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• W2088702923 abstract "After engagement of the B cell receptor for antigen, the Syk protein-tyrosine kinase becomes phosphorylated on multiple tyrosines, some of which serve as docking sites for downstream effectors with SH2 or other phosphotyrosine binding domains. The most frequently identified binding partner for catalytically active Syk identified in a yeast two-hybrid screen was the p85 regulatory subunit of phosphoinositide 3-kinase. The C-terminal SH2 domain of p85 was sufficient for mediating an interaction with tyrosine-phosphorylated Syk. Interestingly, this domain interacted with Syk at phosphotyrosine 317, a site phosphorylated in trans by the Src family kinase, Lyn, and identified previously as a binding site for c-Cbl. This site interacted preferentially with the p85 C-terminal SH2 domain compared with the c-Cbl tyrosine kinase binding domain. Molecular modeling studies showed a good fit between the p85 SH2 domain and a peptide containing phosphotyrosine 317. Tyr-317 was found to be essential for Syk to support phagocytosis mediated by FcγRIIA receptors expressed in a heterologous system. These studies establish a new type of p85 binding site that can exist on proteins that serve as substrates for Src family kinases and provide a molecular explanation for observations on direct interactions between Syk and phosphoinositide 3-kinase. After engagement of the B cell receptor for antigen, the Syk protein-tyrosine kinase becomes phosphorylated on multiple tyrosines, some of which serve as docking sites for downstream effectors with SH2 or other phosphotyrosine binding domains. The most frequently identified binding partner for catalytically active Syk identified in a yeast two-hybrid screen was the p85 regulatory subunit of phosphoinositide 3-kinase. The C-terminal SH2 domain of p85 was sufficient for mediating an interaction with tyrosine-phosphorylated Syk. Interestingly, this domain interacted with Syk at phosphotyrosine 317, a site phosphorylated in trans by the Src family kinase, Lyn, and identified previously as a binding site for c-Cbl. This site interacted preferentially with the p85 C-terminal SH2 domain compared with the c-Cbl tyrosine kinase binding domain. Molecular modeling studies showed a good fit between the p85 SH2 domain and a peptide containing phosphotyrosine 317. Tyr-317 was found to be essential for Syk to support phagocytosis mediated by FcγRIIA receptors expressed in a heterologous system. These studies establish a new type of p85 binding site that can exist on proteins that serve as substrates for Src family kinases and provide a molecular explanation for observations on direct interactions between Syk and phosphoinositide 3-kinase. Syk is a 72-kDa protein-tyrosine kinase that plays a central role in coupling immune recognition receptors to multiple downstream signaling pathways (1Turner M. Schweighoffer E. Colucci F. Di Santo J.P. Tybulewicz V.L. Immunol. Today. 2000; 21: 148-154Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 2Latour S. Veillette A. Curr. Opin. Immunol. 2001; 13: 299-306Crossref PubMed Scopus (171) Google Scholar). This function is a property of both its catalytic activity and its ability to participate in interactions with effector proteins containing SH2 1The abbreviations used are: SH2, Src homology 2; BCR, B cell receptor for antigen; CblN, N-terminal domain of c-Cbl; E3, ubiquitin-protein isopeptide ligase; EGFP, enhanced green fluorescence protein; GST, glutathione S-transferase; His-SykL, linker B region of Syk expressed with a hexahistidine tag; p85CSH2, C-terminal SH2 domain of p85; PDGF, platelet-derived growth factor; PI3K, phosphoinositide 