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Effect of Phosphorylation on Activities of Rap1A to Interact with Raf-1 and to Suppress Ras-dependent Raf-1 Activation

1999; Elsevier BV; Volume: 274; Issue: 1 Linguagem: Inglês

10.1074/jbc.274.1.48

ISSN

1083-351X

Autores

Chang-Deng Hu, Ken-ichi Kariya, Tomoyo Okada, Xiaodong Qi, Chunhua Song, Tohru Kataoka,

Tópico(s)

PI3K/AKT/mTOR signaling in cancer

Resumo

Rap1A is phosphorylated by cAMP-dependent protein kinase (PKA), and this phosphorylation has been shown to modulate its interaction with other proteins. However, it is not known whether Rap1A phosphorylation is involved in regulation of its cellular functions, including suppression of Ras-dependent Raf-1 activation. We have previously shown that this suppressive activity of Rap1A is attributable to its greatly enhanced ability to bind to the cysteine-rich region (CRR, residues 152–184) of Raf-1 compared with that of Ras. Here, we show that phosphorylation of Rap1A by PKA abolished its binding activity to CRR. Furthermore, a mutant Rap1A(S180E), whose sole PKA phosphorylation residue, Ser-180, was substituted by an acidic residue, Glu, to mimic its phosphorylated form, failed to suppress Ras-dependent Raf-1 activation in COS-7 cells. These results indicate that the CRR binding activity and the Ras-suppressive function of Rap1A can be modulated through phosphorylation and suggest that Rap1A may function as a PKA-dependent regulator of Raf-1 activation, not merely as a suppressor. Rap1A is phosphorylated by cAMP-dependent protein kinase (PKA), and this phosphorylation has been shown to modulate its interaction with other proteins. However, it is not known whether Rap1A phosphorylation is involved in regulation of its cellular functions, including suppression of Ras-dependent Raf-1 activation. We have previously shown that this suppressive activity of Rap1A is attributable to its greatly enhanced ability to bind to the cysteine-rich region (CRR, residues 152–184) of Raf-1 compared with that of Ras. Here, we show that phosphorylation of Rap1A by PKA abolished its binding activity to CRR. Furthermore, a mutant Rap1A(S180E), whose sole PKA phosphorylation residue, Ser-180, was substituted by an acidic residue, Glu, to mimic its phosphorylated form, failed to suppress Ras-dependent Raf-1 activation in COS-7 cells. These results indicate that the CRR binding activity and the Ras-suppressive function of Rap1A can be modulated through phosphorylation and suggest that Rap1A may function as a PKA-dependent regulator of Raf-1 activation, not merely as a suppressor. protein kinase A (cAMP-dependent protein kinase) Ral guanine nucleotide dissociation stimulator Ras binding domain cysteine-rich region maltose-binding protein glutathioneS-transferase a kinase negative mutant of extracellular signal-regulated kinase 2 polyacrylamide gel electrophoresis guanosine 5′-O-(3-thiotriphosphate). Rap1A belongs to the Ras family of small GTP-binding proteins. Accumulated evidences indicate that Rap1A is phosphorylated at its C-terminal Ser-180 both in vitro and in vivo by PKA1 (for reviews, see Refs.1Bokoch G.M. Biochem. J. 1993; 289: 17-24Crossref PubMed Scopus (83) Google Scholar and 2Noda M. Biochim. Biophys. Acta. 1993; 1155: 97-109PubMed Google Scholar). Phosphorylation of Rap1A induced by cAMP-elevating agents is observed in a number of cell types including Rat-1 cells (3Lerosey I. Pizon V. Tavitian A. Gunzburg J.D. Biochem. Biophys. Res. Commun. 1991; 175: 430-436Crossref PubMed Scopus (47) Google Scholar), HL-60 cells (4Quilliam L.A. Mueller H. Bohl B.P. Prossnitz V. Sklar L.A. Der C.J. Bokoch G.H. J. Immunol. 1991; 147: 1628-1635PubMed Google Scholar), PC12 cells (5Vossler M.R. Yao H. York R.D. Pan M.