL cells and IL-2-dependent lines. Furthermore, expression of SHP-1 phosphatase which is a negative regulator of cytokine receptor signaling, was lost in most IL-2 independent cell lines (3/4) but not in the leukemic ATLL cells (0/3). Finally, the HTLV-I(+) T-cell lines (313) but not the control, HTLV-I(-) T-cell lines were resistant to rapamycin and its novel analog RAD. We conclude that (1) HTLV-I infection per se does not result in a constitutive phosphorylation of the Jak3, STAT3, and STAT5 proteins; (2) malignant transformation in at least some cases of ATLL does not require the constitutive, but may require IL-2-induced, activation of the IL-2R Jak/STAT pathway; and (3) there are major differences in T-cell immortalization mechanism(s) which appear to involve SHP-1 and target molecules for rapamycin and RAD.     

Involvement of Jak2 tyrosine phosphorylation in Bcr-Abl transformation

We have previously reported that the Jak2 tyrosine kinase but not Jak1 is tyrosine phosphorylated in the absence of IL-3 in Bcr-Abl positive M3.16 cells, which are rendered IL-3 independent by BCR-ABL gene expression. We have explored the involvement of Jak2 tyrosine phosphorylation in Bcr-Abl oncogenic effects. Our results indicate that Jak2 became tyrosine-phosphorylated in a number of cell lines expressing Bcr-Abl, when maintained in medium lacking IL-3, whereas Bcr-Abl negative cells lacked Jak2 tyrosine phosphorylation. Jak2 was poorly tyrosine-phosphorylated in cells expressing the SH2 deletion mutant of Bcr-Abl compared to either wild-type Bcr-Abl or its SH3 deletion mutant. Moreover, tyrosine phosphorylation of Jak2 by Bcr-Abl was inhibited by the Abl tyrosine kinase inhibitor, STI 571, in a dose-dependent manner. This inhibition of Bcr-Abl kinase by the drug did not interfere with the ability of Jak2 and Bcr-Abl to form a complex. Studies with deletion mutants of Bcr-Abl indicated that the C-terminal domain of Abl within Bcr-Abl was involved in complex formation with Jak2. Similarly, GST-Abl pull-down assays confirmed the strong binding to Jak2 by the C-terminus of Abl. Jak2 peptide substrate studies indicated that the Bcr-Abl and Abl tyrosine kinases specifically phosphorylated Y1007 of Jak2 but only poorly phosphorylated Y1008. Phosphorylation of Y1007 of Jak2 is known to be critical for its tyrosine kinase activation. Tyrosine residue 1007 of Jak2 was phosphorylated in 32Dp210 cells as measured by Western blotting with a phosphotyrosine 1007 sequence-specific antibody. A kinase-inactive Jak2 mutant blocked the colony forming ability of K562 cells. Tumor formation of K562 cells in nude mice was similarly inhibited by this kinase-inactive Jak2 mutant. This inhibition was independent of Stat5 tyrosine phosphorylation. Furthermore, tyrosine-phosphorylated Jak2 was detected in blood cells from CML patients in blast crisis but not in a normal marrow sample. In summary, these findings provide strong evidence that the Jak2 tyrosine kinase is a critical factor in Bcr-Abl malignant transformation.     

SH2-containing phosphotyrosine phosphatase SHP-1 is involved in BCR-ABL signal transduction pathways

