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• W2016398041 abstract "The regulatory subunit of phosphatidylinositol 3-kinase, p85, contains a number of well defined domains involved in protein-protein interactions, including an SH3 domain and two SH2 domains. In order to investigate in detail the nature of the interactions of these domains with each other and with other binding partners, a series of deletion and point mutants was constructed, and their binding characteristics and apparent molecular masses under native conditions were analyzed. The SH3 domain and the first proline-rich motif bound each other, and variants of p85 containing the SH3 and BH domains and the first proline-rich motif were dimeric. Analysis of the apparent molecular mass of the deletion mutants indicated that each of these domains contributed residues to the dimerization interface, and competition experiments revealed that there were intermolecular SH3 domain-proline-rich motif interactions and BH-BH domain interactions mediating dimerization of p85α bothin vitro and in vivo. Binding of SH2 domain ligands did not affect the dimeric state of p85α. Recently, roles for the p85 subunit have been postulated that do not involve the catalytic subunit, and if p85 exists on its own we propose that it would be dimeric. The regulatory subunit of phosphatidylinositol 3-kinase, p85, contains a number of well defined domains involved in protein-protein interactions, including an SH3 domain and two SH2 domains. In order to investigate in detail the nature of the interactions of these domains with each other and with other binding partners, a series of deletion and point mutants was constructed, and their binding characteristics and apparent molecular masses under native conditions were analyzed. The SH3 domain and the first proline-rich motif bound each other, and variants of p85 containing the SH3 and BH domains and the first proline-rich motif were dimeric. Analysis of the apparent molecular mass of the deletion mutants indicated that each of these domains contributed residues to the dimerization interface, and competition experiments revealed that there were intermolecular SH3 domain-proline-rich motif interactions and BH-BH domain interactions mediating dimerization of p85α bothin vitro and in vivo. Binding of SH2 domain ligands did not affect the dimeric state of p85α. Recently, roles for the p85 subunit have been postulated that do not involve the catalytic subunit, and if p85 exists on its own we propose that it would be dimeric. The Class IA phosphatidylinositol 3-kinases (PI3K) 1The abbreviations used are: PI3K, phosphatidylinositol 3-kinases; BH, BCR homology; PRM, proline-rich motif; pY, phosphotyrosine; HP-SEC, high performance-size exclusion chromatography; DTT, dithiothreitol; GST, glutathioneS-transferase; PAGE, polyacrylamide gel electrophoresis; SE-AUC, sedimentation equilibrium-analytical ultracentrifugation1The abbreviations used are: PI3K, phosphatidylinositol 3-kinases; BH, BCR homology; PRM, proline-rich motif; pY, phosphotyrosine; HP-SEC, high performance-size exclusion chromatography; DTT, dithiothreitol; GST, glutathioneS-transferase; PAGE, polyacrylamide gel electrophoresis; SE-AUC, sedimentation equilibrium-analytical ultracentrifugationare heterodimeric enzymes with a p110 catalytic subunit and a p85 regulatory subunit (1Zvelebil M.J. MacDougall L. Leevers S. Volinia S. Vanhaesebroeck B. Gout I. Panayotou G. Domin J. Stein R. Pages F. Koga H. Salim K. Linacre J. Das P. Panaretou C. Wetzker R. Waterfield M. Philos. Trans. R. Soc. Lond. Biol. Sci. 1996; 351: 217-223Crossref PubMed Scopus (88) Google Scholar). The p85 subunit is a multidomain protein comprising an amino-terminal SH3 domain, a BCR homology (BH) domain which has homology to the GTPase activating protein domain of the Break-point Cluster Region protein (2Diekmann D. Brill S. Garrett M.D. Totty N. Hsuan J. Monfries C. Hall C. Lim L. Hall A. Nature. 