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• W2060859699 abstract "We demonstrate here that growth hormone (GH) stimulates the activation of Rap1 and Rap2 in NIH-3T3 cells. Full activation of Rap1 and Rap2 by GH necessitated the combined activity of both JAK2 and c-Src kinases, although c-Src was predominantly required. GH-stimulated Rap1 and Rap2 activity was also demonstrated to be CrkII-C3G-dependent. GH stimulated the tyrosine phosphorylation of C3G, which again required the combined activity of JAK2 and c-Src. C3G tyrosine residue 504 was required for GH-stimulated Rap activation. Activated Rap1 inhibited GH-stimulated activation of RalA and subsequent GH-stimulated p44/42 MAP kinase activity and Elk-1-mediated transcription. In addition, we demonstrated that C3G-Rap1 mediated CrkII enhancement of GH-stimulated JNK/SAPK activity. We have therefore identified a linear JAK2-independent pathway switching GH-stimulated p44/42 MAP kinase and JNK/SAPK activities. We demonstrate here that growth hormone (GH) stimulates the activation of Rap1 and Rap2 in NIH-3T3 cells. Full activation of Rap1 and Rap2 by GH necessitated the combined activity of both JAK2 and c-Src kinases, although c-Src was predominantly required. GH-stimulated Rap1 and Rap2 activity was also demonstrated to be CrkII-C3G-dependent. GH stimulated the tyrosine phosphorylation of C3G, which again required the combined activity of JAK2 and c-Src. C3G tyrosine residue 504 was required for GH-stimulated Rap activation. Activated Rap1 inhibited GH-stimulated activation of RalA and subsequent GH-stimulated p44/42 MAP kinase activity and Elk-1-mediated transcription. In addition, we demonstrated that C3G-Rap1 mediated CrkII enhancement of GH-stimulated JNK/SAPK activity. We have therefore identified a linear JAK2-independent pathway switching GH-stimulated p44/42 MAP kinase and JNK/SAPK activities. The Rap proteins (Rap1 and Rap2) belong to the Ras-like small GTPase superfamily, which consists of at least 13 members (1Bos J.L. de Rooij J. Reedquist K.A. Nat. Rev. Mol. Cell. Biol. 2001; 2: 369-377Crossref PubMed Scopus (512) Google Scholar). In this family, Ras has been extensively studied and was originally regarded as a transforming oncogene since Ras genes have been found mutated in about 15% of all human tumors (2Bos J.L. Cancer Res. 1989; 49: 4682-4689PubMed Google Scholar). It possesses an important role in cell growth and differentiation as it has been demonstrated to be required for activation of p44/42 MAP 1The abbreviations used are: MAP, mitogen-activated protein; GEFs, guanine nucleotide exchange factors; JNK, Jun N-terminal kinase; JAK, Janus kinase; GAP, GTPase-activating protein; EGF, epidermal growth factor; GH, growth hormone; GST, glutathione S-transferase; STAT, signal transducers and activators of transcription; ECM, extracellular matrix; IL, interleukin; SAPK, stress-activated protein kinase. kinase activity by a number of growth factors including growth hormone (GH) (3Winston L.A. Hunter T. J. Biol. Chem. 1995; 270: 30837-30840Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 4Burgering B.M. Bos J.L. Trends Biochem. Sci. 1995; 20: 18-22Abstract Full Text PDF PubMed Scopus (292) Google Scholar, 5Marshall C.J. Curr. Opin. Cell Biol. 1996; 8: 197-204Crossref PubMed Scopus (474) Google Scholar). Rap1 is closely related to Ras and was initially identified as a suppressor of the K-ras transformed phenotype (6Kitayama H. Sugimoto Y. Matsuzaki T. Ikawa Y. Noda M. Cell. 1989; 56: 77-84Abstract Full Text PDF PubMed Scopus (763) Google Scholar). Structurally, Rap1 shares striking similarity with Ras in the effector domain. It has been suggested that Rap1 antagonizes Ras activity by sequestration of its target, Raf-1, in the inactive form (7Bos J.L. EMBO J. 1998; 17: 6776-6782Crossref PubMed Scopus (288) Google Scholar). In addition, several studies have demonstrated that both Rap1 and Ras can bind the same effectors, such as Raf-1 and RalGDS (8Okada S. Matsuda M. Anafi M. Pawson T. Pessin J.E. EMBO J. 1998; 17: 2554-2565Crossref PubMed Scopus (82) Google Scholar). Rap2 exhibits 60% identity to Rap1 and shares most of the effector proteins with Ras and Rap1 (9Nancy V. Wolthuis