Etk, a Btk family tyrosine kinase, mediates cellular transformation by linking Src to STAT3 activation

Etk (also called Bmx) is a member of the Btk tyrosine kinase family and is expressed in a variety of hematopoietic, epithelial, and endothelial cells. We have explored biological functions, regulators, and effectors of Etk. Coexpression of v-Src and Etk led to a transphosphorylation on tyrosine 566 of Etk and subsequent autophosphorylation. These events correlated with a substantial increase in the kinase activity of Etk. STAT3, which was previously shown to be activated by Etk, associated with Etk in vivo. To investigate whether Etk could mediate v-Src-induced activation of STAT3 and cell transformation, we overexpressed a dominant-negative mutant of Etk in an immortalized, untransformed rat liver epithelial cell line, WB, which contains endogenous Etk. Dominant-negative inactivation of Etk not only blocked v-Src-induced tyrosine phosphorylation and activation of STAT3 but also caused a great reduction in the transforming activity of v-Src. In NIH3T3 cells, although Etk did not itself induce transformation, it effectively enhanced the transforming ability of a partially active c-Src mutant (c-Src378G). Furthermore, Etk activated STAT3-mediated gene expression in synergy with this Src mutant. Our findings thus indicate that Etk is a critical mediator of Src-induced cell transformation and STAT3 activation. The role of STAT3 in Etk-mediated transformation was also examined. Expression of Etk in a human hepatoma cell line Hep3B resulted in a significant increase in its transforming ability, and this effect was abrogated by dominant-negative inhibition of STAT3. These data strongly suggest that Etk links Src to STAT3 activation. Furthermore, Src-Etk-STAT3 is an important pathway in cellular transformation.     

信号转导与转录活化蛋白STAT3在肿瘤发生中的作用

STAT3是信号转导与转录活化蛋白（STAT）家族的一个重要成员,也是一个可以被多种调控细胞增殖、分化、发育、存活和炎症的细胞因子、生长因子及肿瘤蛋白所激活的关键信号转导蛋白。在多种肿瘤细胞、细胞性状转化过程和肿瘤形成过程中可以检测到组成性激活的STAT3。我们从STAT3在肿瘤细胞中组成性激活的机制、STAT3作为转录因子对肿瘤细胞调控的机制等方面对STAT3在肿瘤发生中的作用进行简要综述。     

Ras-induced activation of Raf-1 is dependent on tyrosine phosphorylation.

Although Rafs play a central role in signal transduction, the mechanism(s) by which they become activated is poorly understood. Raf-1 activation is dependent on the protein's ability to bind Ras, but Ras binding is insufficient to activate Raf-1 tyrosine phosphorylation to this Ras-induced activation, in the absence of an over-expressed tyrosine kinase. We demonstrate that Raf-1 purified form Sf9 cells coinfected with baculovirus Ras but not Src could be inactivated by protein tyrosine phosphatase PTP-1B. 14-3-3 and Hsp90 proteins blocked both the tyrosine dephosphorylation and inactivation of Raf-1, suggesting that Raf-1 activity is phosphotyrosine dependent. In Ras-transformed NIH 3T3 cells, a minority of Raf-1 protein was membrane associated, but essentially all Raf-1 activity and Raf-1 phosphotyrosine fractionated with plasma membranes. Thus, the tyrosine-phosphorylated and active pool of Raf-1 constitute a membrane-localized subfraction which could also be inactivated with PTP-1B. By contrast, B-Raf has aspartic acid residues at positions homologous to those of the phosphorylated tyrosines (at 340 and 341) of Raf-1 and displays a high basal level of activity. B-Raf was not detectably tyrosine phosphorylated, membrane localized, or further activated upon Ras transformation, even though B-Raf has been shown to bind to Ras in vitro. We conclude that tyrosine phosphorylation is an essential component of the mechanism by which Ras activates Raf-1 kinase activity and that steady-state activated Ras is insufficient to activate B-Raf in vivo.     

