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Constitutively Active Gα16 Stimulates STAT3 via a c-Src/JAK- and ERK-dependent Mechanism

2003; Elsevier BV; Volume: 278; Issue: 52 Linguagem: Inglês

10.1074/jbc.m307299200

1083-351X

Rico K.H. Lo, Helen Oi-Lam Cheung, Yung Hou Wong,

PI3K/AKT/mTOR signaling in cancer

The hematopoietic-specific Gα16 protein has recently been shown to mediate receptor-induced activation of the signal transducer and activator of transcription 3 (STAT3). In the present study, we have delineated the mechanism by which Gα16 stimulates STAT3 in human embryonic kidney 293 cells. A constitutively active Gα16 mutant, Gα16QL, stimulated STAT3-dependent luciferase activity as well as the phosphorylation of STAT3 at both Tyr705 and Ser727. Gα16QL-induced STAT3 activation was enhanced by overexpression of extracellular signal-regulated kinase 1 (ERK1), but was inhibited by U0126, a Raf-1 inhibitor, and coexpression of the dominant negative mutants of Ras and Rac1. Inhibition of phospholipase Cβ, protein kinase C, and calmodulin-dependent kinase II by their respective inhibitors also suppressed Gα16QL-induced STAT3 activation. The involvement of tyrosine kinases such as c-Src and Janus kinase 2 and 3 (JAK2 and JAK3) in Gα16QL-induced activation of STAT3 was illustrated by the combined use of selective inhibitors and dominant negative mutants. In contrast, c-Jun N-terminal kinase, p38 MAPK, RhoA, Cdc42, phosphatidylinositol 3-kinase, and the epidermal growth factor receptor did not appear to be required. Similar observations were obtained with human erythroleukemia cells, where STAT3 phosphorylation was stimulated by C5a in a PTX-insensitive manner. Collectively, these results highlight the important regulatory roles of the Ras/Raf/MEK/ERK and c-Src/JAK pathways on the stimulation of STAT3 by activated Gα16. Demonstration of the involvement of different kinases in Gα16QL-induced STAT3 activation supports the involvement of multiple signaling pathways in the regulation of transcription by G proteins. The hematopoietic-specific Gα16 protein has recently been shown to mediate receptor-induced activation of the signal transducer and activator of transcription 3 (STAT3). In the present study, we have delineated the mechanism by which Gα16 stimulates STAT3 in human embryonic kidney 293 cells. A constitutively active Gα16 mutant, Gα16QL, stimulated STAT3-dependent luciferase activity as well as the phosphorylation of STAT3 at both Tyr705 and Ser727. Gα16QL-induced STAT3 activation was enhanced by overexpression of extracellular signal-regulated kinase 1 (ERK1), but was inhibited by U0126, a Raf-1 inhibitor, and coexpression of the dominant negative mutants of Ras and Rac1. Inhibition of phospholipase Cβ, protein kinase C, and calmodulin-dependent kinase II by their respective inhibitors also suppressed Gα16QL-induced STAT3 activation. The involvement of tyrosine kinases such as c-Src and Janus kinase 2 and 3 (JAK2 and JAK3) in Gα16QL-induced activation of STAT3 was illustrated by the combined use of selective inhibitors and dominant negative mutants. In contrast, c-Jun N-terminal kinase, p38 MAPK, RhoA, Cdc42, phosphatidylinositol 3-kinase, and the epidermal growth factor receptor did not appear to be required. Similar observations were obtained with human erythroleukemia cells, where STAT3 phosphorylation was stimulated by C5a in a PTX-insensitive manner. Collectively, these results highlight the important regulatory roles of the Ras/Raf/MEK/ERK and c-Src/JAK pathways on the stimulation of STAT3 by activated Gα16. Demonstration of the involvement of different kinases in Gα16QL-induced STAT3 activation supports the involvement of multiple signaling pathways in the regulation of transcription by G proteins. In addition to their classical roles as second messenger regulators, heterotrimeric G proteins have been implicated as mitogenic signal transmitters. The discovery of activating G protein mutations in various disease states highlights their roles in normal and aberrant growth (1Lyons J. Landis C.A. Harsh G. Vallar L. Grunewald K. Feichtinger H. Duh Q.Y. Clark O.H. Kawasaki E. Bourne H.R. McCormick F. Science. 