R.M. de Tand M.F. Janoueix-Lerosey I. Bos J.L. de Gunzburg J. J. Biol. Chem. 1999; 274: 8737-8745Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The function of Rap2 is more limited compared with Rap1, although an antagonistic effect of Rap2 on Ras-mediated transcription has been reported (10Ohba Y. Mochizuki N. Matsuo K. Yamashita S. Nakaya M. Hashimoto Y. Hamaguchi M. Kurata T. Nagashima K. Matsuda M. Mol. Cell. Biol. 2000; 20: 6074-6083Crossref PubMed Scopus (93) Google Scholar). One common mechanism utilized by small GTPases to regulate cellular function is to cycle between the inactive GDP-bound state and the active GTP-bound state. Guanine nucleotide exchange factors (GEFs) facilitate GDP dissociation and allow the more abundant GTP to rebind, while GTPase-activating proteins (GAPs) accelerate GTP hydrolysis to complete the cycle (1Bos J.L. de Rooij J. Reedquist K.A. Nat. Rev. Mol. Cell. Biol. 2001; 2: 369-377Crossref PubMed Scopus (512) Google Scholar, 7Bos J.L. EMBO J. 1998; 17: 6776-6782Crossref PubMed Scopus (288) Google Scholar). C3G is a Rap-specific GEF since it predominantly catalyzes the guanine nucleotide exchange reaction for Rap1 and Rap2 (10Ohba Y. Mochizuki N. Matsuo K. Yamashita S. Nakaya M. Hashimoto Y. Hamaguchi M. Kurata T. Nagashima K. Matsuda M. Mol. Cell. Biol. 2000; 20: 6074-6083Crossref PubMed Scopus (93) Google Scholar, 11Gotoh T. Hattori S. Nakamura S. Kitayama H. Noda M. Takai Y. Kaibuchi K. Matsui H. Hatase O. Takahashi H. Kurata T. Matsuda M. Mol. Cell. Biol. 1995; 15: 6746-6753Crossref PubMed Scopus (336) Google Scholar). C3G was originally identified as a major protein bound to the SH3 domain of c-Crk (12Zhai B. Huo H. Liao K. Biochem. Biophys. Res. Commun. 2001; 286: 61-66Crossref PubMed Scopus (8) Google Scholar). There are three proline-rich sequences that bind to the SH3 domain of c-Crk in the central region of C3G and one C-terminal CDC25 homology domain catalyzing the GEF reaction of Rap (13Matsuda M. Ota S. Tanimura R. Nakamura H. Matuoka K. Takenawa T. Nagashima K. Kurata T. J. Biol. Chem. 1996; 271: 14468-14472Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The function of the C3G N terminus remains unknown but recent studies have reported the association of p130cas to this region (14Kirsch K.H. Georgescu M.M. Hanafusa H. J. Biol. Chem. 1998; 273: 25673-25679Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). C3G is activated by c-Crk-mediated membrane recruitment. Two isoforms of c-Crk protein are generated by alternative mRNA splicing. The larger form is CrkII, containing one SH2 domain and two SH3 domains, while the smaller form is CrkI, which lacks the C-terminal SH3 domain and one negative regulatory tyrosine residue compared with CrkII. CrkII is more abundant than CrkI in normal cells; therefore, CrkII is the major adaptor for C3G (15Matsuda M. Tanaka S. Nagata S. Kojima A. Kurata T. Shibuya M. Mol. Cell. Biol. 1992; 12: 3482-3489Crossref PubMed Scopus (247) Google Scholar). A number of growth factors and cytokines stimulate the recruitment of the Crk-C3G complex to the membrane where tyrosine kinases are located such that C3G tyrosine residue 504 is phosphorylated with a resultant increase in its GEF activity (16Kiyokawa E. Mochizuki N. Kurata T. Matsuda M. Crit. Rev. Oncog. 1997; 8: 329-342Crossref PubMed Scopus (62) Google Scholar, 17Ichiba T. Hashimoto Y. Nakaya M. Kuraishi Y. Tanaka S. Kurata T. Mochizuki N. Matsuda M. J. Biol. Chem. 1999; 274: 14376-14381Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). GH is the primary regulator of postnatal somatic growth and metabolism (18Carter-Su C. Smit L.S. Rec. Prog. Horm. Res. 1998; 53 (discussion 82-63): 61-82PubMed Google Scholar, 19Zhu T. Goh E.L. Graichen R. Ling L. Lobie P.E. Cell Signal. 2001; 13: 599-616Crossref PubMed Scopus (221) Google Scholar). It utilizes special groups of signaling molecules to regulate the transcription of specific genes required for the above processes. These signaling molecules include: 1) receptor-tyrosine kinases (EGF receptor) (20Yamauchi T. Ueki K. Tobe K. Tamemoto H. Sekine N. Wada M. Honjo M. Takahashi M. Takahashi T. Hirai H. Tushima T. Akanuma Y. Fujita T. Komuro I. Yazaki Y. Kadowaki T. Nature. 