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Intermolecular Interactions of the p85α Regulatory Subunit of Phosphatidylinositol 3-Kinase*

Open AccessPublished:April 30, 1999DOI:https://doi.org/10.1074/jbc.274.18.12323
      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)
      The 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 ultracentrifugation
      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 ultracentrifugation
      are heterodimeric enzymes with a p110 catalytic subunit and a p85 regulatory subunit (
      • Zvelebil 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.
      ). 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 (
      • Diekmann D.
      • Brill S.
      • Garrett M.D.
      • Totty N.
      • Hsuan J.
      • Monfries C.
      • Hall C.
      • Lim L.
      • Hall A.
      ) and Rho subfamily GTPases, and two SH2 domains separated by an inter-SH2 domain through which p85 binds the catalytic subunit (
      • Dhand R.
      • Hara K.
      • Hiles I.
      • Bax B.
      • Gout I.
      • Panayotou G.
      • Fry M.J.
      • Yonezawa K.
      • Kasuga M.
      • Waterfield M.D.
      ). 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 (
      • Otsu 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.
      ), human (
      • Skolnik E.Y.
      • Margolis B.
      • Mohammadi M.
      • Lowenstein E.
      • Fischer R.
      • Drepps A.
      • Ullrich A.
      • Schlessinger J.
      ), and mouse (
      • Escobedo J.A.
      • Navankasattusas S.
      • Kavanaugh W.M.
      • Milfay D.
      • Fried V.A.
      • Williams L.T.
      ) cDNA libraries, whereas only bovine p85β has been identified (
      • Otsu 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.
      ). Two splice variants of p85α, termed p55 and p50, have been identified in the human (
      • Antonetti D.A.
      • Algenstaedt P.
      • Kahn C.R.
      ), the rat (
      • Inukai K.
      • Anai M.
      • Van Breda E.
      • Hosaka T.
      • Katagiri H.
      • Funaki M.
      • Fukushima Y.
      • Ogihara T.
      • Yazaki Y.
      • Kikuchi
      • Oka Y.
      • Asano T.
      ,
      • Inukai 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.
      ), and the mouse (
      • Fruman D.A.
      • Cantley L.C.
      • Carpenter C.L.
      ). 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
      F. Pagès and M. D. Waterfield, unpublished results.
      2F. Pagès and M. D. Waterfield, unpublished results.
      and human (
      • Pons S.
      • Asano T.
      • Glasheen E.
      • Miralpeix M.
      • Zhang Y.
      • Fisher T.L.
      • Myers Jr., M.G.
      • Sun X.J.
      • White M.F.
      ) 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 (
      • Vanhaesebroeck B.
      • Leevers S.J.
      • Panayotou G.
      • Waterfield M.D.
      ). 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 (
      • Pleiman C.M.
      • Hertz W.M.
      • Cambier J.C.
      ), whereas the small G proteins Rac (
      • Tolias K.F.
      • Cantley L.C.
      • Carpenter C.L.
      ,
      • Nobes C.D.
      • Hawkins P.
      • Stephens L.
      • Hall A.
      ) and Cdc42 (
      • Zheng Y.
      • Bagrodia S.
      • Cerione R.A.
      ) 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 (
      • Songyang 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.
      ,
      • Panayotou G.
      • Waterfield M.D.
      ,
      • Backer 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.
      ).
      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 (
      • Inukai 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.
      ) 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 (
      • Shepherd P.R.
      • Nave B.T.
      • Rincon J.
      • Nolte L.A.
      • Bevan A.P.
      • Siddle K.
      • Zierath J.R.
      • Wallberg Henriksson H.
      ). 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.

      DISCUSSION

      The p85 subunit of PI3K may represent yet another intracellular signal transduction protein which utilizes dimerization as a regulatory mechanism, in a similar manner to cell-surface receptors, which are often activated by ligand-induced dimerization or oligomerization (
      • Schlessinger J.
      • Ullrich A.
      ). Several other intracellular signaling proteins, such as the STAT transcription factors (
      • Darnell Jr., J.E.
