Title: Syk Is Required for BCR-mediated Activation of p90Rsk, but Not p70S6k, via a Mitogen-activated Protein Kinase-independent Pathway in B Cells
Abstract: The tyrosine kinases Syk and Lyn are activated in B lymphocytes following antibody induced cross-linking of the B cell receptor for antigen (BCR). It has been suggested that activation of Syk is dependent on Lyn. We tested this hypothesis by comparing the phosphorylation and activation of several downstream effector molecules in parental DT40, DT40Syk− and DT40Lyn−B cells. The phosphorylation and activation of p90Rsk was ablated in Syk-deficient B cells but unaffected in Lyn-deficient B cells while the phosphorylation/activation of Ras GTPase activating protein (Ras GAP) and mitogen activated protein (MAP) kinase required both Syk and Lyn. Thus, these data indicate that Syk can be activated in the absence of Lyn after BCR cross-linking and results in the activation of p90Rsk via a MAP kinase-independent pathway in DT40Lyn− cells. We also demonstrated that BCR mediates the activation of p70S6k. However, activation of p70S6k in DT40Syk− and DT40Lyn−cells was comparable with that observed in parental cells. Thus, either Syk or Lyn may be sufficient for activation of p70S6k, or activation of p70S6k occurs independently of both Syk and Lyn. The kinase activity of Syk was required for the phosphorylation/activation of each of these downstream effector molecules but only the phosphorylation of Ras GAP was affected in cells expressing a mutant of Syk in which tyrosines 525 and 526 were substituted to phenlyalanines. The tyrosine kinases Syk and Lyn are activated in B lymphocytes following antibody induced cross-linking of the B cell receptor for antigen (BCR). It has been suggested that activation of Syk is dependent on Lyn. We tested this hypothesis by comparing the phosphorylation and activation of several downstream effector molecules in parental DT40, DT40Syk− and DT40Lyn−B cells. The phosphorylation and activation of p90Rsk was ablated in Syk-deficient B cells but unaffected in Lyn-deficient B cells while the phosphorylation/activation of Ras GTPase activating protein (Ras GAP) and mitogen activated protein (MAP) kinase required both Syk and Lyn. Thus, these data indicate that Syk can be activated in the absence of Lyn after BCR cross-linking and results in the activation of p90Rsk via a MAP kinase-independent pathway in DT40Lyn− cells. We also demonstrated that BCR mediates the activation of p70S6k. However, activation of p70S6k in DT40Syk− and DT40Lyn−cells was comparable with that observed in parental cells. Thus, either Syk or Lyn may be sufficient for activation of p70S6k, or activation of p70S6k occurs independently of both Syk and Lyn. The kinase activity of Syk was required for the phosphorylation/activation of each of these downstream effector molecules but only the phosphorylation of Ras GAP was affected in cells expressing a mutant of Syk in which tyrosines 525 and 526 were substituted to phenlyalanines. The cytoplasmic protein tyrosine kinase (PTK) 1The abbreviations used are: PTK, protein tyrosine kinase; TCR and BCR, T and B cell receptor for antigen, respectively; FcR, Fc receptor; PLC-γ, phospholipase-Cγ; MAP, mitogen activated protein; Ras Gap, Ras GTPase activating protein; Ab, antibody, mAb, monoclonal Ab; PDBu, phorbol 12,13-dibutyrate; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; MOPS, 4-morpholinepropanesulfonic acid; PI, phosphatidylinositol; WT, wild type. Syk is expressed in a wide variety of hematopoietic cells including B and T lymphocytes, thymocytes, myeloid lineage cells, mast cells, and platelets (1Zioncheck T.F. Harrison M.L. Isaacson C.C. Gaehlen R.L. J. Biol. Chem. 1988; 263: 19195-19202Abstract Full Text PDF PubMed Google Scholar, 2Taniguchi T. Kobayashi T. Kondo J. Takahashi K. Nakamura H. Suzuki J. Nagai K. Yamada T. Nakamura S. Yamamura H. J. Biol. Chem. 1991; 266: 15790-15796Abstract Full Text PDF PubMed Google Scholar, 3Law C.