-G. Rim C.S. Stork P.J.S. Cell. 1997; 89: 73-82Abstract Full Text Full Text PDF PubMed Scopus (944) Google Scholar), and neutrophils (6Quinn M.T. ParKos C.A. Walker L. Orkin S.H. Dinauer M.C. Jesaitis A.J. Nature. 1989; 342: 198-200Crossref PubMed Scopus (181) Google Scholar, 7Bokoch G.M. Quilliam L.A. Bohl B.P. Jesaitis A.J. Quinn M.T. Science. 1991; 254: 1794-1796Crossref PubMed Scopus (98) Google Scholar). The observation in the neutrophils that the association of Rap1A with cytochromeb is dramatically reduced by phosphorylation (7Bokoch G.M. Quilliam L.A. Bohl B.P. Jesaitis A.J. Quinn M.T. Science. 1991; 254: 1794-1796Crossref PubMed Scopus (98) Google Scholar) suggests a regulatory role of Rap1A phosphorylation in its interaction with other proteins. However, there has been no report demonstrating the effect of phosphorylation on cellular functions of Rap1A. Rap1A has a very high homology to Ras and associates with almost all cellular effectors of Ras including Raf-1 (8Nassar N. Horn G. Herrmann C. Scherer A. McCormick F. Wittinghofer A. Nature. 1995; 375: 554-560Crossref PubMed Scopus (558) Google Scholar), B-Raf (5Vossler M.R. Yao H. York R.D. Pan M.-G. Rim C.S. Stork P.J.S. Cell. 1997; 89: 73-82Abstract Full Text Full Text PDF PubMed Scopus (944) Google Scholar), and RalGDS (9Albright C.F. Giddings B.W. Liu J. Vito M. Weinberg R.A. EMBO J. 1993; 12: 339-347Crossref PubMed Scopus (159) Google Scholar, 10Hofer F. Fields S. Schneider C. Martin G.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11089-11093Crossref PubMed Scopus (250) Google Scholar, 11Kikuchi A. Demo S.D. Ye Z.-H. Chen Y.-W. Williams L.T. Mol. Cell. Biol. 1994; 14: 7483-7491Crossref PubMed Scopus (244) Google Scholar) in mammalian cells as well as Caenorhabditis elegansPLC210 (12Shibatohge M. Kariya K. Liao Y. Hu C.-D. Watari Y. Goshima M. Shima F. Kataoka T. J. Biol. Chem. 1998; 273: 6218-6222Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Raf-1 is a serine/threonine protein kinase regulating the mitogen-activated protein kinase cascade (for a review, see Ref. 13Daum G. Eisenmann-Tappe I. Fries H.-W. Troppmair J. Rapp U.R. Trends Biochem. Sci. 1994; 19: 474-480Abstract Full Text PDF PubMed Scopus (485) Google Scholar). Ras activates Raf-1 by physically associating with it at the plasma membrane. This physical association is mediated mainly by the interaction between RBD (residues 51–131) of Raf-1 and the effector region of Ras (residues 32–40 of Ha-Ras) (13Daum G. Eisenmann-Tappe I. Fries H.-W. Troppmair J. Rapp U.R. Trends Biochem. Sci. 1994; 19: 474-480Abstract Full Text PDF PubMed Scopus (485) Google Scholar). In addition to RBD, we and others have recently found that CRR (residues 152–184) of Raf-1 also interacts with Ras and shown that this novel interaction is essential for activation of Raf-1 (Refs. 14Hu C.-D. Kariya K. Tamada M. Akasaka K. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1995; 270: 30274-30277Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 15Ghosh S. Xie W.Q. Quest A.F.G. Mabrouk G.M. Strum J.C. Bell R.M. J. Biol. Chem. 1994; 269: 10000-10007Abstract Full Text PDF PubMed Google Scholar, 16Ghosh S. Bell R.M. J. Biol. Chem. 1994; 269: 30785-30788Abstract Full Text PDF PubMed Google Scholar, 17Brtva T.R. Drugan J.K. Ghosh S. Terrell R.S. Campbell-Burk S. Bell R.M. Der C.J. J. Biol. Chem. 1995; 270: 9809-9812Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 18Luo Z. Dlaz B. Marshall M.S. Avruch J. Mol. Cell. Biol. 1997; 17: 46-53Crossref PubMed Scopus (106) Google Scholar, 19Roy S. Lane A. Yan J. McPherson R. Hancock J.F. J. Biol. Chem. 1997; 272: 20139-20145Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar; for a review, see Ref.20Morrison D.K. Cutler R.E. Curr. Opin. Cell Biol. 1997; 9: 174-179Crossref PubMed Scopus (534) Google Scholar). In contrast to Ras, Rap1A cannot activate Raf-1. Instead, Rap1A inhibits Ras-dependent Raf-1 activation. Because Rap1A has the identical effector region to that of Ras and associates well with RBD (8Nassar N. Horn G. Herrmann C. Scherer A. McCormick F. Wittinghofer A. Nature. 