Distinct Mechanisms of Activation of Stat1 and Stat3 by Platelet-Derived Growth Factor Receptor in a Cell-Free System

Abstract

Ligand-dependent activation of the platelet-derived growth factor receptor (PDGFR) in fibroblasts in culture leads to the activation of the JAK family of protein-tyrosine kinases and of the transcription factors Stat1 and Stat3. To determine the biochemical mechanism of STAT activation by PDGFR, we devised a cell-free system composed of a membrane fraction from cells overexpressing PDGFR. When supplemented with crude cytosol, the membrane fraction supported PDGF-and ATP-dependent activation of both Stat1 and Stat3. However, the extent of Stat3 activation differed depending on the source of the cytosolic fraction. Using purified recombinant STAT proteins produced in Escherichia coli, we found that Stat1 could be activated by immunopurified PDGFR and showed no additional requirement for membrane-or cytosol-derived proteins. In contrast, activation of Stat3 exhibited a strong requirement for the cytosolic fraction. The activity present in the cytosolic fraction could be depleted with antibodies to JAK proteins. We conclude that the mechanisms of activation of Stat1 and Stat3 by PDGFR are distinct. Stat1 activation appears to result from a direct interaction with the receptor, whereas Stat3 activation additionally requires JAK proteins.Proteins of the STAT family of transcription factors are activated by a wealth of cytokines and polypeptide growth factors. Activation by cytokine receptors requires members of the JAK family of protein tyrosine kinases (2-4, 8-10, 15, 17, 24). The general model for STAT activation was based on the paradigm of STAT activation by the interferon receptors (5, 18). Gamma interferon activates both Jak1 and Jak2, which are in turn required for the activation of Stat1 (12,25). Alpha interferon activates Jak1 and Tyk2, leading to the activation of Stat1 and Stat2 (12,21). For interferons and other cytokines that act through cell-surface receptors that lack intrinsic protein-tyrosine kinase activity, receptor-associated JAKs provide the catalytic activity responsible for receptor phosphorylation to create docking sites for signaling proteins such as STATs and presumably for phosphorylation of the proteins recruited to the phosphorylated receptor.Polypeptide growth factors, such as epidermal growth factor and platelet-derived growth factor (PDGF), also activate JAKs and STATs (11,22). Because the receptors for these factors harbor intrinsic protein-tyrosine kinase activity, the role of receptor-activated JAKs, if any, in STAT activation by these receptors remains unclear. We used a cell-free system and partially purified proteins to examine the biochemical steps in STAT activation by the PDGF receptor (PDGFR). We found that the mechanisms of activation of Stat1 and Stat3 are different and that JAKs may be required only for the activation of Stat3. MATERIALS AND METHODSCell cultures and cellular extracts. The cell line 2fTGH.PS1 (22), derived from the human fibrosarcoma HT1080 cells, was grown in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS).Cytosolic ext...

Mechanism of STAT3 Activation by Insulin-like Growth Factor I Receptor

“…STAT1 activation seems to involve its direct interaction with the PDGF receptor, whereas STAT3 activation appears to require JAKs (51). Since multiple members of the JAK family can be activated by PDGF, it was suggested that each JAK is able to mediate STAT3 activation by PDGF independently (51). Our data are in agreement with the previous observation that JAK1 and JAK2 can be phosphorylated upon IGF-I stimulation (41).…”

“…This was shown by using the PDGF receptor mutant that was impaired in binding to Src. Furthermore, using cell-free system, it was demonstrated that the mechanisms of activation of STAT1 and STAT3 by PDGF are distinct (51). STAT1 activation seems to involve its direct interaction with the PDGF receptor, whereas STAT3 activation appears to require JAKs (51).…”

“…Furthermore, using cell-free system, it was demonstrated that the mechanisms of activation of STAT1 and STAT3 by PDGF are distinct (51). STAT1 activation seems to involve its direct interaction with the PDGF receptor, whereas STAT3 activation appears to require JAKs (51). Since multiple members of the JAK family can be activated by PDGF, it was suggested that each JAK is able to mediate STAT3 activation by PDGF independently (51).…”

Shear Flow Attenuates Serum-induced STAT3 Activation in Endothelial Cells

“…STAT3 is activated by tyrosine phosphorylation at a single site (Tyr-705) and by serine phosphorylation at 727 (Ser-727). Tyrosine phosphorylation of STAT3 in response to cytokine stimulation is mediated by a Janus kinase, and Src family members have been widely studied (17)(18)(19). Tyrosine phosphorylation is required for STAT3 dimerization, nuclear translocation, and DNA binding.…”

Suppressor of cytokine signaling 3 expression in anaplastic large cell lymphoma

“…The lack of dependency on ALK for STAT3 activation has been reported in many other human neoplasms that are ALKÀ, and multiple mechanisms of activating STAT3 also have been identified. 19,[36][37][38][39][40] More specifically, previous studies have shown that STAT3 activation may occur in a subset of ALKÀ ALCL 15,17 and in a subset of HL. 41,42 In addition to ALK and JAK3, several other oncogenic kinases, such as those encoded by the v-src, abl, v-fps, and v-sis genes, activate STAT3 in a direct and indirect manner.…”