Interferon-alpha induction of STATs1, -3 DNA binding and growth arrest is independent of Lck and active mitogen-activated kinase in T cells

Type I interferons (IFNs) are a family of cytokines that have antiviral and antiproliferative effects. Data regarding the processes by which these cytokines transduce signals from the cell membrane to the nucleus are becoming increasingly complex. The most characterized pathway is via JAK-STAT signaling. Previous studies established a potential role for the Src-family kinase Lck in JAK-STAT signaling. Therefore, this study was designed to analyze the role of Lck in IFN-alpha signaling by using the Jurkat, JCam (an Lck-defective cell line derived from Jurkat), and JCam/Lck (JCam cells with Lck restored). The results show that IFN-alpha can induce MAPK activity, but only in cells containing Lck. Furthermore, STATs1 and -3 are effectively phosphorylated and activated to bind DNA in the absence of Lck expression in IFN-alpha-treated cells. Finally, the results demonstrate that IFN-alpha exerts an antiproliferative effect in all three cell lines. These data indicate that Lck and active MAPK do not affect IFN-alpha-induced growth arrest or induction of STAT1s1 and -3 DNA binding ability.     

Activation of the lck tyrosine-protein kinase by the binding of the tip protein of herpesvirus saimiri in the absence of regulatory tyrosine phosphorylation.

The Tip protein of herpesvirus saimiri 484 binds to the Lck tyrosine-protein kinase at two sites and activates it dramatically. Lck has been shown previously to be activated by either phosphorylation of Tyr394 or dephosphorylation of Tyr505. We examined here whether a change in the phosphorylation of either site was required for the activation of Lck by Tip. Remarkably, mutation of both regulatory sites of tyrosine phosphorylation did not prevent activation of Lck by Tip either in vivo or in a cell free in vitro system. Tip therefore appears to be able to activate Lck through an induced conformational change that does not necessarily involve altered phosphorylation of the kinase. Tip may represent the prototype of a novel type of regulator of tyrosine-protein kinases.     

Oxidative stress induces protein kinase D activation in intact cells. Involvement of Src and dependence on protein kinase C

Protein kinase D (PKD) is a protein serine kinase that is directly stimulated in vitro by phorbol esters and diacylglycerol in the presence of phospholipids, and activated by phorbol esters, neuropeptides, and platelet-derived growth factor via protein kinase C (PKC) in intact cells. Recently, oxidative stress was shown to activate transfected PKC isoforms via tyrosine phosphorylation, but PKD activation was not demonstrated. Here, we report that oxidative stress initiated by addition of H(2)O(2) (0.15-10 mm) to quiescent Swiss 3T3 fibroblasts activates PKD in a dose- and time- dependent manner, as measured by autophosphorylation and phosphorylation of an exogenous substrate, syntide-2. Oxidative stress also activated transfected PKD in COS-7 cells but not a kinase-deficient mutant PKD form or a PKD mutant with critical activating serine residues 744 and 748 mutated to alanines. Genistein, or the specific Src inhibitors PP-1 and PP-2 (1-10 micrometer) inhibited H(2)O(2)-mediated PKD activation by 45%, indicating that Src contributes to this signaling pathway. PKD activation by H(2)O(2) was also selectively potentiated by cotransfection of PKD together with an active form of Src (v-Src) in COS-7 cells, as compared with PDB-mediated activation. The specific phospholipase C inhibitor, partly blocked H(2)O(2)-mediated but not PDB-mediated PKD activation. In contrast, PKC inhibitors blocked H(2)O(2) or PDB-mediated PKD activation essentially completely, suggesting that whereas Src mediates part of its effects via phospholipase C activation, PKC acts more proximally as an upstream activator of PKD. Together, these studies reveal that oxidative stress activates PKD by initiating distinct Src-dependent and -independent pathways involving PKC.     