1990; 249: 655-659Crossref PubMed Scopus (935) Google Scholar). To date, a number of Gα subunits have been shown to stimulate mitogenesis and induce neoplastic growth via initiation of intracellular signaling cascades that lead to the activation of mitogen-activated protein kinases (MAPKs, 1The abbreviations used are: MAPKsmitogen-activated protein kinasesSTATsignal transducer and activator of transcriptionJAKJanus kinasev-Srcviral Srcc-Srccellular SrcPDGFplatelet-derived growth factorJNKc-Jun N-terminal kinaseGPCRsG protein-coupled receptorsERKextracellular signal-regulated kinasePI3Kphosphatidylinositol 3-kinasePKCprotein kinase CPTXPertussis toxinMEKMAPK/ERK kinasePLCβphospholipase CβCaMKIIcalmodulin-dependent protein kinase IIEGFepidermal growth factor. Refs. 2Pace A.M. Wong Y.H. Bourne H.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7031-7035Crossref PubMed Scopus (136) Google Scholar and 3Heasley L.E. Storey B. Fanger G.R. Butterfield L. Zamarripa J. Blumberg D. Maue R.A. Mol. Cell. Biol. 1996; 16: 648-656Crossref PubMed Google Scholar). In addition to MAPKs, other critical molecules such as signal transducers and activators of transcription (STATs) have also been shown to participate in the transduction of proliferative signals (4Ram P.T. Horvath C.M. Iyengar R. Science. 2000; 287: 142-144Crossref PubMed Scopus (96) Google Scholar). mitogen-activated protein kinases signal transducer and activator of transcription Janus kinase viral Src cellular Src platelet-derived growth factor c-Jun N-terminal kinase G protein-coupled receptors extracellular signal-regulated kinase phosphatidylinositol 3-kinase protein kinase C Pertussis toxin MAPK/ERK kinase phospholipase Cβ calmodulin-dependent protein kinase II epidermal growth factor. STATs are latent cytoplasmic transcription factors that transduce signals from the cell membrane to the nucleus upon tyrosine phosphorylation (5Darnall Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3401) Google Scholar). They were first identified as mediators of cellular responses to cytokines (6Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (5062) Google Scholar), but later it became apparent that they are also involved in mitogenic growth factor signaling (7Schindler C. Darnell Jr., J.E. Annu. Rev. Biochem. 1995; 64: 621-651Crossref PubMed Scopus (1657) Google Scholar). Binding of cytokines or growth factors to their cognate receptors leads to receptor dimerization and activation of receptor-associated Janus kinases (JAKs), resulting in the recruitment and homo- or heterodimerization of STAT proteins. Activated STAT proteins are then translocated to the nucleus to regulate gene expression. STAT activation by other non-receptor tyrosine kinases has also been demonstrated. Transformation of mammalian fibroblasts by viral Src (v-Src) specifically induces constitutive activation of STAT3 (8Cao X. Tay A. Guy G.R. Tan Y.H. Mol. Cell. Biol. 1996; 16: 1595-1603Crossref PubMed Scopus (341) Google Scholar). Cellular Src (c-Src) tyrosine kinase is involved in the activation of both STAT1 and STAT3 in platelet-derived growth factor (PDGF)-stimulated NIH-3T3 cells (9Cirri P. Chiarugi P. Marra F. Raugei G. Camici G. Manao G. Ramponi G. Biochem. Biophys. Res. Commun. 