-L. Sidorenko S.P. Chandran K.A. Draves K.E. Chan A.C. Weiss A. Edelhoff S. Disteche C.M. Clark E.A. J. Biol. Chem. 1994; 269: 12310-12319Abstract Full Text PDF PubMed Google Scholar, 4Chan A.C. van Oers N.S.C. Tran A. Turka L. Law C.L. Ryan J.C. Clark E.A. Weiss A. J. Immunol. 1994; 152: 4758-4766PubMed Google Scholar, 5Ohta S. Taniguchi T. Asahi M. Kato Y. Nakagawara G. Yamamura H. Biochem. Biophys. Res. Commun. 1992; 185: 1128-1132Crossref PubMed Scopus (37) Google Scholar). Syk has been implicated in the signal transduction pathways of several cell surface receptors such as the antigen receptors on B (BCR) (6Hutchcroft J.E. Harrison M.L. Geahlen R.L. J. Biol. Chem. 1991; 266: 14846-14849Abstract Full Text PDF PubMed Google Scholar, 7Hutchcroft J.E. Harrison M.L. Geahlen R.L. J. Biol. Chem. 1992; 267: 8613-8619Abstract Full Text PDF PubMed Google Scholar) and T (TCR) (4Chan A.C. van Oers N.S.C. Tran A. Turka L. Law C.L. Ryan J.C. Clark E.A. Weiss A. J. Immunol. 1994; 152: 4758-4766PubMed Google Scholar, 8Burkhardt A.L. Stealey B. Rowley R.B. Mahajan S. Prendergast M. Fargnoli J. Bolen J.B. J. Biol. Chem. 1994; 269: 23642-23647Abstract Full Text PDF PubMed Google Scholar) lymphocytes, as well as the Fc receptors (FcR) for IgG (FcγRI, FcγRII, and FcγRIII) (9Darby C. Geahlen R.L. Schreiber A.D. J. Immunol. 1994; 152: 5429-5437PubMed Google Scholar, 10Agarwal A. Salem P. Robbins K.C. J. Biol. Chem. 1993; 268: 15900-15905Abstract Full Text PDF PubMed Google Scholar, 11Kiener P.A. Rankin B.M. Burkhardt A.L. Schieven G.L. Gilliland L.K. Rowley R.B. Bolen J.B. Ledbetter J.A. J. Biol. 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Walker J.J. Haskill S. Juliano R.L. J. Cell Biol. 1994; 126: 1585-1593Crossref PubMed Scopus (116) Google Scholar, 18Lin T.H. Rosales C. Mondal K. Bolen J.B. Haskill S. Juliano R.L. J. Cell Biol. 1995; 270: 16186-16197Google Scholar, 19Xiao J. Messinger Y. Jin J. Myers D. Bolen J. Uckun F.M. J. Biol. Chem. 1996; 271: 7659-7664Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Upon ligation of these receptors, Syk is tyrosine phosphorylated and catalytically activated. In contrast, the T cell receptor associated protein tyrosine kinase, ZAP-70, which is structurally homologous to Syk, has a more limited tissue distribution (4Chan A.C. van Oers N.S.C. Tran A. Turka L. Law C.L. Ryan J.C. Clark E.A. Weiss A. J. Immunol. 1994; 152: 4758-4766PubMed Google Scholar, 20Chan A.C. Iwashima M. Turck C.W. Weiss A. Cell. 1992; 71: 649-662Abstract Full Text PDF PubMed Scopus (884) Google Scholar), and to date, it has only been demonstrated to mediate signaling by two receptor complexes, the TCR (4Chan A.C. van Oers N.S.C. Tran A. Turka L. Law C.L. Ryan J.C. Clark E.A. Weiss A. J. Immunol. 1994; 152: 4758-4766PubMed Google Scholar) and CD16 (FcγRIII) on NK cells (13Stahls A. Liwszyc G.E. Couture C. Mustelin T. Andersson L.C. Eur. J. Immunol. 1994; 24: 2491-2496Crossref PubMed Scopus (41) Google Scholar, 21Viver E. da Silva A.J. Ackerly M. Levine H. Rudd C.E. Anderson P. Eur. J. Immunol. 