Activation of the Rap GTPases in B Lymphocytes Modulates B Cell Antigen Receptor-induced Activation of Akt but Has No Effect on MAPK Activation
2003; Elsevier BV; Volume: 278; Issue: 43 Linguagem: Inglês
10.1074/jbc.m303180200
ISSN1083-351X
AutoresSherri L. Christian, Rosaline L. Lee, Sarah J. McLeod, Anita E. Burgess, Anson H.Y. Li, May Dang-Lawson, Kevin B.L. Lin, Michael R. Gold,
Tópico(s)Chronic Lymphocytic Leukemia Research
ResumoSignaling by the B cell antigen receptor (BCR) activates the Rap1 and Rap2 GTPases, putative antagonists of Ras-mediated signaling. Because Ras can activate the Raf-1/ERK pathway and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, we asked whether Rap activation limits the ability of the BCR to signal via these pathways. To do this, we blocked the activation of endogenous Rap1 and Rap2 by expressing the Rap-specific GTPase-activating protein RapGAPII. Preventing Rap activation had no effect on BCR-induced activation of ERK. In contrast, BCR-induced phosphorylation of Akt on critical activating sites was increased 2- to 3-fold when Rap activation was blocked. Preventing Rap activation also increased the ability of the BCR to stimulate Akt-dependent phosphorylation of the FKHR transcription factor on negative regulatory sites and decreased the levels of p27Kip1, a pro-apoptotic factor whose transcription is enhanced by FKHR. Moreover, preventing Rap activation reduced BCR-induced cell death in the WEHI-231 B cell line. Thus activation of endogenous Rap by the BCR limits BCR-induced activation of the PI3K/Akt pathway, opposes the subsequent inhibition of the FKHR/p27Kip1 pro-apoptotic module, and enhances BCR-induced cell death. Consistent with the idea that Rap-GTP is a negative regulator of the PI3K/Akt pathway, expressing constitutively active Rap2 (Rap2V12) reduced BCR-induced phosphorylation of Akt and FKHR. Finally, our finding that Rap2V12 can bind PI3K and inhibit its activity in a manner that depends upon BCR engagement provides a potential mechanism by which Rap-GTP limits activation of the PI3K/Akt pathway, a central regulator of B cell growth and survival. Signaling by the B cell antigen receptor (BCR) activates the Rap1 and Rap2 GTPases, putative antagonists of Ras-mediated signaling. Because Ras can activate the Raf-1/ERK pathway and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, we asked whether Rap activation limits the ability of the BCR to signal via these pathways. To do this, we blocked the activation of endogenous Rap1 and Rap2 by expressing the Rap-specific GTPase-activating protein RapGAPII. Preventing Rap activation had no effect on BCR-induced activation of ERK. In contrast, BCR-induced phosphorylation of Akt on critical activating sites was increased 2- to 3-fold when Rap activation was blocked. Preventing Rap activation also increased the ability of the BCR to stimulate Akt-dependent phosphorylation of the FKHR transcription factor on negative regulatory sites and decreased the levels of p27Kip1, a pro-apoptotic factor whose transcription is enhanced by FKHR. Moreover, preventing Rap activation reduced BCR-induced cell death in the WEHI-231 B cell line. Thus activation of endogenous Rap by the BCR limits BCR-induced activation of the PI3K/Akt pathway, opposes the subsequent inhibition of the FKHR/p27Kip1 pro-apoptotic module, and enhances BCR-induced cell death. Consistent with the idea that Rap-GTP is a negative regulator of the PI3K/Akt pathway, expressing constitutively active Rap2 (Rap2V12) reduced BCR-induced phosphorylation of Akt and FKHR. Finally, our finding that Rap2V12 can bind PI3K and inhibit its activity in a manner that depends upon BCR