1991; 351: 400-402Crossref PubMed Scopus (352) Google Scholar) and Rho subfamily GTPases, and two SH2 domains separated by an inter-SH2 domain through which p85 binds the catalytic subunit (3Dhand R. Hara K. Hiles I. Bax B. Gout I. Panayotou G. Fry M.J. Yonezawa K. Kasuga M. Waterfield M.D. EMBO J. 1994; 13: 511-521Crossref PubMed Scopus (295) Google Scholar). The BH domain is flanked by two proline-rich motifs. To date, five isoforms of p85 have been identified. p85α has been cloned from bovine (4Otsu M. Hiles I. Gout I. Fry M.J. Ruiz Larrea F. Panayotou G. Thompson A. Dhand R. Hsuan J. Totty N. Smith A.D. Morgan S.J. Courtneidge S.A. Parker P.J. Waterfield M.D. Cell. 1991; 65: 91-104Abstract Full Text PDF PubMed Scopus (538) Google Scholar), human (5Skolnik E.Y. Margolis B. Mohammadi M. Lowenstein E. Fischer R. Drepps A. Ullrich A. Schlessinger J. Cell. 1991; 65: 83-90Abstract Full Text PDF PubMed Scopus (435) Google Scholar), and mouse (6Escobedo J.A. Navankasattusas S. Kavanaugh W.M. Milfay D. Fried V.A. Williams L.T. Cell. 1991; 65: 75-82Abstract Full Text PDF PubMed Scopus (372) Google Scholar) cDNA libraries, whereas only bovine p85β has been identified (4Otsu M. Hiles I. Gout I. Fry M.J. Ruiz Larrea F. Panayotou G. Thompson A. Dhand R. Hsuan J. Totty N. Smith A.D. Morgan S.J. Courtneidge S.A. Parker P.J. Waterfield M.D. Cell. 1991; 65: 91-104Abstract Full Text PDF PubMed Scopus (538) Google Scholar). Two splice variants of p85α, termed p55 and p50, have been identified in the human (7Antonetti D.A. Algenstaedt P. Kahn C.R. Mol. Cell. Biol. 1996; 16: 2195-2203Crossref PubMed Scopus (125) Google Scholar), the rat (8Inukai K. Anai M. Van Breda E. Hosaka T. Katagiri H. Funaki M. Fukushima Y. Ogihara T. Yazaki Y. Kikuchi Oka Y. Asano T. J. Biol. Chem. 1996; 271: 5317-5320Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 9Inukai K. Funaki M. Ogihara T. Katagiri H. Kanda A. Anai M. Fukushima Y. Hosaka T. Suzuki M. Shin B.C. Takata K. Yazaki Y. Kikuchi M. Oka Y. Asano T. J. Biol. Chem. 1997; 272: 7873-7882Crossref PubMed Scopus (147) Google Scholar), and the mouse (10Fruman D.A. Cantley L.C. Carpenter C.L. Genomics. 1996; 37: 113-121Crossref PubMed Scopus (99) Google Scholar). p55α lacks the SH3 and BH domains and the first proline-rich motif (PRM1) but retains the second proline-rich motif (PRM2) and has an amino-terminal extension of 34 amino acids. In p50α, this extension comprises only 6 residues. To date, no splice variants of p85β have been identified. A variant known as p55γ or p55PIK has been cloned from bovine 2F. Pagès and M. D. Waterfield, unpublished results.2F. Pagès and M. D. Waterfield, unpublished results. and human (11Pons S. Asano T. Glasheen E. Miralpeix M. Zhang Y. Fisher T.L. Myers Jr., M.G. Sun X.J. White M.F. Mol. Cell. Biol. 1995; 15: 4453-4465Crossref PubMed Scopus (228) Google Scholar) cDNA libraries and is homologous to p55α, but no higher molecular mass isoforms of this protein have yet been identified. PI3K has been implicated in a wide range of signaling pathways including those regulating proliferation and cell migration (12Vanhaesebroeck B. Leevers S.J. Panayotou G. Waterfield M.D. Trends Biochem. Sci. 1997; 22: 267-272Abstract Full Text PDF PubMed Scopus (825) Google Scholar). The modular domains in p85α possess intrinsic signaling functions, in that they bind numerous intracellular ligands and mediate the formation of multiprotein complexes. The proline-rich motifs bind the SH3 domains of the Src family tyrosine kinases, Lyn and Fyn (13Pleiman C.M. Hertz W.M. Cambier J.C. Science. 