3-kinase; pTyr, phosphotyrosine; SykL, Syk linker; TKB, tyrosine kinase binding.1The abbreviations used are: SH2, Src homology 2; BCR, B cell receptor for antigen; CblN, N-terminal domain of c-Cbl; E3, ubiquitin-protein isopeptide ligase; EGFP, enhanced green fluorescence protein; GST, glutathione S-transferase; His-SykL, linker B region of Syk expressed with a hexahistidine tag; p85CSH2, C-terminal SH2 domain of p85; PDGF, platelet-derived growth factor; PI3K, phosphoinositide 3-kinase; pTyr, phosphotyrosine; SykL, Syk linker; TKB, tyrosine kinase binding. domains. For example, after the engagement of antigen receptors on B cells, Syk is phosphorylated on three tyrosines that lie within the linker B region, which separates the N-terminal tandem pair of SH2 domains from the catalytic domain (3Keshvara L.M. Isaacson C. Yankee T.M. Sarac R. Harrison M.L. Geahlen R.L. J. Immunol. 1998; 161: 5276-5283PubMed Google Scholar). Phosphorylation of the first, at Tyr-317 (numbering based on the murine Syk sequence), is catalyzed primarily by Lyn, a Src family kinase. This creates a docking site for c-Cbl, a potential negative regulator of Syk-dependent signaling (4Lupher M.L. Rao N. Lill N.L. Andoniou C.E. Miyake S. Clark E.A. Druker B. Band H. J. Biol. Chem. 1998; 273: 35273-35281Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 5Yankee T.M. Keshvara L.M. Sawasdikosol S. Harrison M.L. Geahlen R.L. J. Immunol. 1999; 163: 5827-5835PubMed Google Scholar). Indeed, mutant forms of Syk containing substitutions of Phe for Tyr at position 317 exhibit enhanced activity in B cells and mast cells (3Keshvara L.M. Isaacson C. Yankee T.M. Sarac R. Harrison M.L. Geahlen R.L. J. Immunol. 1998; 161: 5276-5283PubMed Google Scholar, 5Yankee T.M. Keshvara L.M. Sawasdikosol S. Harrison M.L. Geahlen R.L. J. Immunol. 1999; 163: 5827-5835PubMed Google Scholar, 6Sada K. Zhang J. Siraganian R.P. J. Immunol. 2000; 164: 338-344Crossref PubMed Scopus (55) Google Scholar). To date, c-Cbl is the only protein identified that is capable of binding to Syk at pTyr-317. Phosphorylation of Tyr-342 and 346 forms a docking site for multiple SH2 domain-containing proteins including phospholipase C-γ, Vav, and Fgr (7Law C-L. Chandran K.A. Sidorenko S.P. Clark E.A. Mol. Cell. Biol. 1996; 16: 1305-1315Crossref PubMed Google Scholar, 8Deckert M. Tartare-Deckert S. Couture C. Mustelin T. Altman A. Immunity. 1996; 5: 591-604Abstract Full Text PDF PubMed Scopus (244) Google Scholar, 9Vines C.M. Potter J.W. Xu Y. Geahlen R.L. Costello P.S. Tybulewicz V.L. Lowell C.A. Chang P.W. Gresham H.D. Willman C.L. Immunity. 2001; 15: 507-519Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 10Hong J. Yankee T.M. Harrison M.L. Geahlen R.L. J. Biol. Chem. 2002; 277: 31703-31714Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Mutant forms of Syk containing substitutions of Phe for Tyr-342 or both Tyr-342 and 346 exhibit a reduced ability to couple immune recognition receptors to the activation of downstream effectors such as phospholipase C-γ2 in B cells and mast cells (10Hong J. Yankee T.M. Harrison M.L. Geahlen R.L. J. Biol. Chem. 2002; 277: 31703-31714Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 11Zhang J. Berenstein E. Siraganian R.P. Mol. Cell. Biol. 2002; 22: 8144-8154Crossref PubMed Scopus (50) Google Scholar).Syk also is required for the activation of phosphoinositide 3-kinase (PI3K) in response to a variety of signals (12Beitz L.O. Fruman D.A. Kurosaki T. Cantley L.C. Scharenberg A.M. J. Biol. Chem. 1999; 274: 32662-32666Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 13Crowley M.T. Costello P.S. Fitzer-Attas C.J. Turner M. Meng F. Lowell C. Tybulewicz V.L. DeFranco A.L. J. Exp. Med. 1997; 186: 1027-1039Crossref PubMed Scopus (404) Google Scholar, 14Raeder E.M. Mansfield P.J. Hinkovska-Galcheva V. Shayman J.A. Boxer L.A. J. Immunol. 