1993; 23: 1872-1876Crossref PubMed Scopus (72) Google Scholar). Cross-linking of BCR and TCR induces B and T cell activation. The early signaling pathways involve rapid tyrosine phosphorylation and activation of Src and Syk family kinases. Optimal activation of Syk family kinases, i.e. Syk and ZAP70, in COS cells requires Src family kinases (22Kurosaki T. Takata M. Yamanashi Y. Inazu T. Taniguchi T. Yamamoto T. Yamamura H. J. Exp. Med. 1994; 179: 1725-1729Crossref PubMed Scopus (251) Google Scholar, 23Latour S. Chow L.M.L. Veillette A. J. Biol. Chem. 1997; 271: 22782-22790Abstract Full Text Full Text PDF Scopus (132) Google Scholar). Moreover, the activation of Src family kinases temporally precedes that of Syk family kinases during T cell activation (24Iwashima M. Irving B.A. van Oers N.S.C. Chan A.C. Weiss A. Science. 1994; 263: 1136-1139Crossref PubMed Scopus (2) Google Scholar), suggesting that Src family kinases may be the upstream effectors of Syk family kinases in T cells. On the other hand, Coutureet al. (25Couture C. Baier G. Oetken C. Williams S. Telford D. Cardine A.M. Baier-Bitterlich G. Fischer S. Burn P. Altman A. Mustelin T. Mol. Cell. Biol. 1994; 14: 5249-5258Crossref PubMed Google Scholar) provided evidence for Syk-mediated activation of Lck, a member of the Src family of kinases in T cells. A chicken B cell line, DT40, has been used to examine the hierarchy of activation of Src and Syk family kinases in B cells. This cell line does not express detectable levels of many Src family members, including Src, Fyn, Lck, Blk, Hck, or the Syk-related PTK, ZAP70 (26Takata M. Sabe H. Hata A. Inazu T. Homma Y. Nukada T. Yamamura H. Kurosaki T. EMBO J. 1994; 13: 1341-1349Crossref PubMed Scopus (588) Google Scholar). Thus, the predominant tyrosine kinases expressed in this cell line are Lyn and Syk (22Kurosaki T. Takata M. Yamanashi Y. Inazu T. Taniguchi T. Yamamoto T. Yamamura H. J. Exp. Med. 1994; 179: 1725-1729Crossref PubMed Scopus (251) Google Scholar, 26Takata M. Sabe H. Hata A. Inazu T. Homma Y. Nukada T. Yamamura H. Kurosaki T. EMBO J. 1994; 13: 1341-1349Crossref PubMed Scopus (588) Google Scholar). DT40 cells deficient in Lyn and Syk (DT40Lyn− and DT40Syk−, respectively) were generated by gene interruption (26Takata M. Sabe H. Hata A. Inazu T. Homma Y. Nukada T. Yamamura H. Kurosaki T. EMBO J. 1994; 13: 1341-1349Crossref PubMed Scopus (588) Google Scholar). It was shown that BCR-induced tyrosine phosphorylation and activation of PLC-γ2 was abolished in DT40Syk− cells, indicating Syk is required for this event. In contrast, the phosphorylation of PLC-γ2 was only modestly decreased, and the activation of PLC-γ2 was unaffected in DT40Lyn− cells (22Kurosaki T. Takata M. Yamanashi Y. Inazu T. Taniguchi T. Yamamoto T. Yamamura H. J. Exp. Med. 1994; 179: 1725-1729Crossref PubMed Scopus (251) Google Scholar, 26Takata M. Sabe H. Hata A. Inazu T. Homma Y. Nukada T. Yamamura H. Kurosaki T. EMBO J. 1994; 13: 1341-1349Crossref PubMed Scopus (588) Google Scholar). These data indirectly suggested that Syk can be activated in the absence of Lyn and lead to activation of PLC-γ2. In this study, we compared DT40Syk− and DT40Lyn− cells with parental DT40 cells to determine the requirements for Lyn and Syk for the activation of several downstream effectors of BCR-induced signaling, including Ras GTPase activating protein (Ras GAP) (27Yagura H. Oyaizu N. Pahwah S. Blood. 1993; 81: 1535-1539Crossref PubMed Google Scholar, 28Gold M.R. Crowley M.T. Martin G.A. McCormick F. DeFranco A.L. J. Immunol. 