1995; 375: 554-560Crossref PubMed Scopus (558) Google Scholar), we speculated that it would have a defect in association with CRR. However, to our surprise, we found that Rap1A has a greatly enhanced ability to associate with CRR compared with Ras (21Hu C.-D. Kariya K. Kotani G. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1997; 272: 11702-11705Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Further, the enhanced CRR binding property of Rap1A resulted in formation of a ternary complex with Ras and Raf-1 in which Rap1A and Ras independently associate with CRR and RBD of Raf-1, respectively. Raf-1 in this complex cannot be activated by Ras, presumably because interaction of CRR with Ras is hampered by Rap1A tightly bound to CRR. Association with CRR seems to involve the C-terminal region of Rap1A, because it was shown that the association was dependent on the C-terminal posttranslational modification of Rap1A (21Hu C.-D. Kariya K. Kotani G. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1997; 272: 11702-11705Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). We therefore reasoned that phosphorylation of Rap1A at its C-terminal Ser-180 might affect its ability to associate with CRR. Here we report that the phosphorylation specifically abolished the activity of Rap1A to bind to CRR. Further, a Rap1A mutant mimicking the phosphorylated form was found to have lost the activity to suppress Ras-dependent Raf-1 activation. MBP-Raf-1(51–131), MBP-Raf-1(132–206), and MBP-Raf-1(48–206) are MBP fusion proteins containing the indicated residues of human Raf-1 described before (14Hu C.-D. Kariya K. Tamada M. Akasaka K. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1995; 270: 30274-30277Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). MBP-PLC210 is an MBP fusion protein containing the Ras-associating domain (residues 1570–1670) of PLC210 (12Shibatohge M. Kariya K. Liao Y. Hu C.-D. Watari Y. Goshima M. Shima F. Kataoka T. J. Biol. Chem. 1998; 273: 6218-6222Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). MBP-RalGDS is an MBP fusion protein containing the Ras-interacting domain (residues 724–852) of rat RalGDSb (9Albright C.F. Giddings B.W. Liu J. Vito M. Weinberg R.A. EMBO J. 1993; 12: 339-347Crossref PubMed Scopus (159) Google Scholar, 10Hofer F. Fields S. Schneider C. Martin G.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11089-11093Crossref PubMed Scopus (250) Google Scholar, 11Kikuchi A. Demo S.D. Ye Z.-H. Chen Y.-W. Williams L.T. Mol. Cell. Biol. 1994; 14: 7483-7491Crossref PubMed Scopus (244) Google Scholar). The MBP fusion proteins were expressed inEscherichia coli harboring pMal vectors carrying the corresponding cDNA fragments. The E. coli cells were lysed and centrifuged at 100,000 × g for 30 min. The MBP fusion proteins in the resulting supernatant fractions were immobilized on amylose resin and used for the in vitrobinding assays as described below. cDNAs encoding Rap1AV12(S180E) and Rap1AV12(S180A), carrying a substitution of Glu or Ala, respectively, for Ser at residue 180 in addition to an activating mutation of Gly to Val at residue 12, were prepared by polymerase chain reaction using oligonucleotide primers carrying the corresponding mutations and the Rap1AV12cDNA as a template (21Hu C.-D. Kariya K. Kotani G. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1997; 272: 11702-11705Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 22Saiki R.K. Scharf S. Faloona F. Mullis K.B. Horn G.T. Erlich H.A. Arnheim N. Science. 