1999; 163: 6785-6793PubMed Google Scholar, 15Jiang K. Zhong B. Ritchey C. Gilvary D.L. Hong-Geller E. Wei S. Djeu J.Y. Blood. 2003; 101: 236-244Crossref PubMed Scopus (57) Google Scholar, 16Jiang K. Zhong B. Gilvary D.L. Corliss B.C. Vivier E. Hong-Geller E. Wei S. Djeu J.Y. J. Immunol. 2002; 168: 3155-3164Crossref PubMed Scopus (103) Google Scholar, 17Ding J. Takano T. Gao S. Han W. Noda C. Yanagi S. Yamamura H. J. Biol. Chem. 2000; 275: 30873-30877Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 18Pogue S.L. Kurosaki T. Bolen J. Herbst R. J. Immunol. 2000; 165: 1300-1306Crossref PubMed Scopus (124) Google Scholar) including engagement of the B cell antigen receptor (BCR) (12Beitz L.O. Fruman D.A. Kurosaki T. Cantley L.C. Scharenberg A.M. J. Biol. Chem. 1999; 274: 32662-32666Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) and macrophage or neutrophil Fcγ receptors (13Crowley M.T. Costello P.S. Fitzer-Attas C.J. Turner M. Meng F. Lowell C. Tybulewicz V.L. DeFranco A.L. J. Exp. Med. 1997; 186: 1027-1039Crossref PubMed Scopus (404) Google Scholar, 14Raeder E.M. Mansfield P.J. Hinkovska-Galcheva V. Shayman J.A. Boxer L.A. J. Immunol. 1999; 163: 6785-6793PubMed Google Scholar). Furthermore, the expression of a constitutively active TEL-Syk fusion protein in atypical myelodysplastic syndrome leads to the constitutive activation of PI3K (19Kanie T. Abe A. Matsuda T. Kuno Y. Towatari M. Yamamoto T. Saito H. Emi N. Naoe T. Leukemia. 2004; 18: 548-555Crossref PubMed Scopus (39) Google Scholar). The mechanisms by which Syk is coupled to the activation of PI3K appear to be either direct or indirect. In B cells, the BCR-stimulated activation of PI3K can be accomplished through the phosphorylation of adaptor proteins such as BCAP, CD19, or Gab1, which creates binding sites for the p85 regulatory subunit (20Okada T. Maeda A. Iwamatsu A. Gotoh K. Kurosaki T. Immunity. 2000; 13: 817-827Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 21Buhl A.M. Cambier J.C. J. Immunol. 1999; 162: 4438-4446PubMed Google Scholar, 22Ingham 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). Signals transmitted by many IgG receptors require the activities of both Syk and PI3K and their recruitment to the site of the clustered receptor (23Cooney D.S. Phee H. Jacob A. Coggeshall K.M. J. Immunol. 2001; 167: 844-854Crossref PubMed Scopus (50) Google Scholar). In neutrophils and monocytes, a direct association of p85 with phosphorylated immunoreceptor tyrosine-based activation motif sequences on FcγRIIA was proposed as a mechanism for the recruitment of PI3K to the receptor (24Chacko G.W. Brandt J.T. Coggeshall K.M. Anderson C.L. J. Biol. Chem. 1996; 271: 10775-10781Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), whereas in platelets, an association of PI3K with receptor-bound Syk has been suggested (25Chacko G.W. Duchemin A.M. Coggeshall K.M. Osborne J.M. Brandt J.T. Anderson C.L. J. Biol. Chem. 1994; 269: 32435-32440Abstract Full Text PDF PubMed Google Scholar). A thrombin-stimulated association of Syk with PI3K in platelets also has been reported (26Yanagi S. Sada K. Tohyama Y. Tsubokawa M. Nagai K. Yonezawa K. Yamamura H. Eur. J. Biochem. 1994; 224: 329-333Crossref PubMed Scopus (29) Google Scholar). In cells expressing TEL-Syk, PI3K binds directly to the fusion protein, which is constitutively activated and phosphorylated because of dimerization mediated by the TEL SAM or helix-loop-helix domain (19Kanie T. Abe A. Matsuda T. Kuno Y. Towatari M. Yamamoto T. Saito H. Emi N. Naoe T. Leukemia. 