1997; 239: 493-497Crossref PubMed Scopus (56) Google Scholar). Additionally, it has recently been demonstrated that MAPKs can phosphorylate Ser727 on STAT3 to modulate its transcriptional activity (10Chung J. Uchida E. Grammer T.C. Blenis J. Mol. Cell. Biol. 1997; 17: 6508-6516Crossref PubMed Scopus (556) Google Scholar), while activation of p38 MAPK and c-Jun N-terminal kinase (JNK) is thought to be required for v-Src activation of STAT3 (11Turkson J. Bowman T. Adnane J. Zhang Y. Djeu J.Y. Sekharam M. Frank D.A. Holzman L.B. Wu J. Sebti S. Jove R. Mol. Cell. Biol. 1999; 19: 7519-7528Crossref PubMed Scopus (230) Google Scholar). Although activation of STAT proteins has generally been associated with cytokine and mitogenic growth factor signaling, ligands acting on G protein-coupled receptors (GPCRs) can also activate STAT proteins. Angiotensin II has been shown to induce c-Src-dependent tyrosine (Tyr705) phosphorylation of STAT3 via activation of the G protein-coupled AT1 receptor in vascular smooth muscle cells (12Liang H. Venema V.J. Wang X. Ju H. Venema R.C. Marrero M.B. J. Biol. Chem. 1999; 274: 19846-19851Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). α-Melanocyte-stimulating hormone, which enhances cellular proliferation, has been found to activate JAK2 and STAT1 in B-lymphocytes via stimulation of the melanocortin 5 receptor (13Buggy J.J. Biochem. J. 1998; 331: 211-216Crossref PubMed Scopus (104) Google Scholar). Likewise, activation of α1-adrenoceptors and protease-activated receptor 1 has been shown to induce tyrosine phosphorylation of JAK2, Tyk2, and STAT1 in vascular smooth muscle cells (14Sasaguri T. Teruya H. Ishida A. Abumiya T. Ogata J. Biochem. Biophy. Res. Commun. 2000; 268: 25-30Crossref PubMed Scopus (32) Google Scholar). While activation of STATs in response to GPCR stimulation has been reported, the involvement of STATs in Gα-mediated transformation of cells is beginning to emerge (15Ram P.T. Iyengar R. Oncogene. 2001; 20: 1601-1606Crossref PubMed Scopus (109) Google Scholar). Expression of constitutively active Gαo in NIH-3T3 cells results in Src-dependent activation of STAT3, which leads to cellular transformation. Similarly, expression of constitutively active Gαi2 in NIH-3T3 cells increases STAT3 activity (4Ram P.T. Horvath C.M. Iyengar R. Science. 2000; 287: 142-144Crossref PubMed Scopus (96) Google Scholar). Conversely, expression of a dominant negative mutant of Gαi2 inhibits Src kinase activity and Tyr705 phosphorylation of STAT3, leading to a reduction of v-fms-induced proliferation in NIH-3T3 cells (16Corre I. Baumann H. Hermouet S. Oncogene. 1999; 18: 6335-6342Crossref PubMed Scopus (34) Google Scholar). These data suggest that the STAT3 pathway may play a vital role in the Gα subunit regulation of cell proliferation and transformation. With the recent demonstration of STAT3 involvement in Gαo- and Gαi2-induced cell transformation, it is reasonable to deduce that other Gα subunits may also regulate mitogenesis via STAT3 activation. Gα16, being unique in its restricted expression in hematopoietic cells (17Amatruda T.T. Steele D.A. Slepak V.Z. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5587-5591Crossref PubMed Scopus (240) Google Scholar), is also expressed in poorly differentiated leukemia cells, suggesting an association with hematopoietic cell growth and differentiation. Expression of a constitutively active Gα16 mutant has been shown to induce cell differentiation in rat pheochromocytoma PC12 (3Heasley L.E. Storey B. Fanger G.R. Butterfield L. Zamarripa J. Blumberg D. Maue R.A. Mol. Cell. Biol. 1996; 16: 648-656Crossref PubMed Google Scholar) and aortic vascular smooth muscle cells, although the same mutant was found to inhibit cell growth in Swiss 3T3 cells (18Qian N.X. Russell M. Buhl A.M. Johnson G.L. J. Biol. Chem. 1994; 269: 17417-17423Abstract