1993; 150: 377-386PubMed Google Scholar), MAP kinase (29Tordai A. Franklin R.A. Patel H. Gardner A.M. Johnson G.L. Gelfand E.W. J. Biol. Chem. 1994; 269: 7538-7543Abstract Full Text PDF PubMed Google Scholar), and the serine/threonine kinases, p90Rsk and p70S6k, involved in the regulation of ribosomal S6 protein that functions in the efficient translation of some proteins in mitogen-activated cells (30Kozma S.C. Ferrari S. Bassand P. Siegmann M. Totty N. Thomas G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7365-7369Crossref PubMed Scopus (112) Google Scholar, 31Banerjee P. 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Alternatively, p90Rsk, as well as p70S6k, can be activated independently of MAP kinases (36Kalab P. Kubiak J.Z. Verlhac M.H. Colledge W.H. Maro B. Development. 1996; 122: 1957-1964Crossref PubMed Google Scholar). By comparing anti-Ig-induced responses in the parental and mutant DT40 cell lines, we dissected the multiple signal transduction pathways that lead to the activation of these downstream effectors. Together with previous results, our data indicate that in addition to activation of signal transduction pathways that include both Lyn and Syk and lead to activation, for example of MAP kinase, cross-linking of the BCR also results in activation of Syk that can occur independently of Lyn and that is sufficient for the activation of p90Rsk. We also demonstrated that p70S6k is activated in B cells following cross-linking of the BCR. Finally, either Lyn or Syk is sufficient for activation of p70S6k, or BCR-induced activation of p70S6k does not require Syk or Lyn. The chicken B cell line, DT40, was cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 1% chicken serum, 100 μg/ml penicillin, 100 μg/ml streptomycin, and 2 mm glutamine. DT40Lyn− and DT40Syk− cells were cultured in the same medium containing 2 mg/ml G418 (37Kinch M.S. Sanfridson A. Doyle C. J. Exp. Med. 1994; 180: 1729-1739Crossref PubMed Scopus (13) Google Scholar). Wild type and mutants of Syk were cloned into the expression vector pApuro (38Lankester A.C. van Schijndel G.M.W. Fromme J. Cordell J.L. Van Lier R.A.W. Van Noesel C.J.M. J. Immunol. 1994; 152: 2157-2162PubMed Google Scholar) and transfected into DT40Syk− cells by electroporation using a gene pulser apparatus (Bio-Rad) at 330 V, 250 microfarad, and selected in the presence of 0.5 μg/ml puromycin. Two independent transfectants of each mutant that expressed comparable amounts of Syk by immunoblotting were further selected by Southern blotting of genomic DNA (data not shown). All results are representative of at least three experiments using a minimum of two independent clones expressing wild type or each mutant form of Syk. For induction of tyrosine phosphorylation of Lyn, Syk, and Ras GAP, cells were stimulated with 10 μg/ml anti-chicken IgM mAb (M4) for 3 min. For activation of p90Rsk and p70S6k, cells in an exponential growth phase were serum starved for 8 and 12 h prior to stimulation, respectively. The indicated numbers of cells were stimulated with 10 μg/ml M4 anti-IgM monoclonal Ab or 100 nm phorbol ester (phorbol 12,13-dibutyrate (PDBu), Sigma) for 30 min at 37 °C. Cells were quickly chilled on ice and pelleted by centrifugation immediately after stimulation. Cell pellets were lysed in 500 μl of ice-cold lysis buffer (1 × PBS, 1 mm phenylmethylsulfonyl fluoride, 100 μg/ml soy bean trypsin inhibitor, 20 μg/ml aprotinin, 100 μg/ml leupeptin, 1% Nonidet P-40, 0.1% deoxycholate, 