Constitutive activation of the Janus kinase-STAT pathway in T lymphoma overexpressing the Lck protein tyrosine kinase

The Lck protein, a Src family tyrosine kinase, plays a critical role in T cell maturation and activation. Dysregulation of Lck expression or Lck kinase activity has been implicated in T cell leukemias from mice to humans, although the mechanism underlying Lck-mediated oncogenesis is still largely unclear. We report here that both DNA binding activities and tyrosine phosphorylation of STAT3 and STAT5, but not STAT1, are constitutively enhanced in the mouse T cell lymphoma LSTRA, which is a well-characterized cell line that overexpresses Lck protein and exhibits high levels of Lck kinase activity. Furthermore, Janus kinase 1 (jak1) and Jak2 protein tyrosine kinases are constantly activated in these cells, as determined by their autophosphorylation in an in vitro kinase assay and increased levels of tyrosine phosphorylation on immunoblots. Therefore, like Src-transformed cells, Lck-overexpressing LSTRA cells also exhibit constitutive activation of distinct Jak and STAT proteins.     

Dependence on the motif YIPP for the physical association of Jak2 kinase with the intracellular carboxyl tail of the angiotensin II AT1 receptor

Angiotensin II is the effector molecule of the renin-angiotensin system. Virtually all of its biochemical actions are mediated through a single class of cell-surface receptors called AT1. These receptors contain the structural features of the seven-transmembrane, G-protein-coupled receptor superfamily. Angiotensin II, acting through the AT1 receptor, also stimulates the Jak/STAT pathway by inducing ligand-dependent Jak2 tyrosine phosphorylation and activation. Here, we show that a glutathione S-transferase fusion protein containing the carboxyl-terminal 54 amino acids of the rat AT1A receptor physically binds to Jak2 in an angiotensin II-dependent manner. Deletional analysis, using both in vitro protocols and cell transfection analysis, showed that this association is dependent on the AT1A receptor motif YIPP (amino acids 319-322). The wild-type AT1A receptor can induce Jak2 tyrosine phosphorylation. In contrast, an AT1A receptor lacking the YIPP motif is unable to induce ligand-dependent phosphorylation of Jak2. Competition experiments with synthetic peptides suggest that each of the YIPP amino acids, including tyrosine 319, is important in Jak2 binding to the AT1A receptor. The binding of the AT1A receptor to the intracellular tyrosine kinase Jak2 supports the concept that the seven-transmembrane superfamily of receptors can physically associate with enzymatically active intracellular proteins, creating a signaling complex mechanistically similar to that observed with growth factor and cytokine receptors.     

Mapping of a cytoplasmic domain of the human growth hormone receptor that regulates rates of inactivation of Jak2 and Stat proteins

It has been previously demonstrated that growth hormone (GH)-stimulated tyrosine phosphorylation of Jak2 and Stat5a and Stat5b occurs in FDP-C1 cells expressing either the entire GH receptor or truncations of the cytoplasmic domain expressing only the membrane-proximal 80 amino acids. However, other receptor domains that might modulate rates of GH activation and inactivation of this cascade have not been examined. Here we have defined a region in the human GH receptor between amino acids 520 and 540 in the cytoplasmic domain that is required for attenuation of GH-activated Jak/Stat signaling. Immunoprecipitations with antibodies to Jak2 indicate that the protein tyrosine phosphatase SHP-1 is associated with this kinase in cells exposed to GH. To address the possibility that SHP-1 could function as a negative regulator of GH signaling, liver extracts from motheaten mice deficient in SHP-1 or unaffected littermates were analyzed for activation of Stats and Jak2. Extracts from motheaten mice displayed prolonged activation of the Stat proteins as measured by their ability to interact with DNA and prolonged tyrosine phosphorylation of Jak2. These results delineate a novel domain in the GH receptor that regulates the inactivation of the Jak/Stat pathway and appears to be modulated by SHP-1.     