1994; 263: 1609-1612Crossref PubMed Scopus (393) Google Scholar), whereas the small G proteins Rac (14Tolias K.F. Cantley L.C. Carpenter C.L. J. Biol. Chem. 1995; 270: 17656-17659Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar, 15Nobes C.D. Hawkins P. Stephens L. Hall A. J. Cell Sci. 1995; 108: 225-233Crossref PubMed Google Scholar) and Cdc42 (16Zheng Y. Bagrodia S. Cerione R.A. J. Biol. Chem. 1994; 269: 18727-18730Abstract Full Text PDF PubMed Google Scholar) are potential PI3K regulators or effectors that bind PI3K, presumably via the BH domain. The SH2 domains of p85 bind phosphotyrosine (pY)-containing sequences from a range of receptor tyrosine kinases and docking proteins such as insulin receptor substrate 1 (17Songyang 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 (2371) Google Scholar, 18Panayotou G. Waterfield M.D. BioEssays. 1993; 15: 171-177Crossref PubMed Scopus (114) Google Scholar, 19Backer J.M. Myers Jr., M.G. Shoelson S.E. Chin D.J. Sun X.J. Miralpeix M. Hu P. Margolis B. Skolnik E.Y. Schlessinger J. White M.F. EMBO J. 1992; 11: 3469-3479Crossref PubMed Scopus (811) Google Scholar). The roles of the various isoforms of the adaptor subunits of the Class IA PI3Ks are as yet undefined. Some isoforms, especially the truncated isoforms p55α and p50α, have been shown to have restricted tissue distributions (9Inukai K. Funaki M. Ogihara T. Katagiri H. Kanda A. Anai M. Fukushima Y. Hosaka T. Suzuki M. Shin B.C. Takata K. Yazaki Y. Kikuchi M. Oka Y. Asano T. J. Biol. Chem. 1997; 272: 7873-7882Crossref PubMed Scopus (147) Google Scholar) compared with the 85-kDa isoforms. In addition, the truncated isoforms are clearly unable to interact with proline-rich motif- or SH3 domain-containing proteins or with small G proteins, and it has been shown that there is some selectivity in the recruitment of p85 isoforms by receptor tyrosine kinases (20Shepherd P.R. Nave B.T. Rincon J. Nolte L.A. Bevan A.P. Siddle K. Zierath J.R. Wallberg Henriksson H. J. Biol. Chem. 1997; 272: 19000-19007Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). However, it is not yet understood whether the large number of isoforms of both subunits of PI3K represents a functional redundancy or reflects a form of signaling specificity. We have therefore undertaken a detailed study of the potential interactions of the individual domains of the p85 protein in order to elucidate their roles within the adaptor subunit as a whole. Peptides were synthesized by Zinsser Analytic or Alta Biosciences and were of the following sequences: P1, KISPPTPKPRPPRPLPVAPGPS; P2, WNERQQPAPALPPKPPKPT; Tyr-740/Tyr-751, GGpYMDMSKDESVDpYVPML; Tyr-740, GGpYMDMSKDESVDYVPML; Tyr-751, GGYMDMSKDESVDpYVPML (where pY represents a phosphotyrosine residue). Subcloning of cDNAs encoding p85α and p85β into the pAcC4 baculovirus transfer vector has been previously described (21Gout I. Dhand R. Panayotou G. Fry M.J. Hiles I. Otsu M. Waterfield M.D. Biochem. J. 1992; 288: 395-405Crossref PubMed Scopus (57) Google Scholar). p55γ is the bovine homologue of human p55PIK (11Pons S. Asano T. Glasheen E. Miralpeix M. Zhang Y. Fisher T.L. Myers Jr., M.G. Sun X.J. White M.F. Mol. Cell. Biol. 1995; 15: 4453-4465Crossref PubMed Scopus (228) Google Scholar) and rat p55γ (8Inukai K. Anai M. Van Breda E. Hosaka T. Katagiri H. Funaki M. Fukushima Y. Ogihara T. Yazaki Y. Kikuchi Oka Y. Asano T. J. Biol. Chem. 1996; 271: 5317-5320Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). p49α is the bovine homologue of rat p50α (9Inukai K. Funaki M. Ogihara T. Katagiri H. Kanda A. Anai M. Fukushima Y. Hosaka T. Suzuki M. Shin B.C. Takata K. Yazaki Y. Kikuchi M. Oka