1997; 390: 91-96Crossref PubMed Scopus (256) Google Scholar) and non-receptor-tyrosine kinases (JAK2, Ref. 3Winston L.A. Hunter T. J. Biol. Chem. 1995; 270: 30837-30840Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar; c-Src; c-Fyn, Ref. 21Zhu T. Goh E.L. LeRoith D. Lobie P.E. J. Biol. Chem. 1998; 273: 33864-33875Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar; and FAK, Ref. 22Zhu T. Goh E.L. Lobie P.E. J. Biol. Chem. 1998; 273: 10682-10689Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), although in the case of the EGF receptor it may be used simply as an adaptor protein; 2) members of the MAP kinase family including p44/42 MAP kinase (23Campbell G.S. Pang L. Miyasaka T. Saltiel A.R. Carter-Su C. J. Biol. Chem. 1992; 267: 6074-6080Abstract Full Text PDF PubMed Google Scholar), p38 MAP kinase (24Zhu T. Lobie P.E. J. Biol. Chem. 2000; 275: 2103-2114Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), and JNK/SAPK (21Zhu T. Goh E.L. LeRoith D. Lobie P.E. J. Biol. Chem. 1998; 273: 33864-33875Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) and their respective downstream effectors; 3) members of the insulin receptor substrate (IRS) group including IRS-1, -2, and -3, which may act as docking proteins for further activation of signaling molecules including phosphatidylinositol 3-kinase (25Argetsinger L.S. Hsu G.W. Myers Jr., M.G. Billestrup N. White M.F. Carter-Su C. J. Biol. Chem. 1995; 270: 14685-14692Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar); 4) small Ras-like GTPases (26Vanderkuur J.A. Butch E.R. Waters S.B. Pessin J.E. Guan K.L. Carter-Su C. Endocrinology. 1997; 138: 4301-4307Crossref PubMed Scopus (88) Google Scholar); and 5) STAT family members including STATs 1, 3, 5a, and 5b (27Wood T.J. Sliva D. Lobie P.E. Pircher T.J. Gouilleux F. Wakao H. Gustafsson J.A. Groner B. Norstedt G. Haldosen L.A. J. Biol. Chem. 1995; 270: 9448-9453Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 28Bergad P.L. Shih H.M. Towle H.C. Schwarzenberg S.J. Berry S.A. J. Biol. Chem. 1995; 270: 24903-24910Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), which constitute one group of signaling molecules involved in transcriptional regulation by GH. Although JAK2 is postulated to be required for GH signal transduction, our group has recently identified a JAK2-independent pathway regulating GH-stimulated p44/42 MAP kinase activity (29Zhu T. Ling L. Lobie P.E. J. Biol. Chem. 2002; 277: 45592-45603Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). GH stimulated the formation of GTP-bound RalA and subsequent phospholipase D activation, required for the activation of p44/42 MAP kinase by GH in a c-Src-dependent manner. Here we have demonstrated that cellular stimulation with GH results in the activation of both Rap1 and Rap2 in NIH-3T3 cells. The activation of Rap by GH was achieved by the combined JAK2- and c-Src-dependent tyrosine phosphorylation of C3G. GH-stimulated Rap1 activation was utilized to negatively modulate GH-stimulated p44/42 MAP kinase activity and subsequent Elk-1-mediated transcription through inhibition of RalA activity. Concomitantly, GH stimulated C3G-dependent activation of Rap1-enhanced JNK/SAPK activity and subsequent c-Jun-mediated transcription in response to GH. Rap1 is therefore a GH effector molecule activated in a JAK2-independent manner. Materials—Recombinant human growth hormone (hGH) was a generous gift of Novo Nordisk (Singapore). CrkII monoclonal antibody, Ras monoclonal antibody, RalA monoclonal antibody, and PY20 monoclonal antibody were obtained from Transduction Laboratories (Lexington, KY). JAK2 polyclonal antibody, C3G polyclonal antibody, c-Src polyclonal antiserum, Rap1 polyclonal antibody, Rap2 polyclonal antibody, and protein A/G plus agarose were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary anti-IgG antibodies and the enhanced chemiluminescence (ECL) kit were purchased from Amersham Biosciences. The p44/42 MAP kinase assay kit and SAPK/JNK assay kit were purchased from New England Biolabs (Beverly, MA). The transfection reagent Effectene was purchased from Qiagen (Hilden, Germany). The complete protease inhibitor mixture tablets were purchased from Roche Diagnostics (Mannheim, Germany). All other reagents were obtained from Sigma Chemical. pET15b-GST-RalGDS-RBD construct encoding the 97 amino acids spanning RBD of RalGDS, pGEX4T3-GST-RalBD construct for GST-RLIP-RBD containing amino acids 397–518 of human RLIP76 and pGEX2T-RBD construct for GST-Raf1-RBD containing amino acids 51–131 of Raf-1 (8Okada S. Matsuda M. Anafi M. Pawson T. Pessin J.E. EMBO J. 1998; 17: 2554-2565Crossref PubMed Scopus (82) Google Scholar, 30de Rooij J. Bos J.L. Oncogene. 