10 mm NaF, 10 mm sodium pyrophosphate, and 100 mm sodium orthovanadate) for 15 min on ice. Cell lysates were clarified by centrifugation at 15,000 rpm for 10 min. Clarified cell lysates were normalized based on protein concentration as determined using the BCA® kit (Pierce), and equal amounts of protein were subjected to immunoprecipitation or immunoblotting for each lysate. For Ras GAP immunoprecipitation, the clarified cell lysates (equivalent to ∼1 × 108 cells) were precleared with normal rabbit Ig overnight at 4 °C before polyclonal anti-Ras GAP Ab (Transduction Laboratories, KY) was added. For Lyn, Syk, p90Rsk, and p70S6k immunoprecipitation, the post-nuclear extracts (equivalent to ∼5 × 107 cells) were incubated directly with polyclonal anti-Lyn Ab (a generous gift from Dr. J. Cambier, CO), anti-Syk Ab (a generous gift from Dr. J. Bolen, DNAX, CA), anti-p90Rsk, or anti-p70S6k (Santa Cruz Biotechnology, CA) at 4 °C overnight. Immune complexes were precipitated with 30 μl of protein A/G plus®-agarose beads (Santa Cruz Biotechnology) for p90Rsk immunoprecipitation or protein A-agarose beads (Life Technologies, Inc.) for all other immunoprecipitations for an additional 3-h incubation at 4 °C. For anti-Lyn and anti-Syk immunoprecipitation, the beads were washed twice with lysis buffer followed by two washes with PBS. For anti-Ras GAP, p90Rsk, and p70S6k immunoprecipitation, the precipitates were washed 5 times with RIPA buffer without SDS (1% Triton X-100, 1% deoxycholate, 158 mm NaCl, 10 mm Tris, pH 7.4, 1 mm EGTA, 1 mmphenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, 100 μg/ml leupeptin, 100 μg/ml soy bean trypsin inhibitor) followed by two washes with lysis buffer and then PBS. Bound proteins were eluted by boiling in Laemmli buffer containing 0.4% dithiothreitol and resolved by 8% SDS-PAGE. Proteins with molecular mass less than 45 kDa were run off gels to obtain better separation of the proteins of interest from the Ab or to obtain increased resolution when determining shifts in mobility. The proteins were transferred to polyvinylidene difluoride membrane and subjected to immunoblotting. The blots were blocked with TNB buffer (30 mm Tris, pH 7.6, 75 mm NaCl, 3% bovine serum albumin) overnight at 4 °C. Polyclonal anti-Syk, anti-p90Rsk, anti-p70S6k Ab, monoclonal anti-Ras GAP Ab (Santa Cruz Biotechnology, CA), or monoclonal anti-phosphotyrosine Ab (4G10) was added at the concentrations suggested by the manufacturers. For anti-Ras GAP and phosphotyrosine immunoblotting, the blots were further incubated with 15 μg/ml polyclonal rabbit anti-mouse Ab for 1 h at 4 °C, whereas in p90Rsk immunoblotting, the blots were incubated with 15 μg/ml polyclonal rabbit anti-goat Ab for 1 h at 4 °C. The blots were then incubated with 1 μCi/ml iodinated protein A for 45 min at room temperature followed by autoradiography. For immunoblotting of ERK1 and phosphorylated MAP kinase, cells were stimulated and lysed as described above. Cell lysates were normalized by BCA® kit (Pierce), and 100 μg of cell lysates (equivalent to ∼2 × 106 cells/lane) were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membrane. The blots were blocked and blotted as described above using anti-ERK1 Ab (Transduction Laboratories, KY) or anti-phospho-MAP kinase Ab (New England Biolabs Inc., MA). The blots were developed as described above. Anti-Rsk immunoprecipitates were prepared as above and washed once with kinase buffer (30 mmTris-HCl, pH 7.4, 10 mm MgCl2, 0.1 mm EGTA, 1 mm