Tumor-induced suppression of T lymphocyte proliferation coincides with inhibition of Jak3 expression and IL-2 receptor signaling: role of soluble products from human renal cell carcinomas

The proliferative capacity of T cells infiltrating human tumors is known to be impaired, possibly through their interaction with tumor. Here we demonstrate that soluble products derived from renal cell carcinoma (RCC-S) explants but not normal kidney can inhibit an IL-2-dependent signaling pathway that is critical to T cell proliferation. A major target of the immunosuppression was the IL-2R-associated protein tyrosine kinase, Janus kinase 3 (Jak3). RCC-S suppressed basal expression of Jak3 and its increase following stimulation with anti-CD3/IL-2. Jak3 was most sensitive to suppression by RCC-S; however, reduction in expression of p56(lck), p59(fyn), and ZAP-70 was observed in some experiments. Expression of other signaling elements linked to the IL-2R (Jak1) and the TCR (TCR-zeta, CD3-epsilon, and phospholipase C-gamma) were minimally affected. In naive T cells, RCC-S also partially blocked induction of IL-2R alpha-, beta- and gamma-chain expression when stimulating via the TCR/CD3 complex with anti-CD3 Ab. To determine whether RCC-S suppressed IL-2-dependent signaling, primed T cells were employed since RCC-S had no effect on IL-2R expression but did down-regulate Jak3 expression and, to a lesser degree, p56(lck) and p59(fyn). Reduction in Jak3 correlated with impaired IL-2-dependent proliferation and signal transduction. This included loss of Jak1 kinase tyrosine phosphorylation and no induction of the proto-oncogene, c-Myc. These findings suggest that soluble products from tumors may suppress T cell proliferation through a mechanism that involves down-regulation of Jak3 expression and inhibition of IL-2-dependent signaling pathways.     

Suppression of proliferation and phosphorylation of Jak3 and STAT5 in malignant T-cell lymphoma cells by derivatives of octylamino-undecyl-dimethylxanthine

IL-2R signal transduction involves tyrosine phosphorylation of several proteins including Jak3 and STAT5. In the present study we examined the effect of two octylamino-undecyl-dimethylxanthine (OUDMX) derivatives, designated CT2576 and CT5589, on proliferation and protein tyrosine phosphorylation in human malignant T-cell lymphoma lines. These T-cell lines (PB-1, 2A, and 2B), obtained from a progressive T-cell lymphoma involving skin, are IL-2 independent but have constitutively activated IL-2R-associated signal transduction pathway common to IL-2 and several other cytokines: IL-4, IL-7, IL-9, and IL-15. CT2576, characterized previously on the functional level as an inhibitor of IL-2 signaling and, on the biochemical level, as an inhibitor of phosphatidic acid biosynthesis, suppressed completely growth of the malignant T cell lymphoma lines. CT5589 which is a novel analog of the CT2576, displayed a similar, although weaker, effect. Furthermore, both CT compounds inhibited constitutive tyrosine phosphorylation of two proteins: Jak3 and STAT5 which are key downstream elements in the signal transduction pathway activated by IL-2 and the other cytokines. The CT compounds inhibited also Jak3 phosphorylation induced by IL-2 in the IL-2 dependent SZ-4 cells. Inhibition of phosphorylation by CT2576 and CT5589 was only partially selective since phosphorylation of several other proteins was also affected. Phosphorylation of many others was, however, unaffected. These findings demonstrate that the OUDMX derivatives suppress proliferation of malignant T lymphocytes. Furthermore, they suggest that this suppression may be mediated by inhibition of the IL-2R-associated Jak/STAT signaling pathway. A potential role for OUDMX derivatives in therapy of human T-cell lymphoma should be further explored.     