2004; 18: 548-555Crossref PubMed Scopus (39) Google Scholar).The mechanism by which a direct interaction of Syk with PI3K could occur is not immediately obvious. The SH2 domains of p85 preferentially recognize the sequence pYXXM as a binding motif (27Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2373) Google Scholar). Although Syk has three such motifs, none of these is apparently phosphorylated in vivo or in vitro (3Keshvara L.M. Isaacson C. Yankee T.M. Sarac R. Harrison M.L. Geahlen R.L. J. Immunol. 1998; 161: 5276-5283PubMed Google Scholar, 28Furlong M.T. Mahrenholz A.M. Kim K.H. Ashendel C.L. Harrison M.L. Geahlen R.L. Biochim. Biophys. Acta. 1997; 1355: 177-190Crossref PubMed Scopus (94) Google Scholar). In this study, we report that p85 can, in fact, interact directly with Syk in an interaction mediated by the SH2 domains of p85 binding to specific phosphotyrosines on Syk. In fact, we found that p85 was the major Syk-binding protein identified in yeast two-hybrid screens using libraries from two different sources. Interestingly, the binding of the C-terminal SH2 domain of p85 to pTyr-317 was a major contributor to this interaction. In vitro, the apparent affinity of the p85 C-terminal SH2 domain for pTyr-317 was greater than that of the c-Cbl tyrosine kinase binding (TKB) domain. Molecular modeling was used to investigate the potential structural determinants for recognition between the p85 C-terminal SH2 domain and pTyr-317 of Syk.MATERIALS AND METHODSYeast Two-hybrid Analyses—The cDNAs for wild-type Syk (Syk(WT)), Syk(Y317F), and the Syk catalytic domain (SykK, amino acids 370–629), were amplified by PCR from the corresponding pGEM vectors (3Keshvara L.M. Isaacson C. Yankee T.M. Sarac R. Harrison M.L. Geahlen R.L. J. Immunol. 1998; 161: 5276-5283PubMed Google Scholar) and inserted into the pGBKT7 yeast expression vector (Clontech) to generate the various Syk-GAL4 DNA binding domain fusions. A DNA fragment digested from pGEM-Syk(K396R) with EcoRI and BglII was inserted into predigested pGBKT7-Syk(WT) to generate pGBKT7-Syk(K396R). The cDNA for the human Lck SH2 domain (amino acids 126–234) was PCR amplified and inserted into the yeast expression plasmid pACT2 to generate the LckSH2-GAL4-transcriptional activation domain fusion. Y187 yeast cells transformed with a bone marrow cDNA library were purchased from Clontech and mated with AH109 yeast cells transformed with pGBKT7-Syk(WT). Plasmids were eluted from colonies that grew on stringent selection media (His-, Leu-, Trp-, and Ade-), amplified in competent bacterial cells, isolated, and sequenced. For analysis of interactions between specific proteins, Y187 yeast cells were transformed with the pACT2 plasmid containing the cDNA of interest and mated with AH109 cells transformed with the pGBKT7 plasmid containing the appropriate DNA insert. Mated transformants were grown on stringent selection media.Protein Interactions with GST-Syk—Insect viruses for the expression of GST-Syk fusion proteins were described previously (29Peters J.D. Furlong M.T. Asai D.J. Harrison M.L. Geahlen R.L. J. Biol. Chem. 1996; 271: 4755-4762Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). A baculovirus for the expression of p85α was generously provided by Lewis Cantley (Harvard University). Sf9 cells expressing GST fusion proteins were lysed by sonication in lysis buffer containing 20 mm Hepes, pH 7.5, 150 mm NaCl, 5 mm EDTA, 1% Nonidet P-40, and 10 μg/ml each aprotinin and leupeptin. GST fusion proteins were isolated by adsorption to glutathione-agarose. Immobilized GST-Syk, GST-p42.5, and GST-p35 were autophosphorylated in vitro by incubation at 37 °C for 15 min in kinase buffer (50 mm Hepes, pH 7.5, 10 mm MnCl2, 1 mm Na3VO4, 