dithiothreitol, and 1 mg/ml protein kinase A inhibitor (Santa Cruz Biotechnology)). The immune complexes were resuspended in 12.5 μl of kinase buffer with 0.3 mg/ml S6 peptide (Santa Cruz Biotechnology) and 5 μCi of [γ32P]ATP (6000Ci/mmol), followed by incubation at 30 °C for 10 min. The reactions were terminated by adding 7.5 μl of 3 × Laemmli sample buffer and boiling for 5 min. Samples were resolved on an 18.5% polyacrylamide gel. Gels were dried and subjected to autoradiography followed by quantitation using a PhosphorImager (Molecular Dynamics) and Image QuaNT software. Anti-p70S6k immunoprecipitates were prepared as described above and washed once with kinase buffer (50 mm MOPS, pH 7.2, 1 mm dithiothreitol, 30 μm ATP, 5 mm MgCl2, 1 mg/ml protein kinase A inhibitor). The immune complexes were then resuspended in 12.5 μl of kinase buffer containing 0.3 mg/ml S6 peptide and 5 μCi of [γ-32P]ATP and incubated at 30 °C for 15 min. The reactions were terminated by adding 7.5 μl of 3 × Laemmli buffer and boiled for 5 min. Samples were then resolved on an 18.5% polyacrylamide gel. The gel was then dried and subjected to autoradiography. The relationship between Lyn and Syk in BCR-mediated activation has not been clearly defined. In one study, anti-Ig-induced activation of PLC-γ, a direct substrate of Syk (39Sillman A.L. Monroe J.G. J. Biol. Chem. 1996; 270: 11806-11811Abstract Full Text Full Text PDF Scopus (47) Google Scholar, 40Law C.-L. Chandran K.A. Sidorenko S.P. Clark E.A. Mol. Cell. Biol. 1996; 16: 1305-1315Crossref PubMed Google Scholar), was unaffected in a Lyn-deficient cell line, DT40Lyn−, but abolished in a Syk-deficient cell line, DT40Syk− (26Takata M. Sabe H. Hata A. Inazu T. Homma Y. Nukada T. Yamamura H. Kurosaki T. EMBO J. 1994; 13: 1341-1349Crossref PubMed Scopus (588) Google Scholar). This result suggested that the anti-Ig-induced activation of PLC-γ required Syk activation but could occur in the absence of Lyn. However, it has also been suggested that activation of Syk is abolished in DT40Lyn− cells, suggesting that Lyn is required for activation of Syk (22Kurosaki T. Takata M. Yamanashi Y. Inazu T. Taniguchi T. Yamamoto T. Yamamura H. J. Exp. Med. 1994; 179: 1725-1729Crossref PubMed Scopus (251) Google Scholar). In view of this paradox, we tested the hypothesis that Syk can be activated in the absence of Lyn by directly comparing BCR-mediated signaling in DT40Syk−, DT40Lyn−, and parental DT40 cells. Using an optimal concentration of anti-Ig Ab (10 μg/ml M4), we found that the anti-Ig-induced tyrosine phosphorylation of several species, including proteins with approximate molecular masses of 55 and 65 kDa, was defective in DT40Lyn− cells compared with parental DT40 cells (Fig. 1 A). In contrast, the anti-Ig-induced tyrosine phosphorylation of a different subset of phosphoproteins including species with molecular masses of 87, 100, and 110 kDa was defective in DT40 Syk− cells (Fig.1 A). We have observed these differences consistently in many independent experiments. Consistent with previous reports, the anti-Ig-induced tyrosine phosphorylation of Lyn in DT40Syk− cells was comparable with that in parental cells (Fig. 1 B). Furthermore, we detected anti-Ig-induced tyrosine phosphorylation (Fig. 1 C) and kinase activity (data not shown) of Syk in DT40Lyn− cells, albeit to a lesser extent, compared with parental DT40 cells (Fig. 1 C). Taken together with previous studies, these data indicate that although Lyn may be an upstream effector of Syk in BCR-mediated