Full Text PDF PubMed Google Scholar). Such observations suggest that Gα16 may regulate cell growth and differentiation via activation of cell type-specific signal transduction pathways. As a promiscuous G protein (19Offermanns S. Simon M. J. Biol. Chem. 1995; 270: 15175-15180Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar), Gα16 possesses the ability to link a variety of GPCRs to the regulation of MAPKs. Recently, Gα16 has been shown to activate JNK (3Heasley L.E. Storey B. Fanger G.R. Butterfield L. Zamarripa J. Blumberg D. Maue R.A. Mol. Cell. Biol. 1996; 16: 648-656Crossref PubMed Google Scholar, 20Chan A.S.L. Lai F.P.L. Lo R.K.H. Voyno-Yasenetskaya T.A. Stanbridge E.J. Wong Y.H. Cell. Signal. 2002; 14: 249-257Crossref PubMed Scopus (132) Google Scholar). Interestingly, in addition to its ability to phosphorylate c-Jun, JNK can also phosphorylate STAT3 at Ser727 (21Lim C.P. Cao X. J. Biol. Chem. 1999; 274: 31055-31061Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). It is therefore plausible that activated Gα16 can influence cell differentiation via MAPK-induced STAT3 signaling. Based on the exclusivity of Gα16 expression in hematopoietic cells and the involvement of STAT pathways in both normal and perturbed hematopoiesis (22Ward A.C. Touw I. Yoshimura A. Blood. 2000; 95: 19-29Crossref PubMed Google Scholar), phosphorylation of STAT3 via Gα16 activation may represent an important pathway for cell differentiation and development in the immune system. Indeed, we have recently shown that Gα16 can support receptor-mediated activation of STAT3 in human embryonic kidney 293 (HEK 293) cells (23Wu E.H.T. Lo R.K.H. Wong Y.H. Biochem. Biophy. Res. Commun. 2003; 303: 920-925Crossref PubMed Scopus (47) Google Scholar). In the present study, we examined the mechanism by which Gα16QL, a constitutively active Gα16 mutant, stimulated STAT3 in HEK 293 cells and provide evidence for the involvement of various intermediate signaling molecules including c-Src, JAKs, and extracellular signal-regulated kinase (ERK). Reagents—HEK 293 cells were obtained from the American Type Culture Collection (CRL-1573, Rockville, MD). Human erythroleukemia (HEL) cells were from German Collection of Microorganisms and Cell Cultures (ACC11, Braunschweig, Germany). Cell culture reagents, including LipofectAMINE PLUS, were purchased from Invitrogen. The cDNAs of Ras, Rac1, and Cdc42 were obtained from Guthrie Research Institute (Sayre, PA). Constitutively activated mutants of Rac1 (Rac1G12V) and Cdc42 (Cdc42G12V) were kindly provided by Dr. Christopher L. Carpenter (Harvard Medical School, Boston). Constitutively activated RasG12V was a gift from Dr. Jeffery Field (University of Pennsylvania School of Medicine, Philadelphia, PA). RhoA and its various mutants (RhoAG14V and RhoAT19N) as well as the dominant negative mutant of Cdc42 (Cdc42T17N) were generous gifts from Dr. Marc Symons (Picower Institute for Medical Research, New York). The origins of plasmids encoding the dominant negative mutant of c-Src (c-Src-DN), phosphatidylinositol 3-kinase γ (PI3Kγ) and its dominant negative mutant (PI3Kγ-DN) were as described previously (24Kam A.Y.F. Chan A.S.L. Wong Y.H. J. Neurochem. 2003; 84: 503-513Crossref PubMed Scopus (34) Google Scholar). The p38 MAPK cDNA was obtained from Dr. Zhenguo Wu (Hong Kong University of Science and Technology, Hong Kong, China). The cDNAs of STAT3 and its dominant negative mutants STAT3Y705F and STAT3S727A were kindly donated by Dr. Nancy C. Reich (State University of New York). Protein kinase C (PKC) α and ϵ dominant negative mutant cDNAs, PKCα-KR and PKCϵ-KR, were kindly obtained from Dr. Bernard Weinstein (Columbia University, New York). The construction or sources of other cDNAs encoding wild-type and mutant forms of Gα16, Ras, Rac1, and JNK1 cDNAs were as described previously (20Chan A.S.L. Lai F.P.L. Lo R.K.H. Voyno-Yasenetskaya T.A. Stanbridge E.J. Wong Y.H. Cell. Signal. 