engagement provides a potential mechanism by which Rap-GTP limits activation of the PI3K/Akt pathway, a central regulator of B cell growth and survival. Signaling by the B cell antigen receptor (BCR) 1The abbreviations used are: BCR, B cell antigen receptor; PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase; PKC, protein kinase C; PDK1, 3-phosphoinositide-dependent kinase-1; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PI(3,4)P2, phosphatidylinositol 3,4-bisphosphate; Ig, immunoglobulin; FKHR, Forkhead-related transcription factor; mER-Akt, myristoylated-estrogen receptor-Akt fusion protein; A2-ER-Akt, non-myristoylated estrogen receptor-Akt fusion protein with a glycine to alanine mutation at position 2; 4-HT, 4-hydroxytamoxifen; ECL, enhanced chemiluminescence; GST, glutathione S-transferase; GAP, GTPase-activating protein; 7-AAD, 7-aminoactinomycin D; MEK, MAPK/ERK kinase; RBD, Rap-binding protein. is required for B cell development and survival, for the elimination or silencing of self-reactive B cells, and for the activation of B cells that recognize foreign antigens (1Gold M.R. Trends Pharmacol. Sci. 2002; 23: 316-324Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The BCR activates multiple signaling pathways, including the phospholipase C pathway, the phosphatidylinositol 3-kinase (PI3K) pathway, and the kinase cascades that lead to activation of the mitogen-activated protein kinases (MAPKs) (1Gold M.R. Trends Pharmacol. Sci. 2002; 23: 316-324Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 2Healy J.I. Goodnow C.C. Annu. Rev. Immunol. 1998; 16: 645-670Crossref PubMed Scopus (282) Google Scholar, 3Marshall A.J. Niiro H. Yun T.J. Clark E.A. Immunol. Rev. 2000; 176: 30-46Crossref PubMed Scopus (123) Google Scholar). Downstream targets of the phospholipase C pathway include protein kinase C (PKC) enzymes as well as the NF-AT and NF-κB transcription factors (4Kurosaki T. Maeda A. Ishiai M. Hashimoto A. Inabe K. Takata M. Immunol. Rev. 2000; 176: 19-29Crossref PubMed Scopus (138) Google Scholar). PI3K produces lipid second messengers that regulate a network of protein kinases, including 3-phosphoinositide-dependent kinase-1, PKC-ζ, PKC-ϵ, p70 S6 kinase, the Btk tyrosine kinase (1Gold M.R. Trends Pharmacol. Sci. 2002; 23: 316-324Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 5Ting H.C. Christian S.L. Burgess A.E. Gold M.R. Immunol. Lett. 2002; 82: 205-215Crossref PubMed Scopus (10) Google Scholar), and Akt/protein kinase B, a kinase that plays a key role in B cell survival (6Pogue S.L. Kurosaki T. Bolen J. Herbst R. J. Immunol. 2000; 165: 1300-1306Crossref PubMed Scopus (125) Google Scholar). The three major classes of MAPKs, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, phosphorylate a variety of transcription factors, increasing their ability to promote transcription. The activation of distinct combinations of these signaling pathways may account for the ability of the BCR to promote survival, apoptosis, proliferation, or differentiation depending on the maturation state of the B cell and the nature of the antigen (2Healy J.I. Goodnow C.C. Annu. Rev. Immunol. 