1985; 230: 1350-1354Crossref PubMed Scopus (6750) Google Scholar). Cloning of the mutant Rap1A cDNAs into a baculovirus transfer vector and preparation of the recombinant baculoviruses expressing them were carried out as described before (23Kuroda Y. Suzuki N. Kataoka T. Science. 1993; 259: 683-686Crossref PubMed Scopus (119) Google Scholar). Procedures for purification of the posttranslationally modified forms of Ras and Rap1A from Sf9 cells infected with baculoviruses expressing the respective proteins were described before (21Hu C.-D. Kariya K. Kotani G. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1997; 272: 11702-11705Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Expression of a GST fusion Ha-Ras (GST-Ha-Ras) in Sf9 cells using a baculovirus vector was described before (21Hu C.-D. Kariya K. Kotani G. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1997; 272: 11702-11705Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). The in vitro binding reactions were carried out by incubating 20–30 μl of amylose resin carrying various immobilized MBP fusion proteins with GTPγS- or GDP-loaded Rap1A in a total volume of 100 μl of buffer A (20 mm Tris/HCl, pH 7.4, 40 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, 5 mmMgCl2, and 0.1% Lubrol PX) as described (14Hu C.-D. Kariya K. Tamada M. Akasaka K. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1995; 270: 30274-30277Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 21Hu C.-D. Kariya K. Kotani G. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1997; 272: 11702-11705Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). After incubation at 4 °C for 2 h, the resin was washed, and the bound proteins were eluted with buffer A containing 10 mm maltose and subjected to SDS-PAGE followed by Western immunoblot detection with anti-Rap1A polyclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, California). The ECL system (Amersham Pharmacia Biotech) was used for signal development. For examination of the ternary complex formation, GST-Ha-Ras in Sf9 cell lysate was first immobilized on glutathione-Sepharose resin and then loaded with GTPγS. The resin was subsequently incubated with GTPγS-loaded Rap1A in the presence of MBP-Raf-1(48–206). The experimental condition was the same as described above, except that the bound proteins were eluted with 10 mm glutathione in buffer A. The eluate was probed with anti-Ras monoclonal antibody Y13–259 (Oncogene Science Inc., Manhasset, New York), anti-MBP polyclonal antibody, or the anti-Rap1A antibody. For examination of the effect of phosphorylation of Rap1A, 5–10 pmol of Rap1A were incubated with 5–10 units of recombinant PKA catalytic subunit (Promega, Madison, Wisconsin) at 25 °C for 30 min. After termination of the phosphorylation reaction by the addition of 0.1–0.2 μg of a synthetic peptide (TTYADFIASGRTGRRNAIHD) corresponding to the active site of the rabbit PKA inhibitor (24Cheng H.-C. Kemp B.E. Pearson R.B. Smith A.J. Misconi L. Van Patten S.M. Walsh D.A. J. Biol. Chem. 1986; 261: 989-992Abstract Full Text PDF PubMed Google Scholar) (Sigma), the phosphorylated Rap1A was used for the assays described above. COS-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics. For the activation of Raf-1 by Ras or Rap1A, cells in 60-mm dishes (50% confluency) were cotransfected with a combination of a Raf-1 expression vector pH8-Raf-1 (25Tamada M. Hu C.-D. Kariya K. Okada T. Kataoka T. Oncogene. 