2002; 14: 249-257Crossref PubMed Scopus (132) Google Scholar, 25Ho M.K.C. Yung L.Y. Wong Y.H. J. Neurochem. 1999; 73: 2101-2109PubMed Google Scholar). The luciferase reporter genes, pSTAT3-TA-luc, pGAS-TA-luc and pISRE-TA-luc, were obtained from Clontech laboratories, Inc. (Palo Alto, CA). The luciferase substrate and its lysis buffer were purchased from Roche Diagnostics (Mannheim, Germany). All antibodies were obtained from Cell Signaling and kinase inhibitors were from Calbiochem (Darmstadt, Germany). C5a was purchased from Sigma Aldrich. Cell Culture and Transfection—HEL cells were maintained at 5% CO2, 37 °C in RPMI 1640 with 10% fetal bovine serum, 50 units/ml penicillin and 50 μl/ml streptomycin. HEK 293 cells were maintained at 5% CO2, 37 °C in Eagle's minimum essential medium (growth medium) with 10% fetal bovine serum, 50 units/ml penicillin and 50 μl/ml streptomycin. HEK 293 cells were seeded on 96-well microtiter plates at a density of 15,000 cells/well and were cultured in the growth medium at 18 to 24 h prior to transfection. They were co-transfected with various cDNAs using LipofectAMINE PLUS reagents. The transfection mixtures using 100 μl/well of serum and antibiotics free OPTI-MEM medium contained 10 ng of G proteins, small GTPases or the control vector cDNAs, 0.1 μg of pSTAT3-TA-luc and 0.2 μl of both PLUS and LipofectAMINE reagents. After 3 h of transfection, 50 μl of OPTI-MEM medium containing 30% fetal bovine serum was added into the wells and incubated for another 30 h. Luciferase Assay—30 h after transfection, cells were serum-starved for 24 h. After removal of the medium, 25 μl of lysis buffer from Roche Diagnostics luciferase assay kit was added to the wells and then gently shaken on ice for 30 min. For detection, cell lysates in 25 μl of lysis buffer and 25 μl of luciferase substrate were measured by a microtiter plate luminometer MicroLumatPlus LB96V from EG&G Berthold. Western Blot Analysis—HEK 293 cells were transferred on 6-well plates at a density of 5 × 105 cells/well and were kept in the growth medium the day before transfection. They were co-transfected with various cDNAs using LipofectAMINE PLUS reagents following the supplier's instructions. After 48 h of transfection, the transfected cells were serum starved or treated with different kinase inhibitors overnight. The cells were lysed in 150 μl of lysis buffer (50 mm Tris-HCl, pH 7.5, 100 mm NaCl, 5 mm EDTA, 40 mm NaP2O7, 1% Triton X-100, 1 mm dithiothreitol, 200 μm Na3VO4, 100 μm phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, 4 μg/ml aprotinin, and 0.7 μg/ml pepstatin) and then gently shaken on ice for 30 min. Supernatants were collected by centrifugation at 16,000 × g for 5 min. HEL cells were seeded at a density of 1 × 106 cells using serum-free medium with or without PTX (100 ng/ml) treatment overnight. Cells were treated with different kinase inhibitors for 30 min and then incubated with 100 nm C5a at 37 °C for 20 min. After that, cells were lysed by lysis buffer. Proteins from the cell lysates were resolved by 12% SDS-polyacrylamide gel electrophoresis, and then transferred to Osmonics nitrocellulose membrane (Westborough, MA). Phospho-STAT3-Tyr705, phospho-STAT3-Ser727, STAT3, phospho-p38 MAPK, p38 MAPK, phospho-JNK, JNK, phospho-ERK, and ERK were detected by specific primary antibodies and horseradish peroxidase-conjugated secondary antibodies. The immunoblots were visualized by chemiluminescence with the ECL kit from Amersham Biosciences. Activation of STAT3 by Constitutively Active Gα16QL in HEK 293 Cells—It has previously been shown that expression of constitutively active Gαo and Gαi2 in NIH-3T3 cells induced STAT3 activation (4Ram P.T. Horvath C.M. Iyengar R. Science. 