      • Kerr I.M.
      • Stark G.R.
      ) and c-Raf (
      • Luo Z.
      • Tzivion G.
      • Belshaw P.J.
      • Vavvas D.
      • Marshall M.
      • Avruch J.
      ,
      • Farrar M.A.
      • Alberol I.
      • Perlmutter R.M.
      ), have also been shown to be regulated by dimerization.
      In this study, a number of techniques have been used to demonstrate that the amino-terminal portion of the p85α subunit of PI3K binds to itself in an intermolecular manner (Fig.7), which leads to dimerization of p85α both in vitro and in vivo. Additionally, we determined that the isolated SH3 domain of p85α binds only one of its endogenous proline-rich motifs, PRM1, and the same interaction occurs within the whole protein. In order to determine whether these two interactions were related, we further investigated the binding properties of each of the domains of p85α. However, it became clear that dimerization was not mediated by a single domain, and the involvement of the BH domain in dimerization made interpretation of this data more complicated. However, the ability of the P1 peptide to disrupt both p85αSH3-PRM1 and p85α dimers (Fig. 4) indicated that the SH3 domain of one p85α molecule binds the PRM1 of a second p85α (Fig. 7).
      Figure thumbnail gr7
      Figure 7Proposed model of intermolecular interactions of the p85α regulatory subunit of phosphatidylinositol 3-kinase.As indicated with the arrows, the N-terminal domains of p85α, the SH3, BH, and PRM1 domains, interact in an intermolecular manner leading to the formation of a p85α dimer (as discussed in the text).
      Previous studies have also demonstrated that various fragments of p85α are dimeric. A fragment of p85α encompassing residues 1–101 (similar to p85αSH3-PRM1 in this study) (
      • Chen J.K.
      • Schreiber S.L.
      ) and a fragment similar to p85αSH3-BH (
      • Musacchio A.
      • Cantley L.C.
      • Harrison S.C.
      ) have been shown to be dimers. Crystals of the BH domain of p85α have previously been shown to contain two monomers per asymmetric unit (
      • Musacchio A.
      • Cantley L.C.
      • Harrison S.C.
      ); however, the hydrophobic dimerization interface was very small, involving only four residues of each monomer. In comparison, the three-dimensional structure of the p85αSH3 domain has been determined by both x-ray crystallography and NMR, and no evidence for self-association of the isolated SH3 domain of p85α has been described (
      • Yu H.
      • Chen J.K.
      • Feng S.
      • Dalgarno D.C.
      • Brauer A.W.
      • Schreiber S.L.
      ,
      • Booker G.W.
      • Gout I.
      • Downing A.K.
      • Driscoll P.C.
      • Boyd J.
      • Waterfield M.D.
      • Campbell I.D.
      ).
      The suggestion from the crystal structure of the BH domain that the 4-residue interface represents a small portion of a larger interaction (
      • Musacchio A.
      • Cantley L.C.
      • Harrison S.C.
      ) is confirmed by the contribution of the BH domain to the overall dimeric interface of p85α (Fig. 2 B). The size of the interface shown in the crystal structure suggests that the affinity of dimerization would be low. If monomeric and dimeric BH domain existed in equilibrium, and the interconversion rate was fast, the low affinity for self-association would result in only a small proportion of dimeric BH domain and thus may explain the slightly higher average molecular mass observed by both HP-SEC and SE-AUC (Figs. 2 B and3 A).
      Interactions between SH3 domains and proline-rich motifs have been reported to regulate several intracellular signaling molecules. In p85α, this interaction not only participates in the dimerization interface but may also block the binding of exogenous ligands for the p85α SH3 domain and PRM1. Stimulation of the B cell receptor has been shown to lead to the binding of the SH3 domains of the Src family tyrosine kinases, Lyn and Fyn, to the p85α PRM1 and the up-regulation PI3K activity (
      • Pleiman C.M.
      • Hertz W.M.
      • Cambier J.C.