0.05 mm [γ-32P]ATP, and 10 μg/ml each aprotinin and leupeptin). Immobilized proteins were incubated with lysates of Sf9 cells expressing p85α and then washed extensively with wash buffer containing 20 mm Hepes, pH 7.5, 250 mm NaCl, 5 mm EDTA, 0.5 m LiCl, and 1% Nonidet P-40. Bound proteins were eluted in SDS-sample buffer, separated by SDS-PAGE, and detected by Western blotting with anti-p85 (Upstate Biotechnology, Inc.) or anti-GST (Santa Cruz Biotechnology, Inc.) antibodies. In some assays, resin-bound, phosphorylated GST-Syk fusion proteins were eluted with 20 mm glutathione, and the soluble fusion proteins were incubated with either immune complexes containing p85α bound to anti-p85 immobilized on protein A-Sepharose or His-tagged LckSH2 bound to His-select HC nickel affinity gel (Sigma). The p85-anti-p85 immune complexes were prepared by incubating protein A-Sepharose beads containing anti-p85 antibody with lysates of Sf9 cells expressing p85. The vector for expression of the His-tagged Lck SH2 domain (LckSH2) in bacteria was constructed by insertion of the appropriate PCR-amplified fragment into Ndel and XhoI double digested pRGT7 (provided by Dr. Hyunho Chung, LG Chem). Beads were washed as above. Bound fusion proteins were separated by SDS-PAGE and detected by autoradiography.Interaction of Proteins with Cellular Syk Family Kinases in Cell Lysates—Jurkat T cells, DG75 B cells, and THP-1 monocytes were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 50 μm 2-mercaptoethanol, 2 mm glutamine, 1 mm sodium pyruvate, 100 IU/ml penicillin G, and 100 μg/ml streptomycin. The chicken DT40 B cell line stably expressing a Myc epitope-tagged murine Syk was described previously (3Keshvara L.M. Isaacson C. Yankee T.M. Sarac R. Harrison M.L. Geahlen R.L. J. Immunol. 1998; 161: 5276-5283PubMed Google Scholar). Cells were treated for 15 min at 37 °C in the presence or absence of pervanadate (0.1 mm sodium orthovanadate and 0.5 mm H2O2). THP-1 cells also were preincubated for 30 min at room temperature with 2 mm dithiobis(succinimidyl propionate) (Pierce Biotechnology). Cells (1 × 107) were lysed with 0.5 ml of lysis buffer as described above. For coimmunoprecipitation experiments, lysate supernatants were incubated at 4 °C for 4 h with protein A-Sepharose preadsorbed to the anti-p85α antibody. The protein/bead mixture was washed three times with wash buffer and then incubated in kinase buffer prior to separation by SDS-PAGE. Bound Syk was detected by Western blotting and by autoradiography. Lysates of Jurkat, DG75, or DT40 B cells that had been treated with or without pervanadate also were incubated with fusion proteins of GST linked to the C-terminal SH2 domain of p85α (p85CSH2), the N terminus of c-Cbl (GST-CblN) or an inactive mutant of c-Cbl (CblN(G306E)) immobilized to glutathione-agarose. The pGEX4T-2-p85CSH2 plasmid coding p85CSH2 (amino acids 565–724) for expression in bacteria was constructed by in-frame insertion of PCR-amplified p85CSH2 cDNA into pGEX4T-2 (Amersham Biosciences). Plasmids for the expression of GST-CblN and GST-CblN(G306E) were generously supplied by Hamid Band (Northwestern University).Interactions with the Syk Linker Region—The cDNA for the linker region of Syk (SykL, amino acids 257–363) was amplified by PCR and inserted into the pET15b expression plasmid (Novagen) to generate pET15b-His-SykL. For the purification of His-SykL, transformed bacterial cells (50-ml culture) were lysed on ice by sonication in 10 ml of denaturing cell lysis buffer (0.1 m sodium phosphate, pH 8.0, 8 m urea). 