Y. Asano T. J. Biol. Chem. 1997; 272: 7873-7882Crossref PubMed Scopus (147) Google Scholar) but with the unique 6-residue amino-terminal sequence deleted. cDNAs encoding p110α (22Hiles I.D. Otsu M. Volinia S. Fry M.J. Gout I. Dhand R. Panayotou G. Ruiz Larrea F. Thompson A. Totty N.F. Hsuan J.J. Courtneidge S.A. Parker P.A. Waterfield M.D. Cell. 1992; 70: 419-429Abstract Full Text PDF PubMed Scopus (538) Google Scholar), p85αΔSH3, p85αΔBH, p85αΔPRM1, p85αΔPRM2, p85αΔPRM1:PRM2, and p55γ (Table I) were subcloned into the baculovirus transfer vector, pVL1393 (Invitrogen). A sequence encoding a hexa-histidine tag was added to the 3′ end of the p49α cDNA, which was then subcloned into the baculovirus transfer vector, pBlueBac4 (Invitrogen). cDNAs encoding p85αSH3-BH-SH2, p85αSH3-BH, p85αSH3-PRM1, p85αSH3, p85αcSH2, and p85αBH (Table I) were subcloned into the bacterial expression vector pGEX-2T (Amersham Pharmacia Biotech). Myc epitope-tagged (23Evan G.I. Lewis G.K. Ramsay G. Bishop J.M. Mol. Cell. Biol. 1985; 5: 3610-3616Crossref PubMed Scopus (2151) Google Scholar) p85α, hexa-histidine-tagged p49α and p55γ were also subcloned into pMT-SM (24Kaufman R.J. Davies M.V. Pathak V.K. Hershey J.W. Mol. Cell. Biol. 1989; 9: 946-958Crossref PubMed Scopus (332) Google Scholar) for expression in mammalian cells.Table IAmino acid sequence specifications for p85α deletion and substitution mutantsThe residues derived from the amino acid sequence of full-length p85α (1–742) that comprise each mutant are listed. Single amino acid substitutions are denoted as P96A, indicating that the proline at position 96 was mutated to arginine. At the bottom is a schematic representation of the domain structure of p85α. The positions of the designated domain boundaries are numbered according to the sequence of p85α. Open table in a new tab The residues derived from the amino acid sequence of full-length p85α (1–742) that comprise each mutant are listed. Single amino acid substitutions are denoted as P96A, indicating that the proline at position 96 was mutated to arginine. At the bottom is a schematic representation of the domain structure of p85α. The positions of the designated domain boundaries are numbered according to the sequence of p85α. All cDNAs in pGEX-2T were expressed as glutathioneS-transferase fusion proteins and purified by glutathione affinity chromatography according to the manufacturer's instructions. cDNAs in the baculovirus transfer plasmid pVL1393 were co-transfected into Sf9 cells with BaculoGold-linearized baculovirus DNA (PharMingen), whereas those in the transfer plasmid pBlueBac4 were co-transfected into Sf9 cells with Bac-N-Blue baculovirus DNA (Invitrogen). The recombinant baculovirus was plaque-purified and amplified as described previously (25O'Reilly D.R. Miller L.K. Luckow V.A. Baculovirus Expression Vectors: A Laboratory Manual. Oxford University Press, New York1994Google Scholar). Exponentially growing Sf9 cells at a density of 1.5–2 × 106/ml were infected with recombinant baculoviruses at an multiplicity of infection between 10 and 20 and harvested 2–3 days post-infection. SV40-transformed monkey kidney cells (Cos7) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, at 37 °C and 10% CO2. Transient transfections were performed using DEAE-Dextran (Sigma). Cells (50% confluent) were transfected with 15 μg of plasmid DNA per 15-cm tissue culture dish and harvested 48 h post-transfection. Sf9 cells expressing recombinant p85 variants were lysed in Triton X-100 lysis buffer (50 mm Tris, pH 8.0, 150 mm NaCl, 1% Triton X-100, 5 mm dithiothreitol (DTT), and a range of protease inhibitors (50 μg/ml 4-(2-aminoethyl)-benzenesulphenylfluoride hydrochloride, 16 μg/ml benzamidine, 