1997; 14: 623-625Crossref PubMed Scopus (420) Google Scholar, 31Wolthuis R.M. Franke B. van Triest M. Bauer B. Cool R.H. Camonis J.H. Akkerman J.W. Bos J.L. Mol. Cell. Biol. 1998; 18: 2486-2491Crossref PubMed Scopus (130) Google Scholar) were the generous gifts of Dr. Johannes L. Bos (Utrecht, Netherlands). The wild-type and dominant-negative Rap1A plasmids were kindly provided by Dr. Alfred Wittinghofer (Dortmund, Germany). The dominant-negative c-Src plasmid was obtained from Dr. Joan S. Brugge (Boston, MA). The dominant-negative JAK2 plasmid was a kind gift of Dr. Olli Silvennoinen (Tampere, Finland). The wild-type CrkII expression vector and the wild-type and mutant form of C3G were generously provided by Dr. Michiyuki Matsuda (Tokyo, Japan). The dominant-negative Ras plasmid was purchased from Upstate Biotechnology. The fusion trans-activator plasmid (pFA-Elk-1) consisting of the DNA binding domain of Gal4 (residues 1–147) and the transactivation domain of Elk-1 were purchased from Stratagene (La Jolla, CA). All plasmids were prepared with the plasmid maxiprep kit from Qiagen. Cell Culture and Treatment—NIH-3T3 cells were grown at 37 °C in 5% CO2 in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mm l-glutamine. Prior to treatment, cells were deprived for 20–24 h in medium containing 0.5% fetal bovine serum. Unless otherwise indicated, the concentration of hGH was 50 nm. This concentration of GH is within the physiological range for circulating rodent GH (32Gaur S. Yamaguchi H. Goodman H.M. Am. J. Physiol. 1996; 270: C1485-C1492Crossref PubMed Google Scholar). Rap, Ral, and Ras Activation Assays—Serum-deprived cells were stimulated with hGH as indicated and then lysed on ice for 15 min in Ral buffer (50 mm Tris-HCl, pH 7.5, 200 mm NaCl, 2.5 mm MgCl2, 10% glycerol, 1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 1 mm Na3VO4, and 1 tablet complete protease inhibitor mixture per 50 ml). After lysis samples were centrifuged at 14,000 × g at 4 °C for 10 min, and the protein concentrations of the lysates were measured. 500 μg of cell lysates were immediately affinity-precipitated at 4 °C for 1 h with 50 μg of GST-RalGDS-RBD or 15 μg of GST-RalBP1-RBD or GST-Raf1-RBD fusion proteins that had been freshly precoupled to glutathioneagarose beads. The precipitates were washed three times with Ral buffer, and the bound proteins were eluted in 20 μl of Laemmli sample buffer. Samples were separated by 12% polyacrylamide SDS-PAGE and detected by Western blot analysis. Immunoprecipitation—After treatment as indicated, cells were lysed at 4 °C for 20 min in 1% Nonidet P-40 lysis buffer (50 mm Tris-HCl, pH 7.4, 1% Nonidet P-40, 150 mm NaCl, 1 mm EDTA, 1 mm NaF, 1 mm phenylmethylsulfonyl fluoride, 1 mm Na3VO4, 1 tablet complete protease inhibitor mixture per 50 ml). Cell lysates were centrifuged at 14,000 × g for 15 min, and the supernatants were precleared by protein A/G plus agarose chromatography. The agarose beads were removed by centrifugation, and then the protein concentrations of the resulting supernatants were determined. For each immunoprecipitation, 500–1000 μg of protein was incubated with 4–8 μg of corresponding antibody for 4 h or overnight at 4 °C. Immunocomplexes were collected by incubating with 40 μl of protein A/G plus agarose for 1 h or overnight. Immunoprecipitates were washed three times with IP buffer (10 mm Tris-HCl, pH 7.4, 1% Triton X-100, 1% Nonidet P-40, 150 mm NaCl, 1 mm EDTA, pH 8.0, 1 mm EGTA, pH 8.0, 1 mm phenylmethylsulfonyl fluoride, 1 mm Na3VO4, 1 tablet complete protease inhibitor mixture per 50 ml). The bound proteins were eluted in Laemmli sample buffer and then resolved by 8–10% SDS-PAGE. Western Blot Analysis—After SDS-PAGE, proteins were transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk in phosphate-buffered saline with 0.1% Tween-20 (PBST) for 1 h at 22 °C. Blots were then immunolabeled with the desired antibodies for 1 h at 22 °C. For reblotting, membranes were stripped at 50 °C for 30 min in the solution containing 62.5 mm Tris-HCl, pH 6.7, 2% SDS, and 0.7% mercaptoethanol. Blots were then washed for 30 min with several changes of PBST at 22 °C. Efficacy of stripping was determined by re-exposure of the membranes to ECL. Thereafter, membranes were reblocked and immunolabeled as described above. p44/42 MAP Kinase Assay—p44/42 MAPK kinase assays were performed according to the manufacturer's instructions. Briefly, cells were lysed at 4 °C in lysis buffer provided, and the cell extract containing 200 μg of protein per sample was incubated for 4 h or overnight with 15 μl of immobilized phosphospecific p44/42 MAP kinase (Tyr-202/Tyr-204) monoclonal antibody. The pellets were washed twice with 500 μl of lysis buffer and twice with 500 μl of kinase assay buffer provided. The kinase reactions were performed in the presence of 2 μg of Elk-1 fusion protein and 200 μm ATP at 30 °C for 30 min. Elk-1 phosphorylation was detected by use of a specific phospho-Elk1 (Ser-383) antibody. SAPK/JNK Assay—SAPK/JNK assays were performed according to the manufacturer's instructions. Briefly, cells were lysed at 4 °C in lysis buffer provided and the cell extract containing 250 μg of total protein per sample was incubated overnight at 4 °C with 20 μl of c-Jun fusion protein beads. The pellets were washed twice with 500 μl of lysis buffer and twice with 500 μl of kinase assay buffer provided. The kinase assay was performed in the presence of 100 μm ATP at 30 °C for 30 min. c-Jun phosphorylation was detected by use of a specific phospho-c-Jun (Ser-63) antibody. Elk-1 Reporter Assay—Cells were cultured to 60–80% confluence in 6-well plates and transfected with 0.4 μg of the reporter plasmid pFR-Luc, 8 ng of the fusion trans-activator plasmid pFA-Elk1, and 1 μg of the expression plasmid as indicated or empty vector in each well. 25 μl of Effectene was used for each microgram of DNA according to the manufacturer's instructions. DNA-lipid complex was diluted in medium containing 2% fetal bovine serum for 16–20 h. 50 nm hGH was added for an additional 24 h. The cells were washed in cold phosphate-buffered saline twice and then lysed with 150 μl of 1× lysis buffer (25 mm Tris-phosphate, pH 7.8, 2 mm EDTA, 2 mm EDTA, 2 mm dithiothreitol, 10% glycerol, 1% Triton X-100) for 20 min, and supernatant was collected by centrifugation at 14,000 × g for 15 min. The luciferase activity was detected and normalized by protein content. c-Jun Reporter Assay—Cells were cultured to 60–80% confluence in 6-well plates and transfected with 0.2 μg of the reporter plasmid pFR-Luc, 4 ng of the fusion trans-activator plasmid pFA-cJun, and 1 μg of the expression plasmid as indicated or empty vector in each well. 25 μl of Effectene was used for each microgram of DNA according to the manufacturer's instructions. DNA-lipid complex was diluted in medium containing 5% fetal bovine serum for 12 h. 50 nm hGH was added for an additional 18 h. The cells were washed in cold phosphate-buffered saline twice and then lysed with 150 μl of 1× lysis buffer (25 mm Tris-phosphate, pH 7.8, 2 mm EDTA, 2 mm EDTA, 2 mm dithiothreitol, 10% glycerol, 1% Triton X-100) for 20 min, and supernatant was collected by centrifugation at 14,000 × g for 15 min. The luciferase activity was detected and normalized by protein content. Statistical Analysis and Presentation of Data—All experiments were performed at least three times. Numerical data were expressed as mean ± S.D. Data were analyzed using the two-tailed t test or analysis of variance. GH Stimulation of NIH-3T3 Cells Increases the Level of GTP-bound Rap1 and Rap2—We utilized the GST fused Ral-GDS-RBD (33Franke B. Akkerman J.W. Bos J.L. EMBO J. 1997; 16: 252-259Crossref PubMed Scopus (367) Google Scholar) as a specific probe to determine Rap1 and Rap2 activation in lysates of NIH-3T3 cells stimulated by GH. The GST-fused Ral-GDS-RBD protein recognizes only the active GTP-bound form of Rap1 and Rap2 but not the inactive