Mechanism of inhibition of eosinophil activation by transforming growth factor-beta. Inhibition of Lyn, MAP, Jak2 kinases and STAT1 nuclear factor

The activation of eosinophils by IL-5 plays a crucial role in the pathogenesis of allergic and parasitic disorders. IL-5 has recently been shown to activate Lyn and Jak2 tyrosine kinases, MAP kinases, and STAT1 nuclear factor. We have previously reported that TGF-beta blocks the IL-5-induced activation of eosinophils. In this study, we investigated the effect of TGF-beta on the IL-5-induced signaling molecules in eosinophils. Purified eosinophils from mildly allergic patients were preincubated with TGF-beta and then stimulated with IL-5. The cell lysates were then immunoprecipitated and blotted with antiphosphotyrosine Abs. The activity of the kinases was further studied in the immune-complex kinase assay. We found that TGF-beta inhibited the tyrosine phosphorylation of multiple proteins in eosinophils. The identity of some of the proteins was established by immunoprecipitation. We found that TGF-beta inhibited tyrosine phosphorylation of Lyn, Jak2, and a 44-kDa MAP kinase. In further experiments, it blocked the activation of the above kinases as determined by immune-complex kinase assay. TGF-beta also inhibited phosphorylation of the STAT1 (p91) nuclear protein in eosinophils. We believe that the inhibition of Lyn, Jak2, MAP kinase, and the STAT1 nuclear protein may underlie the inhibitory activity of TGF-beta on eosinophils.     

Physical and functional interaction between p72(syk) and erythropoietin receptor

Erythropoietin (Epo) regulates the proliferation and differentiation of erythroid cells through interaction with a cell surface receptor (EpoR) that belongs to the cytokine receptor family. The Jak2 tyrosine kinase was previously shown to bind to the EpoR, to be activated upon Epo stimulation, and to play a critical role in Epo-induced proliferation. However, little is known about the role of other tyrosine kinases in Epo signaling. In this paper, we examined whether Syk was involved in EpoR activation. Coimmunoprecipitation experiments showed that the phosphorylated EpoR was associated with the Syk kinase in activated UT7 cells. The interaction of Epo with its receptor led to an increased kinase activity. The use of recombinant Syk Src homology 2 (SH2) domains expressed in tandem or individually revealed that both N- and C-SH2 domains of Syk participated in EpoR binding with a major contribution of the C-terminal SH2 domain. Far Western blotting further indicated that Syk directly binds to the EpoR and that the interaction of Syk with EpoR only occurred after Epo activation. These data suggest that phosphorylation of EpoR on tyrosine residues may mediate Syk binding to the receptor through interaction between the two SH2 domains of Syk and tyrosines of the receptor. We propose that in addition to Jak2, Syk protein kinase may be a component of EpoR signaling.     

Requirement of Src kinase Lyn for induction of DNA synthesis by granulocyte colony-stimulating factor

Treatment of cells with granulocyte colony-stimulating factor (G-CSF) leads to tyrosine phosphorylation of cellular proteins. G-CSF stimulates both the activation of protein tyrosine kinases Lyn, Jak1, and Jak2 and the association of these enzymes with the G-CSF receptor. Wild-type, lyn-deficient, and syk-deficient chicken B lymphocyte cell lines were transfected with the human G-CSF receptor, and stable transfectants were studied. G-CSF-dependent tyrosyl phosphorylation of Jak1 and Jak2 occurred in all three cell lines. Wild-type and syk-deficient transfectants responded to G-CSF in a dose-responsive fashion with increased thymidine incorporation, but none of the clones of lyn-deficient transfectants did. Ectopic expression of Lyn, but not that of c-Src, in the lyn-deficient cells restored their mitogenic responsiveness to G-CSF. Ectopic expression in wild-type cells of the kinase-inactive form of Lyn, but not of the kinase-inactive form of Jak2, inhibited thymidine incorporation in response to G-CSF. These studies show that the absence of Lyn results in the loss of mitogenic signaling in the G-CSF signaling pathway and that activation of Jak1 or Jak2 is not sufficient to cause mitogenesis.