The BCR-ABL fusion protein is strongly implicated in the malignant process of Philadelphia (Ph-1)-positive leukemia. The BCR-ABL fusion protein exhibits a deregulated tyrosine kinase activity capable of phosphorylating different cellular substrates in vivo and in vitro. SHP-1 (SHPTP1, PTP1C, HCP, SHP) is an SH2 domain-containing tyrosine phosphatase expressed predominantly in hematopoietic cells. The association of the murine motheaten phenotype of severe hematopoietic dysregulation with loss of SHP-1 tyrosine phosphatase activity indicates a critical role of SHP-1 in the regulation of hematopoietic cell growth and differentiation. Experiments were performed to determine whether SHP-1 might form specific complexes with BCR-ABL signaling pathway. We found that SHP-1 was highly and constitutively tyrosine phosphorylated in 32DCl3 and TF-1 cells transfected with BCR-ABL expression vector. Furthermore, SHP-1 and BCR-ABL formed stable complexes in BCR-ABL expressing cells. Direct binding between SHP-1 and Grb2 was observed in vitro. Expression of BCR-ABL in TF-1 cells resulted in a two-fold increase in SHP-1 phosphatase activity. BCR-ABL tyrosine kinase was able to phosphorylate recombinant SHP-1 protein in vitro. SHP-1 therefore has the capacity to bind and potentially modulate the signaling effectors involved in activation of Ras, and accordingly, regulation of cellular transformation of BCR-ABL.     

Mitogenic synergy through multilevel convergence of hepatocyte growth factor and interleukin-4 signaling pathways

Hepatocyte growth factor (HGF) regulates various physiological and developmental processes in concert with other growth factors, cytokines and hormones. We examined interactions between cell signaling events elicited by HGF and the cytokine interleukin (IL)-4, in the IL-3-dependent murine myeloid cell line 32D transfected with the human HGF receptor, c-Met. HGF was a potent mitogen in these cells, and prevented apoptosis in response to IL-3 withdrawal. IL-4 showed modest anti-apoptotic activity, but no significant mitogenic activity. IL-4 synergistically enhanced HGF-stimulated DNA synthesis, whereas only additive prevention of apoptosis was observed. IL-4 did not enhance HGF-dependent tyrosine phosphorylation of c-Met or Shc. In contrast, HGF-stimulated activation of MAP kinases was enhanced by IL-4, suggesting that the IL-4 and HGF signaling pathways converge upstream of these events. Although phosphatidylinositol 3-kinase (PI3K) inhibitors diminished HGF-induced mitogenesis, anti-apoptosis, and MAP kinase activation, IL-4 enhanced HGF signaling persisted even in the presence of these inhibitors. IL-4 enhancement of HGF signaling was partially blocked in 32D/c-Met cells treated with inhibitors of MEK1 or c-Src kinases, completely blocked by expression of a catalytically inactive mutant of Janus kinase 3 (Jak3), and increased in 32D/c-Met cells overexpressing STAT6. Our results suggest that the IL-4 and HGF pathways converge at multiple levels, and that IL-4-dependent Jak3 and STAT6 activities modulate signaling events independent of PI3K to enhance HGF-dependent mitogenesis in myeloid cells, and possibly other common cellular targets.     

Jak kinase activity is required for lymphoma invasion and metastasis

Jak tyrosine kinases are activated by interleukins and other growth factors, and promote survival and proliferation of cells in multiple tissues. These kinases are constitutively active in many hematopoietic malignancies and certain carcinomas. We have investigated whether Jak kinases play a role in lymphoma invasion and metastasis. Proliferation and survival of a highly metastatic T-lymphoma was made independent of its constitutively active Jak by expression of active forms of both STAT3 and PI3-kinase. Jak activity was then blocked by the isolated JH2 'pseudokinase' domain of Jak2. In vitro invasion was blocked by the JH2 domain, and the metastatic capacity of the JH2-expressing cells was much reduced. The Jak inhibitor AG490 inhibited invasion as well. Invasion and metastasis of these cells requires activation of the integrin LFA-1 by the CXCR4 chemokine receptor. We show that Jak kinases act downstream of LFA-1. We conclude that Jak kinase activity is essential for lymphoma invasion and metastasis, independent of its role in survival and proliferation, and independent of STAT and PI3K signaling. This indicates that Jak kinases contribute in multiple ways to the induction of malignant behavior.     