1997; 15: 2959-2964Crossref PubMed Scopus (34) Google Scholar) and either one of pcDNA3.1-Ha-RasV12, pSRα-Rap1AV12, pSRα-Rap1AV12(S180E), or pSRα-Rap1AV12(S180A) by using SuperFect Transfection Reagent (Qiagen GmbH, Germany). For examination of suppression of the Ras-dependent Raf-1 activation by Rap1A or its mutants, cells were cotransfected with a combination of pcDNA3.1-Ha-RasV12, pH8-Raf-1, and either one of pSRα-Rap1AV12, pSRα-Rap1AV12(S180E), or pSRα-Rap1AV12(S180A). Twenty-four h after transfection, cells were transferred to serum-free media and further cultured for 18 h. The membrane extract in 0.4 ml of the lysis buffer (20 mm Tris/HCl, pH 7.5, 137 mm NaCl, 0.5% Nonidet P-40, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, 20 mm β-glycerophosphate, and 1 mm sodium vanadate) was prepared from each dish as described (25Tamada M. Hu C.-D. Kariya K. Okada T. Kataoka T. Oncogene. 1997; 15: 2959-2964Crossref PubMed Scopus (34) Google Scholar). One-half of the total membrane extract was subjected to immunoprecipitation with the anti-Raf-1 antibody and protein A-agarose. The Raf-1 kinase activity was determined by incubating the immunoprecipitates in the presence of GST-mitogen-activated protein kinase kinase (0.3 μg) and GST-KNERK2 (2 μg) in 30 μl of the kinase reaction mixture {20 mm Tris/HCl, pH 7.5, 10 mm MnCl2, 10 mm MgCl2, 20 mm β-glycerophosphate, and 50 μm[γ-32P]ATP (4,000 cpm/pmol)} for 20 min at 25 °C (25Tamada M. Hu C.-D. Kariya K. Okada T. Kataoka T. Oncogene. 1997; 15: 2959-2964Crossref PubMed Scopus (34) Google Scholar). After the incubation, proteins in the reaction mixture were fractionated by SDS-PAGE. Phosphorylated proteins were either visualized by autoradiograph or quantitated by using BAS2000 bioimaging analyzer (Fujix, Tokyo, Japan). In parallel, a 20-μl aliquot of the membrane extract was used for examination of the amounts of the expressed proteins by Western immunoblotting with the anti-Ras, anti-Rap1A, and anti-Raf-1 antibodies. Signals from the corresponding endogenous proteins were negligible (data not shown). In the previous studies (14Hu C.-D. Kariya K. Tamada M. Akasaka K. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1995; 270: 30274-30277Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 21Hu C.-D. Kariya K. Kotani G. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1997; 272: 11702-11705Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), we found that both Ha-Ras and Rap1A in posttranslationally modified form boundin vitro to MBP-Raf-1(132–206) containing CRR in a GTP-independent manner. This was in contrast to their GTP-dependent binding to MBP-Raf-1(51–131) containing RBD. In the present study, we tested the effect of phosphorylation of Rap1A on its binding activities toward RBD and CRR in the same in vitro binding assay. First, we incubated Rap1A with a recombinant catalytic subunit of PKA and confirmed its quantitative phosphorylation by observing its mobility shift on SDS-PAGE as reported (4Quilliam L.A. Mueller H. Bohl B.P. Prossnitz V. Sklar L.A. Der C.J. Bokoch G.H. J. Immunol. 1991; 147: 1628-1635PubMed Google Scholar) (Fig.1 A). After inactivation of PKA by the protein kinase inhibitor peptide, Rap1A was loaded with GTPγS and incubated with MBP-Raf-1(132–206). As shown in Fig. 1 B, the phosphorylated Rap1A exhibited no detectable activity to bind to CRR in a sharp contrast to the unphosphorylated Rap1A. The same result was obtained with the phosphorylated Rap1A loaded with GDP (data not shown). In contrast, when tested with MBP-Raf-1(51–131), the unphosphorylated and the phosphorylated Rap1A did not exhibit any significant difference in their RBD binding activities (Fig.1 C). GTP dependence of the RBD binding was unaffected also. These results indicate that the phosphorylation of Rap1A specifically impaired the Rap1A-CRR association. In addition to Raf-1, we examined the effect of Rap1A phosphorylation on interaction with other Ras effectors, RalGDS (9Albright C.F. Giddings B.W. Liu J. Vito M. Weinberg R.A. EMBO J. 1993; 12: 339-347Crossref PubMed Scopus (159) Google Scholar, 10Hofer F. Fields S. Schneider C. Martin G.