GDP-bound form of these molecules (33Franke B. Akkerman J.W. Bos J.L. EMBO J. 1997; 16: 252-259Crossref PubMed Scopus (367) Google Scholar). We observed an increased level of GTP-bound Rap1 upon cellular stimulation with GH, first at 2 min after GH addition, sustained to 15 min, and followed by a decline from 30 to 60 min (Fig. 1A). GH stimulation of NIH-3T3 cells also resulted in the formation of GTP-bound Rap2 within 2 min. However, GH-stimulated formation of GTP-bound Rap2 was not sustained, as observed for Rap1, and the level of GTP-bound Rap2 decreased immediately after 5 min (Fig. 1C). GH stimulation of NIH-3T3 cells did not alter Rap1 or Rap2 protein levels over the examined time period of stimulation (Fig. 1, B and D). The GH-stimulated formation of Rap1-GTP and Rap2-GTP was also dose-dependent with the enhancement of Rap1-GTP and Rap2-GTP levels first observed at 0.5 nm GH. The maximal stimulation of Rap1-GTP was from 5 to 50 nm GH (Fig. 1E) while that of Rap2-GTP was at 5 nm GH (Fig. 1G). Thus Rap1 and Rap2 are two signaling molecules utilized by GH to exert its effect on cellular function. GH-stimulated Activation of Rap1 and Rap2 Are Cell Density-dependent—During the course of experimentation, an effect of cell density in monolayer culture on the ability of GH to stimulate Rap1 and Rap2 activity was noticed. We therefore compared Rap activity under conditions of increasing cell density that approximated 40, 70, and 100% cell confluence, respectively. A decrease of both basal and GH-stimulated Rap1 and Rap2 activity was observed with increasing cell density. Thus, GH stimulation of NIH-3T3 cells at 100% confluence failed to stimulate the formation of GTP-bound Rap1 and only minimal activation of Rap2 by GH under these conditions was observed (Fig. 2, A and C). This effect of cell density on the ability of GH to stimulate the formation of Rap1-GTP and Rap2-GTP was not due to decreased Rap protein as the total cellular level of both Rap1 and Rap2 was equivalent at different cell densities (Fig. 2, B and D). GH activation of Rap1 and Rap2 was therefore cell density-dependent. Full Activation of Rap1 and Rap2 by GH Requires both JAK2 and c-Src—GH activates both JAK2 and c-Src kinases independent of the other (29Zhu T. Ling L. Lobie P.E. J. Biol. Chem. 2002; 277: 45592-45603Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). We have previously demonstrated that two other small Ras-like GTPases, RalA and RalB, require the activity of both c-Src and JAK2 to be fully activated by GH (29Zhu T. Ling L. Lobie P.E. J. Biol. Chem. 2002; 277: 45592-45603Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). We therefore next examined the requirement of JAK2 and c-Src for GH-stimulated activation of Rap1 and Rap2. Upon forced expression of the JAK2 kinase-deficient mutant (K882E) (34Rui L. Gunter D.R. Herrington J. Carter-Su C. Mol. Cell. Biol. 2000; 20: 3168-3177Crossref PubMed Scopus (51) Google Scholar), both the basal and GH-stimulated formation of Rap1-GTP and Rap2-GTP were diminished, but GH stimulation of cells still resulted in increased Rap1 and Rap2 activity (Fig. 3, A and C). We have previously demonstrated the efficacy of forced expression of JAK2-K882E to prevent GH-stimulated activation of JAK2- and JAK2-dependent signal transduction (29Zhu T. Ling L. Lobie P.E. J. Biol. Chem. 2002; 277: 45592-45603Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Forced expression of the c-Src kinase inactive mutant (K295R/Y527F) (35Rahimi N. Hung W. Tremblay E. Saulnier R. Elliott B. J. Biol. Chem. 1998; 273: 33714-33721Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) also abrogated the ability of GH to stimulate the formation of GTP bound Rap1 and Rap2 (Fig. 3, A and C) to a greater extent than that observed with JAK2-K882E. Co-transfection of cDNA for both JAK2-K882E and c-Src-K295R/Y527F completely prevented the ability of GH to stimulate the formation of GTP-bound Rap1 and Rap2 (Fig. 3, A and C). Forced expression of either JAK2-K882E or c-Src-K295R/Y527F or both did not alter the total cellular level of Rap1 or Rap2 (Fig. 3, B and D). Forced expression of the kinase-deficient JAK2 and c-Src mutants were verified by Western blot analysis (Fig. 3, E