10 ml of cell lysate was loaded on the nickel-resin column. After washing with the same buffer, target proteins bound to the resin were renatured with buffer containing 50 mm sodium phosphate, pH 7.5, and 150 mm sodium chloride. The resin-bound His-SykL was phosphorylated in vitro by incubation in kinase buffer containing a mixture of GST-Syk and Lck. Lck was isolated by immunoprecipitation from lysates of Sf9 cells infected with a baculovirus directing the expression of Lck (30Nadler M.J.S. Harrison M.L. Ashendel C.L. Cassady J.M. Geahlen R.L. Biochemistry. 1993; 32: 9250-9255Crossref PubMed Scopus (42) Google Scholar). Phosphorylated His-SykL was finally eluted from the resin with elution buffer (50 mm sodium phosphate, pH 8.0, 0.3 m sodium chloride, 250 mm imidazole) and incubated with GST-p85CSH2, GST-FgrSH2, or GST-CblN immobilized onto glutathione-agarose. Bound SykL was detected by autoradiography after SDS-PAGE or by Western blotting with a phosphospecific antibody recognizing Syk phosphorylated on Y317 (Cell Signaling Technologies). A bacterial expression plasmid for the GST-Fgr SH2 domain was generously provided by Dr. Cheryl L. Willman (University of New Mexico).Interactions with Synthetic Peptides—Synthetic polypeptides corresponding to sequences surrounding Y317, TVSFNPYEPTGGP, TVSFNPpYEPTGGP, and TVSFNPpYEPELAP, were purchased from Advanced ChemTech. Peptides were covalently coupled to Affi-Gel 10 (Bio-Rad) by incubating 15 mg of each peptide with 500 μl of resin in 10 mm Hepes, pH 7.5, at 4 °C for 12 h. The coupling reaction was stopped by the addition of 0.1 ml of 1 m ethanolamine HCl, pH 8.0. For binding studies, 100 μl of the peptide-resin suspension was mixed with purified GST-p85CSH2, GST-FgrSH2, or GST-CblN at 4 °C for 2 h and washed three times with buffer containing 10 mm Hepes, pH 7.5, 150 mm NaCl, and 5 mm EDTA. Bound proteins were separated by SDS-PAGE and detected by Western blotting with an anti-GST antibody.Phosphopeptide Mapping—For the identification of Syk-derived phosphopeptides, Syk or SykL, phosphorylated in vitro, were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The radiolabeled bands were excised and digested with trypsin. Phosphopeptides were either separated by electrophoresis on an alkaline 40% polyacrylamide gel or were first adsorbed to GST-p85CSH2 immobilized on glutathione-agarose as described previously prior to separation. The phosphopeptides were detected by autoradiography and their identities determined by their migration positions as described previously (3Keshvara L.M. Isaacson C. Yankee T.M. Sarac R. Harrison M.L. Geahlen R.L. J. Immunol. 1998; 161: 5276-5283PubMed Google Scholar).Molecular Modeling—The peptide pYEPTG, derived from residues 317–321 of Syk linker B, was modeled in the phosphopeptide binding site of p85CSH2 based on the crystal structure (PDB entry 1H9O) for the complex of this SH2 domain with the peptide pYVPML from the platelet-derived growth factor (PDGF) receptor. Initial heavy atom coordinates were obtained for the Syk-derived peptide by retaining coordinates from 1H90 for all atoms common to both peptides. Initial positions for heavy atoms unique to pYEPTG were built without steric overlap using the molecular graphics program QUANTA. The peptide N terminus was acetylated, and the C terminus was amidated. Hydrogen atom positions were built with the HBUILD facility of CHARMM. The p85CSH2·pYEPTG complex was conformationally relaxed using molecular dynamics and energy minimization calculated with the CHARMM program and the all hydrogen CHARMM27 force field. To preserve the effects of solvation during the relatively short time period of the conformational relaxation, the crystallographic water molecules were included in the molecular mechanics calculations using the TIP3P water model. The molecular mechanics protocol included 