1998; 16: 645-670Crossref PubMed Scopus (282) Google Scholar). A key feature of signaling by the BCR and many other receptors is the use of monomeric GTPases as molecular switches that cycle between an inactive GDP-bound state and an active GTP-bound state. Activated GTPases bind downstream effector proteins and thereby regulate their subcellular localization and activity. Previous work has shown that BCR engagement results in the activation of the Ras and Rac1 GTPases (7Hashimoto A. Okada H. Jiang A. Kurosaki M. Greenberg S. Clark E.A. Kurosaki T. J. Exp. Med. 1998; 188: 1287-1295Crossref PubMed Scopus (183) Google Scholar). Ras controls the kinase cascade leading to the activation of the ERK1 and ERK2 MAPKs, whereas Rac1 regulates the kinase cascades leading to the activation of the JNK and p38 MAPKs. We have shown that the BCR also activates the Rap1 GTPase (8McLeod S.J. Ingham R.J. Bos J.L. Kurosaki T. Gold M.R. J. Biol. Chem. 1998; 273: 29218-29223Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), and in this report we show that the BCR activates the Rap2 GTPase. Recent work has shown that the Rap GTPases play a key role in receptor-induced integrin activation and, hence, cell adhesion in many cell types (9Shimizu Y. Immunol. Today. 2000; 21: 597Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar), including B cells. 2S. J. McLeod, A. Shum, A. E. Burgess, M. Dang-Lawson, A. H. Y. Li, and M. R. Gold, submitted for publication. However, the Rap GTPases were first described as potential antagonists of Ras-mediated signaling. The idea that Rap is an antagonist of Ras-mediated signaling originated with experiments showing that overexpressing Rap1 caused Ras-transformed 3T3 cells to assume a more normal fibroblast-like morphology (10Kitayama H. Sugimoto Y. Matsuzaki T. Ikawa Y. Noda M. Cell. 1989; 56: 77-84Abstract Full Text PDF PubMed Scopus (763) Google Scholar). Similarly, activated Rap1 can oppose Ras-dependent Xenopus oocyte differentiation (11Campa M.J. Chang K.J. Molina y Vedia L. Reep B.R. Lapetina E.G. Biochem. Biophys. Res. Commun. 1991; 174: 1-5Crossref PubMed Scopus (36) Google Scholar). Because Ras and Rap1 have identical core effector binding domains (residues 32–40) and activated Rap1 can bind in vitro to Ras effectors, including Raf-1 (12Nassar N. Horn G. Herrmann C. Scherer A. McCormick F. Wittinghofer A. Nature. 1995; 375: 554-560Crossref PubMed Scopus (561) Google Scholar, 13Herrmann C. Horn G. Spaargaren M. Wittinghofer A. J. Biol. Chem. 1996; 271: 6794-6800Abstract Full Text PDF PubMed Scopus (301) Google Scholar), it was proposed that activated Rap1 limits Ras-mediated signaling by sequestering Ras effectors in inactive complexes. Although Rap2 has a phenylalanine at position 39 instead of the serine present in Rap1, this substitution does not appear to have any functional consequences (14Zwartkruis F.J. Bos J.L. Exp. Cell Res. 1999; 253: 157-165Crossref PubMed Scopus (151) Google Scholar, 15Ohba 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 (94) Google Scholar), suggesting that activated Rap2 can also bind Ras effectors and act as a Ras antagonist. Indeed, several groups have shown that both activated Rap1 and activated Rap2 can inhibit Ras-dependent activation of ERK (15Ohba 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 (94) Google Scholar, 16Cook S.J. Rubinfeld B. Albert I. McCormick F. EMBO J. 1993; 12: 3475-3485Crossref PubMed Scopus (335) Google Scholar, 17Buensuceso C.S. O'Toole T.E. J. Biol. Chem. 2000; 275: 13118-13125Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 18Kawasaki H. Springett G.M. Toki S. Canales J.J. Harlan P. Blumenstiel J.P. Chen E.J. Bany I.A. Mochizuki N. Ashbacher A. Matsuda M. Housman D.E. Graybiel A.