10 μg/ml 1,10-phenanthroline, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 5 μg/ml pepstatin A)). p85 variants were purified by affinity chromatography using a phosphotyrosine-affinity column that was synthesized by immobilizing 2 mg of phosphotyrosine (Sigma) per ml of Actigel (Sterogene) according to the manufacturer's instructions. Briefly, up to 50 ml of cell lysate was applied to a 14 × 1-cm column of phosphotyrosine-Actigel equilibrated in Buffer A (20 mm Tris, pH 8.0, containing 5 mmDTT) and eluted with 50 ml of Buffer A followed by 50 ml of Buffer A containing 150 mm NaCl and 50 ml of Buffer A containing 2m NaCl. Fractions of 2.5 ml were collected at a flow rate of 2.5 ml/min and exchanged into 3.5 ml of Buffer A using pre-packed Sephadex G-25 columns (PD10, Amersham Pharmacia Biotech). If a second purification step was required, fractions containing p85 variants were pooled and applied to a 0.46 × 10-cm POROS 20 HQ column (Perspective Biosystems) equilibrated in Buffer A. Elution was carried out using 17 ml of Buffer A followed by a 75-ml linear gradient to 1m NaCl in the same buffer. Fractions of 2.5 ml were collected at a flow rate of 2.5 ml/min. Transfected or wild type Cos7 cells were harvested by trypsinization and Dounce-homogenized in hypotonic lysis buffer (5 mm Tris, pH 7.5, 2.5 mm KCl, 1 mmDTT, 1 mm EDTA, 1 mm phenylmethylsufonyl fluoride, 10 μm leupeptin, 10 μm pepstatin A, 1 mm 1,10-phenanthroline, 1 mm sodium orthovanadate). The cytoplasmic fraction was clarified by centrifugation at 100,000 × g at 4 °C for 45 min and filtered through a 0.22-μm membrane (Ultrafree-MC, Millipore). Aliquots of 200 μl or less of molecular mass standards (Bio-Rad), recombinant proteins, or cell lysates were applied to a Superose 12/30 HR column (Amersham Pharmacia Biotech) equilibrated in TBS (20 mm Tris, pH 8.0, 150 mm NaCl). Samples were eluted isocratically with TBS at a flow rate of 0.3 ml/min and 0.3- or 0.15-ml fractions were collected where necessary. All SE-AUC experiments were carried out using an Optima XL-A analytical ultracentrifuge (Beckman) equipped with absorbance optics and an An60Ti rotor. Protein samples were buffer exchanged into SE-AUC buffer (50 mm Tris, pH 7.4, 50 mm NaCl, 7 mm β-mercaptoethanol, and 0.02% NaN3) using either pre-packed Sephadex G-25 columns (NAP5, Amersham Pharmacia Biotech) or by dialysis overnight. Three samples of 110 μl of protein at approximately 0.6, 0.3 and 0.15 mg/ml were analyzed, and the reference cells contained 125 μl of SE-AUC buffer. Experiments were performed at 4 °C at an optimized range of speeds between 4000 and 28,000 rpm. Equilibrium data were collected at 280 nm in step scan mode with a radial increment of 0.001 cm between data points. Five readings were averaged each scan, from which a base-line scan taken at 360 nm was subtracted in order to correct for optical imperfections. Readings were taken at 8-h intervals until no difference could be detected between consecutive scans. The equilibrium distributions from three different loading concentrations and up to three rotor speeds were analyzed both individually and simultaneously using the Nonlin curve-fitting algorithm supplied with the ultracentrifuge (Beckman). The procedure for measuring interactions between domains and peptides using the BIAcore biosensor (Biacore AB) has been previously described (26Panayotou G. Gish G. End P. Truong O. Gout I. Dhand R. Fry M.J. Hiles I. Pawson T. Waterfield M.D. Mol. Cell. Biol. 1993; 13: 3567-3576Crossref PubMed Google Scholar). Briefly, biotinylated peptides were captured on immobilized avidin, and then sample was injected in running buffer (20 mm Hepes, pH 7.4, 150 mm NaCl, 3.4 mm EDTA, 4 mmDTT, 0.005% Tween 