2000; 287: 142-144Crossref PubMed Scopus (96) Google Scholar, 16Corre I. Baumann H. Hermouet S. Oncogene. 1999; 18: 6335-6342Crossref PubMed Scopus (34) Google Scholar). In the present study, we sought to investigate the ability of constitutively active Gα16 to stimulate STAT3 phosphorylation and activation. Mutation of glutamine 212 to leucine (Q212L) in the conserved GTP/GDP binding domain of Gα16 inhibits its intrinsic GTPase activity and results in constitutive activation of Gα16 (3Heasley L.E. Storey B. Fanger G.R. Butterfield L. Zamarripa J. Blumberg D. Maue R.A. Mol. Cell. Biol. 1996; 16: 648-656Crossref PubMed Google Scholar, 25Ho M.K.C. Yung L.Y. Wong Y.H. J. Neurochem. 1999; 73: 2101-2109PubMed Google Scholar). To investigate the effect of Gα16QL mutant on the phosphorylation state of STAT3, HEK 293 cells were transiently transfected with cDNAs encoding pcDNA1 (vector control), Gα16 or Gα16QL. Total cell lysates prepared from the transfected cells were probed with anti-STAT3, anti-phospho-STAT3-Tyr705 and anti-phospho-STAT3-Ser727 antisera. Expression of either Gα16 or Gα16QL in HEK 293 cells did not affect the expression of total STAT3 as compared with the vector control (Fig. 1A). Further studies using anti-phospho-STAT3-Tyr705 and anti-phospho-STAT3-Ser727 antisera revealed basal levels of Ser727 phosphorylation of STAT3 protein in both the vector control and Gα16-expressing cells, while there was little or no Tyr705 phosphorylation of STAT3 in these cells. In contrast, expression of Gα16QL led to a striking increase in both Tyr705 and Ser727 phosphorylation of STAT3 protein (Fig. 1A). The observed Gα16QL-induced phosphorylation of STAT3 is likely to induce STAT3 transcriptional activity, as demonstrated with GαoQL-induced phosphorylation and activation of STAT3 (4Ram P.T. Horvath C.M. Iyengar R. Science. 2000; 287: 142-144Crossref PubMed Scopus (96) Google Scholar). In order to verify a similar correlation between Gα16QL-induced STAT3 phosphorylation and modulation of STAT3 transcriptional activity, we performed reporter gene assays using a pSTAT3-TA-luc construct in combination with various cDNAs (pcDNA1, Gα16 and Gα16QL). As indicated in Fig. 1B, the magnitude of STAT3-mediated gene expression was unaffected by control vector or Gα16 expression. On the contrary, expression of Gα16QL induced an increased level of STAT3 activation as evidenced by a significant elevation of reporter gene expression. These results suggest that activation of Gα16 can indeed lead to the phosphorylation of STAT3 at both Tyr705 and Ser727 as well as the induction of STAT3 transcriptional activity. Using two other reporter gene constructs (pGAS-TA-luc and pISRE-TA-luc), we examined the ability of Gα16QL to similarly activate STAT1 and STAT1/2 transcriptional activities. STAT1-mediated gene expression was significantly elevated in Gα16QL-expressing cells, but not in Gα16 or vector-transfected cells (Fig. 1B). In contrast, STAT1/2-mediated gene expression was not enhanced in Gα16QL-expressing cells (Fig. 1B). The specificity of Gα16QL-induced STAT3 activation was further characterized by the dose-dependent relationship between the amount of Gα16QL expression and STAT3 stimulation. As shown in Fig. 1C, the magnitude of STAT3-dependent luciferase activity was positively correlated to the concentration of Gα16QL cDNA transfected into the HEK 293 cells, whereas no correlation with activity could be observed for the cDNA of Gα16. The progressive increase in STAT3-dependent luciferase activity was eminent at the concentration range of 3–30 ng/ml of Gα16QL cDNA. Increasing the Gα16QL cDNA