and F). Therefore, we conclude that although the combined activities of both JAK2 and c-Src kinases are required for full activation of Rap1 and Rap2 by GH, c-Src is predominantly utilized by GH to activate these molecules. Overexpression of CrkII and C3G Enhances GH-stimulated Rap1 and Rap2 Activity—We have previously demonstrated that GH stimulates the formation of a multiprotein signaling complex centered around CrkII (21Zhu T. Goh E.L. LeRoith D. Lobie P.E. J. Biol. Chem. 1998; 273: 33864-33875Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). CrkII and C3G are constitutively associated within this complex although hGH stimulation of cells results in tyrosine phosphorylation of CrkII (21Zhu T. Goh E.L. LeRoith D. Lobie P.E. J. Biol. Chem. 1998; 273: 33864-33875Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). CrkII is an adaptor protein and has been reported to recruit C3G to the vicinity of kinase molecules (17Ichiba T. Hashimoto Y. Nakaya M. Kuraishi Y. Tanaka S. Kurata T. Mochizuki N. Matsuda M. J. Biol. Chem. 1999; 274: 14376-14381Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). C3G is a Rap-specific GEF that accelerates the replacement of GDP by GTP so as to increase Rap activity (11Gotoh T. Hattori S. Nakamura S. Kitayama H. Noda M. Takai Y. Kaibuchi K. Matsui H. Hatase O. Takahashi H. Kurata T. Matsuda M. Mol. Cell. Biol. 1995; 15: 6746-6753Crossref PubMed Scopus (336) Google Scholar). We therefore proceeded to examine the involvement of CrkII and C3G in the GH stimulated formation of GTP-bound Rap1 and Rap2. Forced expression of CrkII enhanced the ability of GH to activate both Rap1 and Rap2 (Fig. 4, A and C). Forced expression of C3G, or CrkII together with C3G, resulted in a dramatic enhancement of GH-stimulated formation of GTP-bound Rap1 and Rap2 (Fig. 4, A and C). Forced expression of CrkII and C3G was verified by Western blot analysis (Fig. 4, E and F) and expression of these proteins did not alter the total cellular level of either Rap1 or Rap2 (Fig. 4, B and D). Thus, GH activation of Rap1 and Rap2 is CrkII-C3G-dependent. C3G Tyrosine Phosphorylation Is Required for GH-stimulated Rap1 and Rap2 Activation—Tyrosine phosphorylation of C3G has been reported to be required for the GEF activity necessary for Rap1 activation (17Ichiba T. Hashimoto Y. Nakaya M. Kuraishi Y. Tanaka S. Kurata T. Mochizuki N. Matsuda M. J. Biol. Chem. 1999; 274: 14376-14381Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). We therefore first examined whether GH stimulation of NIH-3T3 cells resulted in tyrosine phosphorylation of C3G. As observed in Fig. 5A, GH indeed stimulated the tyrosine phosphorylation of C3G. GH-stimulated tyrosine phosphorylation of C3G was first observed at 1 min, persisted to 15 min, and then declined at 30–60 min after stimulation with GH. Equivalent loading of immunoprecipitated C3G was demonstrated by reprobing of the membrane for C3G (Fig. 5B). To determine the kinases responsible for hGH-stimulated tyrosine phosphorylation of C3G we utilized the kinase-deficient mutants of both JAK2 (JAK2-K882E) and c-Src (c-Src-K295R/Y527F). Similar to the pattern observed with GH-stimulated formation of GTP-bound Rap1 and Rap2 (above), removal of the activities of both kinases was required for complete prevention of GH-stimulated C3G tyrosine phosphorylation (Fig. 5C). Equivalent loading of immunoprecipitated C3G was demonstrated by reprobing of the membrane for C3G (Fig. 5D). Forced expression of the kinase-deficient mutants of both JAK2 and c-Src was demonstrated by Western blot analysis (Fig. 5, E and F). Thus GH-stimulated tyrosine phosphorylation of C3G required the combined activities of both JAK2 and c-Src. We next examined whether tyrosine phosphorylation of C3G was required for GH stimulated Rap1 and Rap2 activation. It has been reported that tyrosine 504 of C3G is the critical tyrosine residue required for guanine nucleotide exchange activity for Rap1 (17Ichiba T. Hashimoto Y. Nakaya M. Kuraishi Y. Tanaka S. Kurata T. Mochizuki N. Matsuda M. J. Biol. Chem. 1999; 274: 14376-14381Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). To determine if GH-stimulated C3G tyrosine phosphorylation was required for" @default.