20), and the response at equilibrium was recorded. For competition experiments, the response at equilibrium was compared with that obtained upon preincubation of the protein or isolated domain solution with increasing amounts of free peptide. In order to calculate IC50 values, the results were plotted as resonance unitsversus peptide concentration, and the curve obtained was fitted with the equation R = (R max/1 + (C/IC50)P), where R is the response, R max is the response obtained in the absence of competitor, C is the concentration of competitor, and P is the Hill coefficient. Transfected Cos7 cells were lysed on ice in Nonidet P-40 lysis buffer (1% Nonidet P-40, 20 mm Tris, pH 7.4, 50 mm NaCl, 50 mm NaF, 1 mm EDTA, 500 μm sodium orthovanadate, 2 mm phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, and 1 mm aprotinin), and cell debris and nuclei were removed by centrifugation at 10,000 × g for 10 min at 4 °C. Immunoprecipitations from Cos7 cell lysates were performed for 2 h using p85α monoclonal antibodies (U9, U13, and U14 (27End P. Gout I. Fry M.J. Panayotou G. Dhand R. Yonezawa K. Kasuga M. Waterfield M.D. J. Biol. Chem. 1993; 268: 10066-10075Abstract Full Text PDF PubMed Google Scholar)) or an anti-Myc epitope monoclonal antibody (23Evan G.I. Lewis G.K. Ramsay G. Bishop J.M. Mol. Cell. Biol. 1985; 5: 3610-3616Crossref PubMed Scopus (2151) Google Scholar) immobilized using protein G-Sepharose Fast Flow (Amersham Pharmacia Biotech) or a metal-chelate affinity matrix (Talon, CLONTECH). Precipitated proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting onto polyvinylidene difluoride membrane (Gelman Sciences), probing with an appropriate mouse monoclonal antibody, and detection of bound horseradish peroxidase-conjugated goat anti-mouse antibody (Bio-Rad) using enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech). Protein in fractions from HP-SEC was precipitated using 10% trichloroacetic acid and analyzed by SDS-PAGE and Western blotting as described above. Proteins were precipitated from Sf9 cell lysates using GST fusion proteins captured on glutathione-Sepharose CL 4B (Amersham Pharmacia Biotech) and analyzed for lipid kinase assay as described below. PI3K assays of glutathione-Sepharose CL 4B-precipitated proteins were carried out essentially as described previously (28Whitman M. Kaplan D.R. Schaffhausen B. Cantley L. Roberts T.M. Nature. 1985; 315: 239-242Crossref PubMed Scopus (544) Google Scholar). Lipid kinase assays contained 2 mm MgCl2, 1 mm ATP, 20 μCi of [γ-32P]ATP, and 200 μg/ml phosphatidylinositol. Extracted phospholipids were analyzed by thin layer chromatography in 65% 1-propanol, 0.7 m acetic acid, 50 mmphosphoric acid. It has previously been reported that both proline-rich motifs in p85α conform to the consensus ligand for the SH3 domain of p85α (29Rickles R.J. Botfield M.C. Weng Z. Taylor J.A. Green O.M. Brugge J.S. Zoller M.J. EMBO J. 1994; 13: 5598-5604Crossref PubMed Scopus (223) Google Scholar). In order to determine which of the two proline-rich motifs was the preferred ligand for the isolated p85α SH3 domain (p85αSH3), we compared its ability to bind to peptides derived from the sequences of the first (P1) or second (P2) proline-rich motifs of p85α, using an optical biosensor (Fig.1 A). Binding of p85αSH3 to immobilized P1 was observed in this system, producing a response of greater than 400 resonance units (Fig. 1 A), whereas little or no p85αSH3 injected at the same concentration bound to P2 (Fig.1 A), suggesting that P1 is the preferred ligand for p85αSH3. In contrast, wild type p85α, which contains the SH3 domain, did not bind immobilized P1 to a significant extent (Fig. 1 B). The small amount of binding observed was equivalent