Persistent activation of STAT3 by latent Kaposi's sarcoma-associated herpesvirus infection of endothelial cells

Kaposi's sarcoma-associated herpesvirus (KSHV) is the infectious cause of Kaposi's sarcoma, primary effusion lymphoma, and plasmablastic multicentric Castleman's disease. STAT3 has been shown to be important for the maintenance of primary effusion lymphoma cells in culture and is chronically activated in many tumor cell lines. However, little is known about the role of KSHV in the activation of STAT3 or the role of STAT3 in KS tumors. We demonstrate that STAT3 is activated by KSHV infection of endothelial cells, the KS tumor cell type, in a biphasic fashion. Viral binding and entry activate STAT3 in the first 2 h after infection, but this activation dissipates by 4 h postinfection. By 12 h after KSHV infection, concomitant with the expression of latent genes, STAT3 is once again activated, and this activation persists for as long as latent infection is maintained. Activated STAT3 translocates to the nucleus, where it can bind to STAT3-specific DNA elements and can activate STAT3-dependent promoter activity. Conditioned medium from KSHV-infected endothelial cells is able to transiently activate STAT3, indicating the involvement of a secreted factor and that a latency-associated factor in KSHV-infected cells is necessary for sustained activation. KSHV upregulates gp130 receptor expression, and both gp130 and JAK2 are required for the activation of STAT3. However, neither human nor viral interleukin-6 is required for STAT3 activation. Persistent activation of the oncogenic signal transducer, STAT3, by KSHV may play a critical role in the viral pathogenesis of Kaposi's sarcoma, as well as in primary effusion lymphomas.     

RalA mediates v-Src, v-Ras, and v-Raf regulation of CD44 and fibronectin expression in NIH3T3 fibroblasts

Oncogenic transformation of fibroblasts by v-Src and v-Ras is often associated with downregulation of fibronectin (FN) and increased expression of CD44, a receptor for hyaluronan. Both v-Src and v-Ras as well as v-Raf activate phospholipase D through the small GTPase, RalA, an important mediator of transformation and tumorigenesis in vivo. We have therefore investigated whether RalA is involved in the downregulation of FN and overproduction of CD44 upon oncogenic transformation. We report here that compared to untransfected cells NIH3T3 cells transformed by v-Src, v-Ras, or v-Raf have reduced levels of FN and increased levels of CD44. Moreover, the ability to form extracellular FN fibrils was significantly reduced in the oncogene-transformed cells compared to parental controls. Coexpression of the dominant negative S28N-RalA mutant restored the levels of CD44 and FN and the capacity of v-Src-, v-Ras-, and v-Raf-expressing cells to form extracellular FN fibrils, to those observed in NIH3T3 cells. The data presented here show a novel regulatory role for RalA, which is required for tumor formation in transformed NIH3T3 cells, in mediating the signal transduction pathway activated by v-Src, v-Ras, and v-Raf, that leads to FN downregulation and CD44 overexpression.     

FAK regulates tyrosine phosphorylation of CAS, paxillin, and PYK2 in cells expressing v-Src, but is not a critical determinant of v-Src transformation.

FAK (focal adhesion kinase) is a nonreceptor protein-tyrosine kinase activated by tyrosine phosphorylation following integrin-mediated cell adhesion. Oncogenic Src promotes enhanced and deregulated FAK tyrosine phosphorylation which has been proposed to contribute to altered cell growth and/or morphological properties associated with transformation. In this study, an inducible FAK expression system was used to study the potential role of FAK in v-Src transformation. Our results portray FAK as a major v-Src substrate that also plays a role in recruiting v-Src to phosphorylate substrates CAS (Crk-associated substrate) and paxillin. The FAK Tyr-397 autophosphorylation site was necessary for this scaffolding function, but was not required for v-Src to stably interact with and phosphorylate FAK. FAK was also shown to negatively regulate v-Src mediated phosphorylation of the FAK-related kinase PYK2. Despite these effects, FAK does not play an essential role in targeting v-Src to major cellular substrates including CAS and paxillin. Nor is FAK strictly required to achieve the altered morphological and growth characteristics of v-Src transformed cells.