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11089-11093Crossref PubMed Scopus (250) Google Scholar, 11Kikuchi A. Demo S.D. Ye Z.-H. Chen Y.-W. Williams L.T. Mol. Cell. Biol. 1994; 14: 7483-7491Crossref PubMed Scopus (244) Google Scholar) and PLC210 (12Shibatohge M. Kariya K. Liao Y. Hu C.-D. Watari Y. Goshima M. Shima F. Kataoka T. J. Biol. Chem. 1998; 273: 6218-6222Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). As shown in Fig. 1, D and E, we observed GTP-dependent binding of Rap1A to both the Ras-interacting domain of rat RalGDSb and the Ras-associating domain of C. elegans PLC210, which were produced as MBP fusions. However, we did not observe any difference of binding between the phosphorylated and the unphosphorylated forms of Rap1A (Fig. 1, D andE). These results further indicate that the effect of Rap1A phosphorylation is specific to Raf-1 CRR. We next examined the effect of Rap1A phosphorylation on its ability to form a ternary complex with Ras and Raf-1. In this complex, Ras and Rap1A were independently associated with RBD and CRR, respectively. When Rap1A in the unphosphorylated form was incubated with GST-Ha-Ras immobilized on glutathione-Sepharose resin in the presence of MBP-Raf-1(48–206) containing both RBD and CRR, all three proteins were trapped on the resin and thereby co-eluted with 10 mm glutathione (Fig. 1 F, lane 1). In contrast, when the phosphorylated Rap1A was used in the same reaction in place of the unphosphorylated form, no Rap1A was detectable in the eluate even though a similar amount of MBP-Raf-1(48–206) was co-eluted with GST-Ha-Ras (Fig. 1 F,lane 2). These results indicate that the phosphorylation of Rap1A abrogates its ability to form a ternary complex with Ha-Ras and Raf-1. Accumulated evidences indicate that Ser-180 is a sole phosphorylation site of Rap1A by PKA examined both in vivo and in vitro (1Bokoch G.M. Biochem. J. 1993; 289: 17-24Crossref PubMed Scopus (83) Google Scholar, 2Noda M. Biochim. Biophys. Acta. 1993; 1155: 97-109PubMed Google Scholar). To prove that phosphorylation at Ser-180 was responsible for the observed effect of PKA treatment of Rap1A, we examined the effect of substitution of Ala for Ser-180. Rap1AV12(S180A) exhibited an activity to associate with Raf-1 CRR comparable with wild-type Rap1AV12 (Fig.2 A). In contrast to the case with wild-type Rap1AV12, no visible mobility shift was observed after treatment of this Rap1A mutant by PKA, suggesting that it was not phosphorylated by PKA (Fig. 2 B). Furthermore, the PKA treatment of Rap1AV12(S180A) did not affect its activity to bind to CRR (Fig. 2 C). This result indicates that Ser-180 is indeed the sole phosphorylation site responsible for the loss of the CRR binding activity of Rap1A. Because we previously found that the tight association of Rap1A with CRR is responsible for the Ras-suppressive activity of Rap1A, the above findings prompted us to test the effect of Rap1A phosphorylation on its cellular function, in particular suppression of Ras-dependent Raf-1 activation (1Bokoch G.M. Biochem. J. 1993; 289: 17-24Crossref PubMed Scopus (83) Google Scholar, 2Noda M. Biochim. Biophys. Acta. 1993; 1155: 97-109PubMed Google Scholar). For this purpose, we could not employ the conventional approach of stimulating cells with cAMP-elevating agents or overexpressing PKA catalytic subunit, because it has been shown that Raf-1 is phosphorylated by PKA and thereby rendered insensitive to activation by Ras (26Burgering B.M.T. Bos J.L. Trends Biochem. Sci. 1995; 20: 18-22Abstract Full Text PDF PubMed Scopus (290) Google Scholar, 27Mischak H. Seitz T. Janosch P. Eulitz M. Steen H. Schellerer M. Philipp A. Kolch W. Mol. Cell. Biol. 1996; 16: 5409-5418Crossref PubMed Scopus (178) Google Scholar). To circumvent this problem, we turned to test the feasibility of using a mutant Rap1AV12(S180E) in which the phosphorylation residue Ser-180 was replaced by an acidic residue Glu. This mutant is similar to the reported mutant Rap1A(S180D), which was shown to mimic the phosphorylated Rap1A (5Vossler M.R. Yao H. York R.D. Pan M.