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13278-13283Crossref PubMed Scopus (313) Google Scholar). Other studies, however, did not find that Rap activation impaired Ras-dependent ERK activation (19Sato K.Y. Polakis P.G. Haubruck H. Fasching C.L. McCormick F. Stanbridge E.J. Cancer Res. 1994; 54: 552-559PubMed Google Scholar, 20Zwartkruis F.J. Wolthuis R.M. Nabben N.M. Franke B. Bos J.L. EMBO J. 1998; 17: 5905-5912Crossref PubMed Scopus (191) Google Scholar, 21Enserink J.M. Christensen A.E. de Rooij J. van Triest M. Schwede F. Genieser H.G. Doskeland S.O. Blank J.L. Bos J.L. Nat. Cell Biol. 2002; 4: 901-906Crossref PubMed Scopus (618) Google Scholar), suggesting that this could a cell type-specific effect. Signaling pathways regulated by Ras play an essential role in BCR signaling. Loss-of-function studies have shown that activation of the Ras/Raf-1/MEK/ERK signaling pathway is essential for B cell development (22Iritani B.M. Forbush K.A. Farrar M.A. Perlmutter R.M. EMBO J. 1997; 16: 7019-7031Crossref PubMed Scopus (133) Google Scholar) and for BCR-induced proliferation of mature B cells (23Richards J.D. Dave S.H. Chou C.H. Mamchak A.A. DeFranco A.L. J. Immunol. 2001; 166: 3855-3864Crossref PubMed Scopus (111) Google Scholar). In addition to activating the Raf-1/MEK/ERK pathway, Ras can also promote signaling via PI3K. Activated Ras can bind directly to the p110 catalytic subunit of PI3K (24Rodriguez-Viciana P. Warne P.H. Dhand R. Vanhaesebroeck B. Gout I. Fry M.J. Waterfield M.D. Downward J. Nature. 1994; 370: 527-532Crossref PubMed Scopus (1731) Google Scholar, 25Pacold M.E. Suire S. Perisic O. Lara-Gonzalez S. Davis C.T. Walker E.H. Hawkins P.T. Stephens L. Eccleston J.F. Williams R.L. Cell. 2000; 103: 931-943Abstract Full Text Full Text PDF PubMed Google Scholar), thereby recruiting PI3K to the plasma membrane where it can produce the lipid second messengers phosphatidylinositol 3,4,5-trisphosphate (PIP3) and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2), which activate downstream targets such as Akt. Akt plays a critical role in regulating survival versus apoptosis in B cells (6Pogue S.L. Kurosaki T. Bolen J. Herbst R. J. Immunol. 2000; 165: 1300-1306Crossref PubMed Scopus (125) Google Scholar). Consistent with the idea that Ras regulates the PI3K/Akt pathway in B cells, recent work has shown that expressing constitutively active Ras leads to Akt activation in the A20 B cell line (26Jacob A. Cooney D. Pradhan M. Coggeshall K.M. J. Biol. Chem. 2002; 277: 23420-23426Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The BCR can also initiate PI3K-dependent signaling by inducing the SH2 domain-dependent binding of PI3K to membrane-associated docking proteins such as CD19, Gab1, BCAP, and Cbl that are tyrosine-phosphorylated after BCR engagement (27Tuveson D.A. Carter R.H. Soltoff S.P. Fearon D.T. Science. 1993; 260: 986-989Crossref PubMed Scopus (284) Google Scholar, 28Ingham R.J. Holgado-Madruga M. Siu C. Wong A.J. Gold M.R. J. Biol. Chem. 1998; 273: 30630-30637Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 29Okada T. Maeda A. Iwamatsu A. Gotoh K. Kurosaki T. Immunity. 2000; 13: 817-827Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 30Panchamoorthy G. Fukazawa T. Miyake S. Soltoff S. Reedquist K. Druker B. Shoelson S. Cantley L. Band H. J. Biol. Chem. 1996; 271: 3187-3194Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Thus there may be two distinct mechanisms by which the BCR initiates PI3K signaling. Nevertheless, if activated Rap were to act as an antagonist of Ras-mediated signaling in B cells, it could limit the activation of two critical BCR signaling pathways, the Ras/Raf-1/MEK/ERK pathway and the Ras/PI3K/Akt pathway. In contrast to the ability of activated Rap to inhibit Ras-dependent ERK activation in some cell types, Rap-GTP can in fact act as a positive regulator of both the ERK and p38 MAPKs in some situations. In neuronal cells such as the PC12 cell line, Stork and colleagues (31Vossler M.R. Yao H. York R.D. Pan M.G. Rim C.S. Stork P.J. Cell. 1997; 89: 73-82Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar, 32York R.D. Yao H. Dillon T. Ellig C.L. Eckert S.P. McCleskey E.W. Stork P.J. Nature. 