concentration beyond 30 ng/ml failed to induce further increase in luciferase activity, presumably because Gα16QL was no longer a limiting factor. The observed saturation of STAT3-dependent luciferase activity was likely to be caused by the limited amount of pSTAT3-TA-luc available in the transfected cells. As both Tyr705 and Ser727 of STAT3 appeared to be phosphorylated in Gα16QL-expressing cells, we examined whether these modifications can occur independently. Using the reporter gene assay, the ability of Gα16QL to induce the phosphorylation of wild-type and phosphorylation-resistant mutants of STAT3 (STAT3Y705F and STAT3S727A) was examined in HEK 293 cells. Coexpression of STAT3 and Gα16QL resulted in an elevation of luciferase activity that was significantly higher than those obtained with Gα16 and vector control, and the magnitude of the response was identical to that of cells lacking recombinant STAT3 (Fig. 1D). In cells coexpressing the Y705F mutant of STAT3, although Gα16QL was still capable of inducing STAT3-dependent luciferase activity beyond the controls, such stimulation was significantly attenuated as compared with cells coexpressing wild-type STAT3 (Fig. 1D). Similar results were obtained with the S727A mutant of STAT3. Overexpression of STAT3 and its mutants was detected with anti-STAT3 antiserum (Fig. 1D). Lack of phosphorylation of the Y705F and S727A mutants of STAT3 at Tyr705 and Ser727, respectively, was confirmed with anti-phospho-STAT3 antisera (Fig. 1D). Basal as well as Gα16QL-dependent Tyr705 phosphorylation were prominently observed in STAT3- and S727A-mutant expressing cells, whereas a reduction in Tyr705 phosphorylation was seen with the Y705F mutant. Conversely, elevations in the levels of Ser727 phosphorylation were detected in cells expressing the wild-type and Y705F mutant of STAT3, while little or no phosphorylation at this site was detectable in S727A-expressing cells. This indicated that Gα16QL-induced phosphorylation of STAT3 at Tyr705 and Ser727 could occur independently. Effects of MAPKs on STAT3 Phosphorylation—The MAPK pathway has been shown to play an important role in the regulation of STAT3 signaling. Both ERKs and JNK1 have been shown to phosphorylate STAT3 at Ser727 (10Chung J. Uchida E. Grammer T.C. Blenis J. Mol. Cell. Biol. 1997; 17: 6508-6516Crossref PubMed Scopus (556) Google Scholar, 21Lim C.P. Cao X. J. Biol. Chem. 1999; 274: 31055-31061Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Likewise, p38 MAPK has been shown to play a key role in the Ser727 phosphorylation of STAT3 (11Turkson J. Bowman T. Adnane J. Zhang Y. Djeu J.Y. Sekharam M. Frank D.A. Holzman L.B. Wu J. Sebti S. Jove R. Mol. Cell. Biol. 1999; 19: 7519-7528Crossref PubMed Scopus (230) Google Scholar). Since Gα16 has been shown to activate JNK (4Ram P.T. Horvath C.M. Iyengar R. Science. 2000; 287: 142-144Crossref PubMed Scopus (96) Google Scholar, 24Kam A.Y.F. Chan A.S.L. Wong Y.H. J. Neurochem. 2003; 84: 503-513Crossref PubMed Scopus (34) Google Scholar), we asked if it can similarly stimulate ERK and p38 MAPK. Anti-phospho-MAPK antisera were used to probe the activities of MAPKs in Gα16QL-transfectants. The activities of all three branches of MAPKs were stimulated by Gα16QL, but not Gα16 (Fig. 2A). Next, we investigated if one or more of these MAPKs were required for Gα16QL-induced STAT3 activation. Firstly, we examined the effect of MAPKs expression on STAT3 activity. As illustrated in Fig. 2B, overexpression of ERK1 significantly stimulated STAT3 activation. In contrast, overexpression of JNK1 or p38 MAPK had no effect on STAT3 stimulation, suggesting that these two pathways did not play a significant role in STAT3 regulation in HEK 293 cells. We used a panel of kinase inhibitors to further