to that observed for binding of the same concentration of p85α to a surface on which P1 was not immobilized (data not shown). A mutant of p85α, in which two proline residues in PRM1 that have been shown to be important for p85α SH3 domain binding (30Yu H. Chen J.K. Feng S. Dalgarno D.C. Brauer A.W. Schreiber S.L. Cell. 1994; 76: 933-945Abstract Full Text PDF PubMed Scopus (868) Google Scholar) were changed to alanine (p85αΔPRM1), was able to bind P1; however, a variant of p85α with mutations at the equivalent residues in PRM2 (p85αΔPRM2) was not. A variant of p85α in which both proline-rich motifs contained mutations (p85αΔPRM1:PRM2) bound to P1 to a similar extent as p85αΔPRM1 (Fig. 1 B). Mutations in PRM1 therefore allowed p85αSH3 to bind exogenous P1 peptide, whereas the SH3 domain in the context of wild type p85α could not bind to exogenous P1. Mutations in PRM2 did not affect binding to P1 peptide, confirming that the first proline-rich motif was the preferred ligand for the p85α SH3 domain. Thus, mutation of key residues in PRM1 of p85α not only affected this site but also the binding characteristics of the SH3 domain. These mutations increased the ability of the SH3 domain to bind exogenous ligands, indicating that in wild type p85α the SH3 domain interacts with PRM1 and that when this interaction is disrupted the SH3 domain is free to bind exogenous peptide. Similarly, deletion of the SH3 domain of p85α would be expected to free PRM1 and allow it to bind exogenous SH3 domains. When wild type p85α was co-expressed with the p110 catalytic subunit in Sf9 cells, a p85αSH3 GST fusion protein (GST-p85αSH3) immobilized on glutathione-Sepharose CL4B was poorly able to co-precipitate PI3K lipid kinase activity from Sf9 cell lysates. However, a monoclonal antibody directed against the SH2 domain of p85α was able to immunoprecipitate PI3K activity; thus both p85α and p110α were expressed in these cells. The same antibody was able to immunoprecipitate PI3K activity from lysates of Sf9 cells co-infected with p110α and a mutant of p85α in which the SH3 domain had been deleted (p85αΔSH3). Immobilized GST-p85αSH3 was able to co-precipitate the p110α-p85αΔSH3 complex to a much greater extent than the p110α-p85α complex (Fig. 1 C); thus deletion of the SH3 domain increased the ability of an exogenous SH3 domain to bind PRM1, confirming that the SH3 domain and PRM1 are bound to each other in both p85α and the p110α-p85α complex. Examination of purified, recombinant p85α and p85β by HP-SEC under native conditions showed that they had apparent molecular masses that were approximately double (162 ± 14 and 151 ± 12 kDa, respectively) that expected from their amino acid sequences (Fig.2, A and B). In contrast, the apparent molecular masses of the truncated p85α isoforms, p55γ and p49α (82 ± 4 kDa and 52 ± 2 kDa), were much closer to their predicted molecular masses (55 and 49 kDa; Fig. 2, A and B). There are several mechanisms by which a protein under native conditions can have an apparent molecular mass different from that predicted from its amino acid sequence. HP-SEC measures the hydrodynamic volume of a protein, which is defined as the spherical volume occupied by that protein as it tumbles rapidly in solution. Non-spherical proteins often elute with an abnormally high apparent molecular mass compared with the globular proteins used to calibrate the column. Proteins can also interact nonspecifically with the column matrix, impeding their progress through the column and resulting in a later elution time, leading to an underestimate of the molecular mass of a protein. High apparent molecular masses may also result from dimerization or oligomerization of the protein; thus we investigated