1,000 steps of Powell energy minimization, followed by 50 ps of molecular dynamics with a temperature ramp from 400 to 100 K and 1000 steps of Powell energy minimization. The root mean square coordinate difference between the initial structure modeled directly from 1H90 and the final structure from conformational relaxation was 1.2 Å for main chain atoms and 2.0 Å for all heavy atoms.Phagocytosis Assay—COS-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Phagocytosis assays in COS-7 cells transfected with expression vectors coding for FcγRIIA and the various Syk mutants were as described previously (31Kim J.S. Peng X. De P.K. Geahlen R.L. Durden D.L. Blood. 2002; 99: 694-697Crossref PubMed Scopus (36) Google Scholar). In brief, cells were transiently transfected using Lipofectamine with plasmids coding for FcγRIIA and either enhanced green fluorescence protein (EGFP) or wild-type or mutant Syk fused to EGFP (Syk-EGFP). The expression levels of EGFP, Syk-EGFP, and FcγRIIA were compared by flow cytometry with FcγRIIA levels measured using an allophycocyanin-conjugated anti-CD32 monoclonal antibody (FLI8.26) (Pharmingen). Transfected cells were incubated with sheep red blood cells coated with IgG at a subagglutinating concentration at a target to effector ratio of 200:1 for 2 h. After uningested red blood cells were lysed in water, COS-7 cells were spun onto glass slides, fixed, stained with Wright Giemsa stain, and counted for internalized red blood cells. Alternatively, cell lysates were prepared and immunoblotted for activated Akt using an anti-phospho-Akt antibody (Cell Signaling Technologies).RESULTSThe p85 Regulatory Subunit of PI3K Interacts with Syk in a Yeast Two-hybrid Screen—To identify proteins capable of interacting directly with Syk, we screened a human bone marrow cDNA library using the yeast two-hybrid method. We reasoned that proteins capable of binding to sites of tyrosine-phosphorylation on Syk would interact with the catalytically active form of the kinase because Syk can catalyze the autophosphorylation of all of the relevant tyrosines that also are phosphorylated in vivo (3Keshvara L.M. Isaacson C. Yankee T.M. Sarac R. Harrison M.L. Geahlen R.L. J. Immunol. 1998; 161: 5276-5283PubMed Google Scholar, 28Furlong M.T. Mahrenholz A.M. Kim K.H. Ashendel C.L. Harrison M.L. Geahlen R.L. Biochim. Biophys. Acta. 1997; 1355: 177-190Crossref PubMed Scopus (94) Google Scholar). This approach requires that a fusion protein of Syk with the GAL4 DNA binding domain be active when expressed in yeast and capable of autophosphorylation. To explore this, we first examined the ability of Syk to interact in the two-hybrid screen with a protein consisting of the GAL4 transactivation domain fused to the SH2 domain derived from Lck, a Src family kinase. The Lck SH2 domain has been shown previously to bind to phosphorylated forms of Syk (32Couture C. Deckert M. Williams S. Russo F.O. Altman A. Mustelin T. J. Biol. Chem. 1996; 271: 24294-24299Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) and the Syk family kinase, Zap-70 (33Williams B.L. Irvin B.J. Sutor S.L. Chini C.C.S. Yacyshyn E. Wardenburg J.B. Dalton M. Chan A.C. Abraham R.T. EMBO J. 1999; 18: 1832-1844Crossref PubMed Google Scholar). We expressed in yeast fusion proteins containing either catalytically active, wild-type Syk, or a catalytically inactive mutant (Syk(K396R)). In a two-hybrid screen, wild-type Syk, but not Syk(K396R), interacted with the Lck SH2 domain fusion protein (Fig. 1). This is consistent with a model in which Syk can undergo autophosphorylation in yeast to create a site for interaction with proteins containing SH2 domains. The two-hybrid screen was then carried out using