signaling, Syk can also clearly be phosphorylated via a Lyn-independent pathway in B cells. We then sought to establish whether the suboptimal phosphorylation of Syk in DT40Lyn− resulted in activation of Syk and to identify potential downstream effectors in addition to PLC-γ that could be activated via a Lyn-independent but Syk-dependent pathway. Although we have not yet identified the tyrosine phosphorylated proteins, pp87, pp100, and pp110, present in DT40Lyn− cells but absent in DT40Syk−cells, we have established that these protein species are neither the p85 or p110 subunits of PI3-kinase nor p120c-cbl (data not shown). Since it has been suggested that BCR-induced tyrosine phosphorylation leads to activation of a series of serine/threonine kinases (29Tordai A. Franklin R.A. Patel H. Gardner A.M. Johnson G.L. Gelfand E.W. J. Biol. Chem. 1994; 269: 7538-7543Abstract Full Text PDF PubMed Google Scholar), we investigated whether the phosphorylation and activation of any serine/threonine phosphorylated proteins/kinases requires Syk but could occur in the absence of Lyn. One of the major serine/threonine kinases activated in B cells is p90Rsk (29Tordai A. Franklin R.A. Patel H. Gardner A.M. Johnson G.L. Gelfand E.W. J. Biol. Chem. 1994; 269: 7538-7543Abstract Full Text PDF PubMed Google Scholar). p90Rsk is one member of a serine/threonine kinase family that like the S6 kinase, p70S6k, regulates the phosphorylation and activity of ribosomal S6 protein and that is thought to be involved in translational control during the cell cycle (41Price D.J. Grove J.R. Calvo V. Avruch J. Bierer B.E. Science. 1992; 257: 973-977Crossref PubMed Scopus (589) Google Scholar, 42Edelmann H.M. Kuhne C. Petritsch C. Ballou L.M. J. Biol. Chem. 1996; 271: 963-971Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Based on mobility shift assays (Fig.2 A) and in vitro kinase assays (Fig. 2, B and C), we found that ligation of surface Ig induced activation of p90Rsk in parental DT40 cells as well as DT40Lyn− cells. However, the anti-Ig-induced activation of p90Rsk was abolished in DT40Syk− cells, indicating that Syk is required for anti-Ig-induced activation of p90Rsk (Fig. 2,B and C). The failure to activate p90Rsk in anti-Ig-stimulated DT40Syk− cells was not due to an intrinsic defect in p90Rsk, since stimulation with phorbol ester (PDBu) resulted in similar activation of p90Rsk in DT40Syk−cells, as observed in DT40Lyn− cells or parental DT40 cells. Thus, these data provide additional evidence supporting the hypothesis that Syk can be functionally activated via a Lyn-independent pathway that is sufficient for activation of the pathway leading to the activation of p90Rsk. To date, two mitogen-induced S6 kinase families have been identified, the Rsk family (including p90Rsk) and p70/85 S6 kinases. It was shown that the members of these two families are regulated by different mechanisms (25Couture C. Baier G. Oetken C. Williams S. Telford D. Cardine A.M. Baier-Bitterlich G. Fischer S. Burn P. Altman A. Mustelin T. Mol. Cell. Biol. 1994; 14: 5249-5258Crossref PubMed Google Scholar). It is well documented that cross-linking of BCR induces activation of p90Rsk (29Tordai A. Franklin R.A. Patel H. Gardner A.M. Johnson G.L. Gelfand E.W. J. Biol. Chem. 1994; 269: 7538-7543Abstract Full Text PDF PubMed Google Scholar), but only CD38-induced activation of p70S6k in B cells has been reported (43Patel H.R. Terada N. Gelfand E.W. Biochem. Biophys. Res. Commun. 