confirm the functional role of various MAPKs on Gα16QL-induced STAT3 phosphorylation and activation. The MAPK/ERK kinase 1/2 (MEK1/2) inhibitor, U0126 (10 μm), inhibited Gα16QL-induced STAT3 activation whereas the p38 MAPK/JNK inhibitors, SB202190 (10 μm) and SB203580 (10 μm), did not affect STAT3-dependent luciferase activity (Fig. 2C). The ineffectiveness of both SB202190 and SB203580 further substantiated the lack of involvement of p38 MAPK and JNK in STAT3 activation. The inhibitory effect of U0126 reflects a possible involvement of MEK1/2 in STAT3 regulation. Inhibition of the upstream activator of MEK1/2, Raf-1, by its specific inhibitor (10 μm) also suppressed the Gα16QL-induced STAT3 activation (Fig. 2C). These observations provided additional evidence to support the regulatory role of ERK on STAT3 activation. The effects of the various inhibitors on the state of phosphorylation of STAT3 were examined with anti-phospho-STAT3 antisera. Both U0126 and the Raf-1 inhibitor, but not SB202190 and SB203580, attenuated the Gα16QL-induced STAT3 phosphorylation at Ser727 (Fig. 2C). Interestingly, Gα16QL-induced STAT3 phosphorylation at Tyr705 was also suppressed by U0126 and the Raf-1 inhibitor, but not by SB202190 and SB203580 (Fig. 2C). Since ERK is a Ser/Thr-specific kinase, these results implied that the ERK pathway might indirectly activate STAT3 by phosphorylation at Tyr705. Roles of Small GTPases on Gα16QL-induced STAT3 Activation—Next, we attempted to identify other intracellular signaling molecules involved in Gα16QL-induced STAT3 activation. Small GTPases have been shown to play critical roles in growth regulation and there is considerable evidence to suggest that GPCR activation can regulate cell growth through the engagement of these monomeric GTPases. In order to determine the role of small GTPases on STAT3 phosphorylation, constitutively activated mutants of Ras (RasG12V), Cdc42 (Cdc42G12V), Rac1 (Rac1G12V), and RhoA (RhoAG14V) were utilized to examine their effects on STAT3 activity. STAT3-dependent luciferase activity was significantly stimulated in cells expressing constitutively active RasG12V or Rac1G12V (Fig. 3A). In contrast, neither Cdc42G12V nor RhoAG14V was able to stimulate STAT3 activity (Fig. 3A). The functional role of Ras and Rac1 in the regulation of Gα16QL-induced STAT3 activation was further examined in HEK 293 cells. If these small GTPases serve as intermediate signaling molecules for Gα16-mediated STAT3 activation, their dominant negative mutants should inhibit Gα16QL-induced STAT3 activity. Using STAT3 driven luciferase reporter gene assays, we coexpressed the various mutants of Ras and Rac1 with either Gα16 or Gα16QL in HEK 293 cells and determined the luciferase activity of the transfectants. In the Gα16-transfectants, the presence of RasG12V significantly elevated STAT3 activity as compared with either the vector control or wild-type Ras (Fig. 3B). The dominant negative mutant of Ras (RasS17N) slightly suppressed the STAT3 activity in Gα16-expressing cells. In HEK 293 cells expressing Gα16QL and a vector control or together with wild-type Ras, STAT3 activities were significantly higher than the corresponding cells expressing Gα16 (Fig. 3B). Expression of constitutively active RasG12V did not further enhance the Gα16QL-induced STAT3 activity, whereas the dominant negative mutant RasS17N almost completely inhibited the Gα16QL-induced STAT3 activity, to a level similar to that seen with Gα16-expressing cells (Fig. 3B). The inhibitory effect of RasS17N on Gα16QL-induced STAT3 phosphorylation was also demonstrated by immunoblotting with anti-phospho-STAT3 antisera. As compared with the vector control or Gα16-transfectants, much higher levels of Tyr705 and Ser727 phosphorylation