Roles for the protein tyrosine phosphatase SHP-2 in cytoskeletal organization, cell adhesion and cell migration revealed by overexpression of a dominant negative mutant

SHP-2, a SRC homology 2 domain-containing protein tyrosine phosphatase, mediates activation of Ras and mitogen-activated protein kinase by various mitogens and cell adhesion. Inhibition of endogenous SHP-2 by overexpression of a catalytically inactive (dominant negative) mutant in Chinese hamster ovary cells or Rat-1 fibroblasts has now been shown to induce a marked change in cell morphology (from elongated to less polarized) that is accompanied by substantial increases in the numbers of actin stress fibers and focal adhesion contacts. Overexpression of the SHP-2 mutant also increased the strength of cell-substratum adhesion and resulted in hyperphosphorylation of SHPS-1, a substrate of SHP-2 that contributes to cell adhesion-induced signaling. Inhibition of SHP-2 also markedly increased the rate of cell attachment to and cell spreading on extracellular matrix proteins such as fibronectin and vitronectin, effects that were accompanied by enhancement of adhesion-induced tyrosine phosphorylation of paxillin and p130Cas. In addition, cell migration mediated by fibronectin or vitronectin, but not that induced by insulin, was impaired by overexpression of the SHP-2 mutant. These results suggest that SHP-2 plays an important role in the control of cell shape by contributing to cytoskeletal organization, and that it is an important regulator of integrin-mediated cell adhesion, spreading, and migration as well as of tyrosine phosphorylation of focal adhesion contact-associated proteins.     

Expression of the GPI-anchored receptor Prv-1 enhances thrombopoietin and IL-3-induced proliferation in hematopoietic cell lines

Prv-1 is a hematopoietic cell surface receptor that has been shown to be overexpressed in patients diagnosed with polycythemia vera (PV) and essential thrombocythemia (ET), yet its cellular function remains unclear. In this study, we assessed the role of Prv-1 in thrombopoietin (Tpo)/Mpl signaling with the goal of identifying molecular mechanisms which augment Tpo-induced proliferation. By engineering the cytokine-dependent hematopoietic cell line BaF3 to express both Prv-1 and wild-type or mutant forms of Mpl, we were able to follow the time course of Tpo-dependent proliferation. We report that the overexpression of Prv-1 increased Tpo as well as IL-3-induced proliferation of BaF3/Mpl and BaF3 cells. Cells co-expressing Prv-1 and an Mpl receptor containing a Box 1 motif mutation, which fails to activate Jak2, was completely deficient in Tpo-dependent proliferation. In addition, BaF3 and BaF3/Prv-1 cells stimulated with IL-3 in the presence of the Jak2 inhibitor, AG490, abrogated the proliferative response, indicating that Prv-1 requires a functional Jak2 for its signaling activities. Western blot analysis showed an increase in Tpo and IL-3-induced Stat3 and Stat5 tyrosine phosphorylation in BaF3/Mpl and BaF3 cells expressing Prv-1. These results indicate a novel function for Prv-1 as a signaling molecule in cytokine signaling cascades and may lead to a greater understanding of the mechanism of overexpression of Prv-1 in myeloproliferative disorders.     

Src transduces signaling via growth hormone (GH)-activated GH receptor (GHR) tyrosine-phosphorylating GHR and STAT5 in human leukemia cells

Most human leukemia cells are shown to express growth hormone receptor (GHR) and some of them proliferate in response to GH. We demonstrate that Src contributes to GHR-mediated signal transduction via STAT5 activation in F-36P human leukemia cells stimulated with GH. The tyrosine phosphorylation levels of GHR and STAT5 induced by GH decreased in the presence of PP2 Src kinase inhibitor. When GHR and wild-type Src were co-expressed in COS7 cells, GHR was markedly tyrosine phosphorylated as well as when Jak2 was co-expressed with GHR, but not when kinase-inactive Src co-expressed. The treatment of F-36P cells with the antisense src oligonucleotides, which selectively decreased the Src expression, reduced the rhGH-induced tyrosine phosphorylation of the STAT5 activation sites.