1998; 392: 622-626Crossref PubMed Scopus (762) Google Scholar) have shown that Rap1-GTP activates B-Raf, leading to sustained activation of ERK. Brummer et al. (33Brummer T. Shaw P.E. Reth M. Misawa Y. EMBO J. 2002; 21: 5611-5622Crossref PubMed Scopus (68) Google Scholar) have recently shown that B-Raf plays a significant role in the activation of ERK by the BCR, suggesting that a Rap/B-Raf pathway could contribute to BCR-induced ERK activation. Thus, depending on the cell type, the Rap GTPases may act as either positive or negative regulators of ERK activation. There is also some evidence that Rap activation is important for activation of the p38 MAPK (34Sawada Y. Nakamura K. Doi K. Takeda K. Tobiume K. Saitoh M. Morita K. Komuro I. De Vos K. Sheetz M. Ichijo H. J. Cell Sci. 2001; 114: 1221-1227Crossref PubMed Google Scholar). Because the Rap GTPases can act as negative regulators of Ras-dependent signaling and, depending on the cell type, as positive regulators of MAPK signaling pathways, we investigated whether Rap activation in B cells modulates the activation of the PI3K/Akt pathway or the MAPK pathways, critical mediators of BCR signaling. We show that Rap is neither a positive nor negative regulator of BCR-induced ERK activation and that Rap activation has no effect on the ability of the BCR to activate the JNK and p38 MAPKs. In contrast, we found that activation of the endogenous Rap GTPases limits the activation of Akt by the BCR. Preventing the activation of endogenous Rap by the BCR enhanced BCR-induced Akt phosphorylation, whereas expressing Rap2V12, a constitutively active form of Rap2, inhibited BCR-induced Akt phosphorylation. Consistent with the idea that Rap-GTP opposes Akt activation, we found that Rap activation inhibits Akt-dependent signaling events, in particular phosphorylation of the FKHR transcription factor. We also found that the ability of Rap-GTP to limit BCR-induced signaling via Akt correlated with effects on B cell survival. Finally, we show that Rap2V12-GTP can bind to PI3K and inhibit its enzymatic activity. Thus, the Rap GTPases act as negative regulators of the PI3K/Akt pro-survival signaling pathway in B cells and may do so via a mechanism that involves the binding of Rap-GTP to PI3K, the upstream activator of Akt. Antibodies—Goat antibodies against mouse immunoglobulin (Ig) G (γ chain-specific) or mouse IgM (μ chain-specific) were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Rabbit antibodies specific for Akt, Akt phosphorylated on serine 473 (anti-P-Ser-473 Akt), Akt phosphorylated on threonine 308 (anti-P-Thr-308 Akt), and p38 were purchased from Cell Signaling Technologies (Beverly, MA) as were the antibodies specific for the phosphorylated forms of ERK, JNK, p38, and the Forkhead-related transcription factor FKHR/FOXO1. The antibodies specific for ERK, JNK, Rap1, and p85α were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Monoclonal antibodies to Rap2, PDK1, and p27Kip1 were obtained from BD Transduction Laboratories (Mississauga, Ontario, Canada). The M2 anti-FLAG monoclonal antibody and the monoclonal antibody to actin were purchased from Sigma-Aldrich (Oakville, Ontario, Canada). B Cell Lines—The A20 and WEHI-231 murine B cell lines were obtained from the American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 50 μm 2-mercaptoethanol, 1 mm pyruvate, 2 mm glutamine, 15 units/ml penicillin, and 50 μg/ml streptomycin (complete medium). A20 cells expressing RapGAPII, Rap2V12, or the empty pMSCVpuro vector (BD Biosciences Clontech, Palo Alto, CA) were generated by electroporation (400 V, 975 microfarads) followed by selection in medium containing 4 μg/ml puromycin (Calbiochem, La Jolla, CA). The cDNAs encoding FLAG-tagged RapGAPII (35Mochizuki N. Ohba Y. Kiyokawa E. Kurata T. Murakami T. Ozaki T. Kitabatake A. Nagashima K. Matsuda M. Nature. 