the bone marrow cDNA library, which produced a number of positive clones of which nearly 50% could be accounted for by one of eight different cDNAs coding for various regions of either the α or β isoforms of p85 (Fig. 1C). Similar results were observed when a human mammary gland library was substituted for the bone marrow library (data not shown). These results indicate that p85 is capable of a direct physical interaction with Syk.Fig. 1The p85 subunit of PI3K interacts with catalytically active Syk in yeast.A, yeast transformants expressing the Lck SH2 domain fused to the GAL4 transcriptional activation domain (LckSH2) and either wild-type Syk or catalytically inactive Syk(K396R) fused to the GAL4 DNA binding domain were grown on plates in media deficient in adenine and histidine. B, growth of transformants described above in liquid selection media. C, schematic diagram of eight independent clones of p85 identified in a yeast two-hybrid screen using a bone marrow cDNA library. D, yeast transformants expressing Syk, Syk(K396R), or p53 fused to the GAL4 DNA binding domain and the indicated proteins fused to the GAL4 transcriptional activation domain were grown on plates containing media deficient in adenine and histidine.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The Binding of p85 Requires the Tyrosine Phosphorylation of Syk—Because the ability of Syk to bind p85 physically had not been described in detail previously, we explored the mechanism of this interaction. To test for a role for protein phosphorylation, we used the yeast two-hybrid screen to compare the binding of p85 SH2 domains with wild-type or catalytically inactive Syk (Syk(K396R)). Both the Lck SH2 domain and Fgr were included as prey for positive controls and p53 as bait to confirm specificity. Fgr is an Src family kinase that also was positively identified in our bone marrow two-hybrid screen and was recently identified as a protein that interacts selectively with Syk to negatively regulate the integrin-mediated spreading of macrophages on ICAM-1 (9Vines C.M. Potter J.W. Xu Y. Geahlen R.L. Costello P.S. Tybulewicz V.L. Lowell C.A. Chang P.W. Gresham H.D. Willman C.L. Immunity. 2001; 15: 507-519Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The small ribosomal subunit protein, S10, which was also identified in the screen as a protein that reacts with multiple baits in an apparently nonspecific fashion, was also included. Neither the C-terminal SH2 domain nor constructs containing both the N + C-terminal SH2 domains of either the α or β isoforms of p85 were capable of interacting with either Syk(K396R) or p53 (Fig. 1D). Similarly, neither the Lck SH2 domain nor Fgr interacted with Syk(K396R) or p53 as expected. This result suggested that the interaction of Syk with p85 was mediated by SH2 domains interacting with sites of tyrosine phosphorylation on Syk.To confirm an interaction biochemically, we examined the ability of a GST-Syk fusion protein to bind to p85. GST-Syk was adsorbed to glutathione-agarose and then was left either untreated or was incubated with [γ-32P]ATP to allow autophosphorylation. Resin-bound Syk proteins were then incubated with lysates of insect cells expressing full-length, untagged p85" @default.
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• W2088702923 date "2005-01-01" @default.
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• W2088702923 title "Molecular Basis for a Direct Interaction between the Syk Protein-tyrosine Kinase and Phosphoinositide 3-Kinase" @default.
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• W2088702923 doi "https://doi.org/10.1074/jbc.m407805200" @default.
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