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• W2060859699 title "Src-CrkII-C3G-dependent Activation of Rap1 Switches Growth Hormone-stimulated p44/42 MAP Kinase and JNK/SAPK Activities" @default.
• W2060859699 cites W1534981615 @default.
• W2060859699 cites W1579739997 @default.
• W2060859699 cites W164957562 @default.
• W2060859699 cites W1667204010 @default.
• W2060859699 cites W1780670592 @default.
• W2060859699 cites W1963659433 @default.
• W2060859699 cites W1965571070 @default.
• W2060859699 cites W1973755084 @default.
• W2060859699 cites W1977302628 @default.
• W2060859699 cites W1985922043 @default.
• W2060859699 cites W1987184179 @default.
• W2060859699 cites W1989839956 @default.
• W2060859699 cites W1989987282 @default.
• W2060859699 cites W1992975639 @default.
• W2060859699 cites W1995363965 @default.
• W2060859699 cites W1995654085 @default.
• W2060859699 cites W1997878751 @default.
• W2060859699 cites W1998873842 @default.
• W2060859699 cites W1999754601 @default.
• W2060859699 cites W2001230322 @default.
• W2060859699 cites W2001617515 @default.
• W2060859699 cites W2004126223 @default.
• W2060859699 cites W2005082414 @default.
• W2060859699 cites W2012711566 @default.
• W2060859699 cites W2012806962 @default.
• W2060859699 cites W2013381560 @default.
• W2060859699 cites W2015535533 @default.
• W2060859699 cites W2016955301 @default.
• W2060859699 cites W2021296418 @default.
• W2060859699 cites W2025381000 @default.
• W2060859699 cites W2027277506 @default.
• W2060859699 cites W2029462030 @default.
• W2060859699 cites W2030986942 @default.
• W2060859699 cites W2031330547 @default.
• W2060859699 cites W2032229470 @default.
• W2060859699 cites W2034900375 @default.
• W2060859699 cites W2040989466 @default.
• W2060859699 cites W2041578282 @default.
• W2060859699 cites W2042260090 @default.
• W2060859699 cites W2044052218 @default.
• W2060859699 cites W2052051648 @default.
• W2060859699 cites W2052222199 @default.
• W2060859699 cites W2055737181 @default.
• W2060859699 cites W2056031476 @default.
• W2060859699 cites W2060141022 @default.
• W2060859699 cites W2060878329 @default.
• W2060859699 cites W2061013276 @default.
• W2060859699 cites W2066254967 @default.
• W2060859699 cites W2068994296 @default.
• W2060859699 cites W2069556423 @default.
• W2060859699 cites W2070564840 @default.
• W2060859699 cites W2073790149 @default.
• W2060859699 cites W2076261991 @default.
• W2060859699 cites W2076897294 @default.
• W2060859699 cites W2078757969 @default.
• W2060859699 cites W2083063047 @default.
• W2060859699 cites W2099290597 @default.
• W2060859699 cites W2107796077 @default.
• W2060859699 cites W2111072594 @default.
• W2060859699 cites W2112294647 @default.
• W2060859699 cites W2118399632 @default.
• W2060859699 cites W2132948080 @default.
• W2060859699 cites W2134537331 @default.
• W2060859699 cites W2141263471 @default.
• W2060859699 cites W2156875265 @default.
• W2060859699 cites W2159972238 @default.
• W2060859699 cites W2162876383 @default.
• W2060859699 cites W2169932670 @default.
• W2060859699 cites W2313522156 @default.
• W2060859699 cites W2329515361 @default.
• W2060859699 cites W2416639895 @default.
• W2060859699 cites W4243003607 @default.
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