1999; 400: 891-894Crossref PubMed Scopus (192) Google Scholar) or Rap2V12 in pMSCVpuro were gifts from Dr. M. Matsuda (Osaka University, Osaka, Japan). A20 clones expressing RapGAPII were obtained by single-cell cloning. Expression of FLAG-RapGAPII or FLAG-Rap2V12 was detected by immunoblotting with the M2 anti-FLAG monoclonal antibody. All experiments were performed using clone 16, and results were confirmed using clone 3. The Rap2V12-expressing A20 cells were an oligoclonal population that was not subjected to single-cell cloning. Bulk populations of WEHI-231 cells expressing RapGAPII, Rap2V12, or the empty pMSCVpuro vector were generated by retrovirus-mediated gene transfer (36Krebs D.L. Yang Y. Dang M. Haussmann J. Gold M.R. Methods Cell Sci. 1999; 21: 57-98Crossref PubMed Scopus (35) Google Scholar) followed by selection in medium containing 0.25 μg/ml puromycin. A20 cells expressing a myristoylated Akt-estrogen receptor fusion protein (mER-Akt) or a non-myristoylated version with a glycine to alanine substitution at position 2 (A2-ER-Akt) were generated by electroporation and single cell cloning in the presence of puromycin. The cDNAs encoding the mER-Akt and A2-ER-Akt proteins (37Kohn A.D. Barthel A. Kovacina K.S. Boge A. Wallach B. Summers S.A. Birnbaum M.J. Scott P.H. Lawrence Jr., J.C. Roth R.A. J. Biol. Chem. 1998; 273: 11937-11943Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar) (gifts from Dr. R. Roth, Stanford University, Stanford, CA) were subcloned into the pMXpuro-IRES-EGFP vector, a derivative of pMXpuro (DNAX, Palo Alto, CA) (38Onishi M. Kinoshita Y. Morikawa A. Shibuya J. Phillips L.L. Lanier D.M. Gorman G.P. Nolan A. Kitamura M.T. Exp. Hematol. 1996; 24: 324-329PubMed Google Scholar). B Cell Stimulation and Preparation of Cell Lysates—Cells were washed once with modified HEPES-buffered saline (39Gold M.R. Scheid M.P. Santos L. Dang-Lawson M. Roth R.A. Matsuuchi L. Duronio V. Krebs D.L. J. Immunol. 1999; 163: 1894-1905PubMed Google Scholar), resuspended in this buffer at 1 × 107 or 2 × 107 per ml, and warmed to 37 °C for 10–30 min. The cells were then stimulated with anti-Ig antibodies for the indicated times. Activation of the mER-Akt fusion protein was achieved by treating the cells with 2 μm 4-hydroxytamoxifen (4-HT, Sigma-Aldrich). For stimulation reactions lasting longer than 1 h, the cells were resuspended at 5 × 105 per ml in complete medium and treated with anti-Ig antibodies at 10 μg/ml for the indicated times. The reactions were terminated by adding ice-cold phosphate-buffered saline containing 1 mm Na3VO4 and then centrifuging the cells for 1 min at 1100 × g in a microcentrifuge at 4 °C. For analyzing the phosphorylation of Akt, ERK, JNK, and p38, the cell pellets were solubilized in Triton X-100 lysis buffer (20 mm Tris-HCl (pH 8.0), 1% Triton X-100, 137 mm NaCl, 2 mm EDTA, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mm Na3VO4, 25 mm β-glycerophosphate, 1 μg/ml microcystin-LR). For Rap activation assays, immunoprecipitation assays, PI3K enzyme assays, and detection of p27Kip1 protein levels, the cell pellets were solubilized in Rap lysis buffer (50 mm Tris-HCl (pH 7.5), 1% Igepal (ICN, Costa Mesa, CA), 200 mm NaCl, 2 mm MgCl2, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mm Na3VO4, 25 mm β-glycerophosphate). For ERK in vitro kinase assays, the cell pellets were solubilized in ERK assay buffer (50 mm Tris-HCl (pH 7.5), 1% Nonidet P-40, 