-G. Rim C.S. Stork P.J.S. Cell. 1997; 89: 73-82Abstract Full Text Full Text PDF PubMed Scopus (944) Google Scholar). Rap1AV12(S180E) was expressed in Sf9 cells, purified, and examined for its activity to associate with CRR. As shown in Fig. 2 A, the mutant exhibited no binding activity toward CRR, which is similar to the case with the phosphorylated Rap1A. This convinced us that Rap1AV12(S180E) can mimic the phosphorylated Rap1AV12. As expected, PKA treatment of this mutant did not alter its electrophoretic mobility (Fig. 2 B) or its CRR binding property (Fig. 2 C), further supporting that Ser-180 is the sole phosphorylation site. Then, we first examined the activities of the Rap1A mutants to stimulate the kinase activity of Raf-1 (Fig.3 A). Rap1AV12(S180E) as well as Rap1AV12(S180A) were found to be incapable of activating Raf-1 like wild-type Rap1AV12. This result further supports our hypothesis that association of Ras with CRR at an appropriate strength is required for Raf-1 activation. Abnormally enhanced or attenuated binding to CRR is detrimental to Raf-1 activation (14Hu C.-D. Kariya K. Tamada M. Akasaka K. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1995; 270: 30274-30277Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 21Hu C.-D. Kariya K. Kotani G. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1997; 272: 11702-11705Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Next, we examined the activities of these Rap1A mutants to antagonize the Ras-dependent Raf-1 activation. When Rap1AV12(S180E) was co-expressed in COS-7 cells with Raf-1 and Ha-RasV12, no significant suppression of the Ras-dependent Raf-1 activation was observed, in sharp contrast to nearly 70% suppression observed with wild-type Rap1AV12 (Fig. 3 B) even though they were expressed in a similar amount (Fig. 3 C). In contrast, Rap1AV12(S180A) was as active as wild-type Rap1AV12 in suppressing the Ras-dependent Raf-1 activation. Considering that Rap1AV12(S180E) had its CRR binding activity greatly attenuated and that Rap1AV12(S180A) retained the greatly enhanced CRR binding activity, these results are consistent with our hypothesis that the tight association of Rap1A with CRR, which results in blockade of Ras access to CRR, accounts for the Ras-suppressive activity of Rap1A (21Hu C.-D. Kariya K. Kotani G. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1997; 272: 11702-11705Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Further, the data obtained with Rap1AV12(S180E), taken together with the loss of CRR binding activity of the phosphorylated Rap1A, strongly suggest that the phosphorylation of Rap1A abrogates its activity to suppresses the Ras-dependent Raf-1 activation. Rap1A has been reported to associate with almost all Ras effectors and other proteins in cells. Although it is assumed that phosphorylation of Rap1A may be a physiological event, regulating its interaction with other proteins based on the observation that phosphorylated Rap1A does not associate with cytochrome b(7Bokoch G.M. Quilliam L.A. Bohl B.P. Jesaitis A.J. Quinn M.T. Science. 1991; 254: 1794-1796Crossref PubMed Scopus (98) Google Scholar), there has been no report that phosphorylation also affects the association of Rap1A with Ras effectors. In the present study, we have shown that the phosphorylated Rap1A lacks its ability to bind to CRR of Raf-1. This represents the second direct evidence that phosphorylation of Rap1A affects its interaction with other proteins. Consistent with our previous finding that CRR binding requires the C-terminal lipid modification of Rap1A (21Hu C.