1996; 227: 507-512Crossref PubMed Scopus (10) Google Scholar, 44Kitanaka A. Ito C. Nishigaki H. Campana D. Blood. 1996; 88: 590-598Crossref PubMed Google Scholar). We sought to determine whether p70S6k could also be activated by BCR-mediated signaling by comparing the mobility of p70S6k on SDS-PAGE and kinase activity of p70S6k in DT40 cells with that in DT40Syk− and DT40Lyn−cells. Cross-linking of surface Ig induced a shift in mobility of p70S6k on SDS-PAGE in parental DT40 cells (Fig. 3,bottom panel). Furthermore, the shift in mobility corrrelated with the activation of p70S6k kinase activity (Fig. 3,top panel). Interestingly, both anti-Ig and PDBu-induced activation of p70S6k were unaffected in DT40Syk− and DT40Lyn− cells compared with parental cells, indicating that either Lyn or Syk is sufficient for anti-Ig-induced activation of p70S6k or that neither kinase mediates the activation of the signal transduction pathway that leads to activation of p70S6k. We next compared the protein tyrosine kinases required for anti-Ig-induced activation of MAPK and Ras GAP since the Ras/Raf/Mek/MAPK pathway has been implicated in activation of p90Rsk in B cells (29Tordai A. Franklin R.A. Patel H. Gardner A.M. Johnson G.L. Gelfand E.W. J. Biol. Chem. 1994; 269: 7538-7543Abstract Full Text PDF PubMed Google Scholar). We measured the anti-Ig-induced phosphorylation of MAPK in DT40Syk− and DT40Lyn− cells since activation of MAPK involves its phosphorylation on tyrosine and threonine residues. Interestingly, we observed that the anti-Ig-induced phosphorylation of MAPK, unlike p90Rsk, is abolished in both DT40Syk− and DT40Lyn− cells compared with parental cells (Fig.4 A). This result indicated that activation of MAPK depends on Syk and Lyn, and thus the BCR-induced activation of p90Rsk in the Lyn-deficient DT40 cells (Fig. 2) must be mediated by an MAPK-independent pathway. These data do not, however, exclude the possibility that MAPK can also mediate anti-Ig-induced activation of p90Rsk in parental DT40 cells. Overall, these data are consistent with previous reports that p90Rsk can be activated with or without activation of MAPK in Xenopus oocytes (36Kalab P. Kubiak J.Z. Verlhac M.H. Colledge W.H. Maro B. Development. 1996; 122: 1957-1964Crossref PubMed Google Scholar). We then examined the anti-Ig-induced tyrosine phosphorylation of Ras GAP, which negatively regulates Ras by converting the GTP-bound Ras (the active form) into the GDP-bound Ras (the inactive form) (34Trahey M.T. Wong G. Halenbeck R. Rubinfeld B. Martin G.A. Ladner M. Long C.M. Crosier W.J. Watt K. Koths K. McCormick F. Science. 1988; 335: 1697-1699Crossref Scopus (304) Google Scholar, 35Vogel Dixon R.A.F. Schaber M.D. Diehl R.E. Marshall M.S. Scolnick E.M. Sigal I.S. Gibbs J.B. Nature. 1988; 335: 90-93Crossref PubMed Scopus (399) Google Scholar). It was previously shown that cross-linking of BCR induced the rapid tyrosine phosphorylation of Ras GAP (27Yagura H. Oyaizu N. Pahwah S. Blood. 1993; 81: 1535-1539Crossref PubMed Google Scholar, 28Gold M.R. Crowley M.T. Martin G.A. McCormick F. DeFranco A.L. J. Immunol. 1993; 150: 377-386PubMed Google Scholar). However, the upstream effectors of this event in B cells were not determined. We found that the anti-Ig-induced tyrosine phosphorylation of Ras GAP is ablated in DT40Syk− and DT40Lyn− cells, suggesting again that both Syk and Lyn are required for this event (Fig.4 B). To map the structural elements required for the activation of Syk-dependent substrates, we transfected DT40Syk− cells with either wi