150 mm NaCl, 5 mm EDTA, 1 mm MoO4, 0.2 mm Na3VO4, 1 mm dithiothreitol, 10 μg/ml aprotinin, 2 μg/ml leupeptin, 0.7 μg/ml pepstatin, 40 μg/ml phenylmethylsulfonyl fluoride, 10 μg/ml soybean trypsin inhibitor). In all cases, the samples were left on ice for 10 min, and then insoluble material was removed by centrifugation. Protein concentrations were determined using the bicinchoninic acid assay (Pierce, Rockford, IL). Immunoblotting—Cell lysates (20 μg of protein) or immunoprecipitated proteins were separated on 12% SDS-PAGE gels and transferred to nitrocellulose membranes. The membranes were blocked for 1–2 h with 5% (w/v) skim milk powder in TBST (10 mm Tris-HCl (pH 7.5), 150 mm NaCl, 0.1% Tween 20) and then incubated overnight at 4 °C or 1 h at room temperature with the primary antibody. All antibodies were diluted in TBST/1% bovine serum albumin. The membranes were then washed with TBST and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Bio-Rad, Hercules, CA) for 1 h at room temperature. Immunoreactive bands were visualized using enhanced chemiluminescence (ECL, Amersham Biosciences, Baie d'Urfe, Quebec, Canada). To re-probe membranes, bound antibodies were eluted by incubating the membrane in 10 mm Tris-HCl (pH 2), 150 mm NaCl for 30 min. The membranes were then re-blocked and probed with antibodies as described above. To quantitate results, scans of ECL exposures were saved as TIFF files and analyzed using ImageQuaNT 1.2 software (Amersham Biosciences). Rap Activation Assays—Cells were stimulated as above, solubilized in Rap lysis buffer, and then assayed for Rap activation as described previously (8McLeod S.J. Ingham R.J. Bos J.L. Kurosaki T. Gold M.R. J. Biol. Chem. 1998; 273: 29218-29223Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Briefly, a glutathione S-transferase (GST) fusion protein containing the Rap1/2-binding domain of the RalGDS protein (GST-RalGDS(RBD)) was used to selectively precipitate the active GTP-bound forms of Rap1 and Rap2, which were then detected by immunoblotting with antibodies specific for either Rap1 or Rap2. In some experiments the filters were probed with anti-Rap2 antibodies, stripped, and then re-probed with anti-Rap1 antibodies. Immunoprecipitation—Cell lysates were pre-cleared with Sepharose-CL-4B beads (Sigma-Aldrich) for 30 min at 4 °C. For anti-FLAG immunoprecipitation, cell lysates were transferred to tubes containing 10 μl of agarose beads covalently coupled with the M2 anti-FLAG-monoclonal antibody (Sigma-Aldrich) plus 10 μl of Sepharose-CL-4B beads as filler. After mixing for 1 h at 4 °C, the beads were pelleted and washed three times with Rap lysis buffer, and bound proteins were eluted using SDS-PAGE sample buffer. ERK and PI3K in Vitro Kinase Assays—ERK2 was immunoprecipitated using monoclonal anti-ERK2 antibodies conjugated to agarose beads (Santa Cruz Biotechnology). ERK in vitro kinase assays were performed as described previously (40Sutherland C.L. Heath A.W. Pelech S.L. Young P.R. Gold M.R. J. Immunol. 1996; 157: 3381-3390PubMed Google Scholar) using either 30 μg of myelin basic protein (Sigma-Aldrich) or 10 μg of GST-ELK-1 (Cell Signaling Technologies) as the substrate. PI3K enzyme assays were performed on anti-FLAG immunoprecipitates as described by Gold et al. (41Gold M.R. Chan V.W. Turck C.W. DeFranco A.L. J. Immunol. 