-D. Kariya K. Kotani G. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1997; 272: 11702-11705Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), this result provides further support to the idea that the C-terminal region of Rap1A is involved in CRR binding. Significantly, we have shown that the S180E mutant, mimicking the phosphorylated Rap1A, can no longer suppress Ras-dependent Raf-1 activation. This result not only agrees well with our hypothesis that the enhanced CRR binding is involved in suppression of Ras-dependent Raf-1 activation by Rap1A but also, to our knowledge, represents the first clear demonstration that the cellular function of Rap1A is controlled by phosphorylation. The first indication of Ras-antagonizing activity of Rap1A came from its ability to induce reversion of Ras-transformed fibroblasts (28Kitayama H. Sugimoto Y. Matsuzaki T. Ikawa Y. Noda M. Cell. 1989; 56: 77-84Abstract Full Text PDF PubMed Scopus (760) Google Scholar). Subsequently, this antagonizing activity of Rap1A was also observed in nontransformed cells (1Bokoch G.M. Biochem. J. 1993; 289: 17-24Crossref PubMed Scopus (83) Google Scholar, 2Noda M. Biochim. Biophys. Acta. 1993; 1155: 97-109PubMed Google Scholar). Although these studies employed expression of a large amount of Rap1A, it has recently been shown that expression of Rap1AV12 at levels similar to the endogenous wild-type protein also efficiently inhibits activation of mitogen-activated protein kinase by growth factors (29Cook S.J. Rubinfeld B. Albert I. McCormick F. EMBO J. 1993; 12: 3475-3485Crossref PubMed Scopus (333) Google Scholar). Because this mitogen-activated protein kinase activation was found dependent on Ras, the observation suggested that a physiological amount of endogenous Rap1A may be sufficient for its antagonistic effect on Ras during normal cell growth (29Cook S.J. Rubinfeld B. Albert I. McCormick F. EMBO J. 1993; 12: 3475-3485Crossref PubMed Scopus (333) Google Scholar). Based on the present results that the antagonizing activity of Rap1A can be controlled by phosphorylation, we propose further that Rap1A may function as a PKA-dependent regulator of Raf-1 activation, not merely as a suppressor. In addition to Rap1A, PKA phosphorylates many cellular proteins including Raf-1 (26Burgering B.M.T. Bos J.L. Trends Biochem. Sci. 1995; 20: 18-22Abstract Full Text PDF PubMed Scopus (290) Google Scholar, 27Mischak H. Seitz T. Janosch P. Eulitz M. Steen H. Schellerer M. Philipp A. Kolch W. Mol. Cell. Biol. 1996; 16: 5409-5418Crossref PubMed Scopus (178) Google Scholar). Phosphorylation of Raf-1 by PKA renders it incapable of associating with Ras, thereby precluding its activation by Ras. Taken together with our present finding, one can hypothesize that PKA may exert both positive and negative regulatory effects on Ras-dependent Raf-1 activation. Although these ambivalent effects appear puzzling, they may be required for fine-tuning the signal output from Raf-1 in different cell-type or developmental contexts. It should also be pointed out that there exists a possibility that a protein kinase other than PKA may specifically phosphorylate Rap1A, but not Raf-1, under control of a certain extracellular signal. Identification of such a protein kinase might provide a further insight into the regulatory mechanism of Raf-1 activity by Rap1A. We thank A. Kikuchi at Hiroshima University School of Medicine for providing GST-mitogen-activated protein kinase kinase, GST-KNERK, and rat RalGDSb cDNA and X.-H. Deng for her skillful technical assistance. We also thank A. Seki and A. Kawabe for help in preparation of this manuscript.

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