whether the higher than expected molecular mass of p85α was due to dimerization by determining the molecular masses of p85α and p49α under different conditions. HP-SEC was carried out in buffers containing either 5 mmDTT or 8 m urea in order to determine whether the putative p85α dimeric interaction could be disrupted. The apparent molecular masses of p85α and p55γ were unaltered in the presence of 5 mm DTT (Fig. 2 B), suggesting that dimerization of p85α was not due to the presence of an intermolecular disulfide bond. In contrast, p85α had an apparent molecular mass of 89 ± 5 kDa in the presence of 8 m urea. High concentrations of urea disrupt non-covalent, but not covalent, bonds, indicating that p85α forms a dimer via a non-covalent interaction. The apparent molecular mass of p49α was relatively unaffected in the presence of 8m urea however (Fig. 2 B), suggesting that it is monomeric under native conditions. Sedimentation Equilibrium-Analytical Ultracentrifugation (SE-AUC) was employed to determine the apparent molecular masses of a number of p85α variants, as the equilibrium distribution of a solute in a gravitational field is dependent on the molecular mass of the solute, but independent of its shape, whereas HP-SEC is dependent on both mass and shape. Analysis of the equilibrium distribution of the concentration of protein with respect to radial position in the centrifuge demonstrated the occurrence of self-association in some samples. Self-association was defined as a lack of adherence to the Lamm equation, which describes the distribution of ideal, non-associating solute particles in a gravitational field. The occurrence of self-association manifests as a non-random distribution of residuals to the fit of the experimental (absorbanceversus radius) data to a derivative of the Lamm equation. The equilibrium distribution of p85α did not fit that of an ideal, non-associating solute, as the residuals were non-random (Fig.3 D). The apparent molecular mass of p85α was dependent on protein concentration and ranged between 80 and 110 kDa (Fig. 3 D), suggesting that p85α exists as an equilibrium of monomers and dimers under these conditions. In contrast, p49α was monomeric under the conditions examined. The residuals of the fit for an ideal, non-associating solute were distributed randomly around zero, and p49α had an apparent molecular mass of approximately 47 kDa (Fig. 3 C). Thus, p85α, which was apparently dimeric by HP-SEC, fitted a model for self-association, although the equilibrium dissociation constant for p85α dimer formation (estimated from the plot of apparent molecular massversus protein concentration; Fig. 3 D) was in the micromolar range, which is a lower affinity than that expected for a protein that was determined to be constitutively dimeric by HP-SEC. Given that p49α was shown to be monomeric using both techniques, this suggested that the dimerization of p85 was mediated by domains that are not present in p49α. The observation that p85α and p85β were dimeric under native conditions, but p55γ and p49α were not, suggested that the amino-terminal half of the higher molecular mass regulatory subunits was involved in intermolecular interactions that caused them to dimerize. Given that the SH3 domain and the first proline-rich motif reside in this region of the protein, and interact with each other (Fig. 1), we investigated whether this interaction was involved in the dimerization of p85α. Indeed, expression of the amino" @default.
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• W2016398041 title "Intermolecular Interactions of the p85α Regulatory Subunit of Phosphatidylinositol 3-Kinase" @default.
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