1992; 148: 2012-2022PubMed Google Scholar). The immunoprecipitates were incubated with phosphatidylinositol (Avanti Polar Lipids, Alabaster, AL) and [γ-32P]ATP for 20 min at room temperature. The resulting 32P-labeled phosphatidylinositol 3-phosphate was separated from the [γ-32P]ATP by thin layer chromatography and quantified using a PhosphorImager (Amersham Biosciences). Anti-IgM-induced Cell Death—WEHI-231 cells were plated at 2 × 105 per ml in complete medium and cultured for 48–72 h at 37 °C. The cells were then pelleted and resuspended in fluorescence-activated cell sorting buffer (phosphate-buffered saline supplemented with 2% fetal calf serum) containing 4 μg/ml 7-AAD (Calbiochem), a membrane-impermeable DNA-binding compound that is excluded from live cells with intact membranes. After staining the cells in the dark for 10 min, the cells were pelleted and resuspended in fluorescence-activated cell sorting buffer. Flow cytometry was used to determine the percentage of cells that had taken up significant amounts of 7-AAD. Expressing RapGAPII Inhibits BCR-induced Activation of Rap1 and Rap2—We have previously shown that BCR engagement results in an increase in the amount of active GTP-bound Rap1 (8McLeod S.J. Ingham R.J. Bos J.L. Kurosaki T. Gold M.R. J. Biol. Chem. 1998; 273: 29218-29223Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Because Rap2 has a nearly identical effector region to that of Rap1 (14Zwartkruis F.J. Bos J.L. Exp. Cell Res. 1999; 253: 157-165Crossref PubMed Scopus (151) Google Scholar), can bind Ras effectors, and can inhibit Ras-dependent ERK activation (15Ohba 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 (94) Google Scholar), we investigated whether the BCR also activates Rap2. We found that inducing BCR signaling with anti-Ig antibodies resulted in the activation of both Rap1 and Rap2 in the WEHI-231 and A20 murine B cell lines (Fig. 1A). WEHI-231 is an IgM+ B cell line that resembles an immature/transitional B cell that can undergo antigen-induced clonal deletion, whereas A20 is an IgG+ B cell line that resembles a mature B cell that has undergone Ig class switching. The activation of Rap1 and Rap2 occurred with similar kinetics, consistent with the idea that they are regulated by the same exchange factors and GTPase-activating proteins (GAPs). To investigate whether activated Rap GTPases modulate BCR-signaling pathways we employed a loss-of-function approach in which we blocked the activation of endogenous Rap1 and Rap2. To do this, we expressed in both WEHI-231 and A20 cells a Rap-specific GAP called RapGAPII (Fig. 1B) that converts Rap1 and Rap2 to their inactive GDP-bound forms. RapGAPII is normally expressed in brain (35Mochizuki N. Ohba Y. Kiyokawa E. Kurata T. Murakami T. Ozaki T. Kitabatake A. Nagashima K. Matsuda M. Nature. 1999; 400: 891-894Crossref PubMed Scopus (192) Google Scholar) but not in B cells (42McLeod S.J. Li A.H. Lee R.L. Burgess A.E. Gold M.R. J. Immunol. 2002; 169: 1365-1371Crossref PubMed Scopus (98) Google Scholar) and has been shown to specifically inhibit the activation of Rap1 and Rap2 (15Ohba 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 (94) Google Scholar, 35Mochizuki N. Ohba Y. Kiyokawa E. Kurata T. Murakami T. Ozaki T. Kitabatake A. Nagashima K. Matsuda M. Nature. 1999; 400: 891-894Crossref PubMed Scopus (192) Google Scholar), but has no effect on the activation of other closely related GTPases such as Ha-Ras, R-Ras (35Mochizuki N. Ohba Y. Kiyokawa E. Kurata T. Murakami T. Ozaki T. Kitabatake A. Nagashima K. Matsuda M. Nature. 1999; 400: 891-894Crossref PubMed Scopus (192) Google Scholar), RhoA (43Polakis P.G. Rubinfeld B. Evans T. McCormick F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 239-243Crossref PubMed Scopus (74) Google Scholar), and Rac1 (42McLeod S.J. Li A.H. Lee R.L. Burgess A.E. Gold M.R. J. Immunol. 2002; 169: 1365-1371Crossref PubMed Scopus (98) Google Scholar). We found that expressing RapGAPII in B
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