      ). The endogenous, intermolecular interaction may therefore also regulate the binding of exogenous ligands to the SH3 domain and PRM1. A similar mechanism has been demonstrated in Itk, a member of the Tec family of cytoplasmic tyrosine kinases (
      • Andreotti A.H.
      • Bunnell S.C.
      • Feng S.
      • Berg L.J.
      • Schreiber S.L.
      ), although the SH3 domain proline-rich motif interaction was intramolecular in this protein. Interaction of the SH3 domain with an adjacent proline-rich motif in Itk prevented the binding of the SH3 domain to proline-rich motifs in Sam-68 and Grb2. Tyrosine phosphorylation within the SH3 domain of another Tec family tyrosine kinase, Btk, was shown to disrupt an intramolecular SH3 domain proline-rich motif interaction and release the Btk SH3 domain to allow it to recruit substrates of the Btk kinase domain (
      • Park H.
      • Wahl M.I.
      • Afar D.E.
      • Turck C.W.
      • Rawlings D.J.
      • Tam C.
      • Scharenberg A.M.
      • Kinet J.P.
      • Witte O.N.
      ). Intramolecular SH3 domain binding to proline-rich motifs may therefore be a general mechanism for the regulation of SH3 domain function, although there seems to be a number of ways in which the SH3 domain can be released to bind its exogenous ligands. The interaction of the p85α SH3 domain and p85α PRM1 seems to be a special case of this general mechanism in which an intermolecular SH3-PRM1 interaction occurs between the two units of a dimer.
      Recent evidence has suggested that the p85 subunit may have functions that are independent of the p110 catalytic subunit. A truncated form of p85α, lacking the carboxyl-terminal SH2 domain and 52 residues of the inter-SH2 region, was identified as a potential oncogene in a number of murine lymphomas (
      • Jimenez C.
      • Jones D.R.
      • Rodriguez Viciana P.
      • Gonzalez Garcia A.
      • Leonardo E.
      • Wennstrom S.
      • von Kobbe C.
      • Toran J.R.-L.
      • Borlado L.
      • Calvo V.
      • Copin S.G.
      • Albar J.P.
      • Gaspar M.L.
      • Diez E.
      • Marcos M.A.
      • Downward J.
      • Martinez A.C.
      • Merida I.
      • Carrera A.C.
      ). Overexpression of this mutant protein (p65) caused transformation of NIH-3T3 fibroblasts, and p65 and v-Raf had synergistic transforming activities. Murine embryonic fibroblasts in which the gene for p85α was deleted by homologous recombination had a defect in a p53-mediated apoptotic pathway (
      • Yin Y.
      • Terauchi Y.
      • Solomon G.G.
      • Aizawa S.
      • Rangarajan P.N.
      • Yazaki Y.
      • Kadowaki T.
      • Barrett J.C.
      ). Apoptosis caused by oxidative stress was reduced in cells lacking p85α. Apoptosis was not inhibited by the PI3K inhibitor, wortmannin, suggesting that the lipid kinase activity of the p110 subunit is not a requisite component of the p53- and p85α-mediated pathways.
      One of the best defined functions of the regulatory subunit of PI3K is the down-regulation of p110-mediated lipid kinase activity (
      • Yu J.
      • Zhang Y.
      • McIlroy J.
      • Rordorf Nikolic T.
      • Orr G.A.
      • Backer J.M.
      ,
      • Kodaki T.
      • Woscholski R.
      • Hallberg B.
      • Rodriguez Viciana P.
      • Downward J.
      • Parker P.J.
      ), suggesting that populations of PI3K comprising a complex of the p110 and p85 subunits or the p110 subunit alone would have different activities. A 100-kDa PI3K activity has been detected in lysates of bovine thymus by size-exclusion chromatography (SEC) (
      • Shibasaki F.
      • Fukui Y.
      • Takenawa T.
      ) suggesting that the p110 subunit may exist on its own and implying that the p85 regulatory subunit may also exist as a separate entity with a biological function distinct from its role in the regulation of p110 activity. Another potential role of a pool of dimeric p85 in vivo could be to bind and stabilize the p110 catalytic subunit as soon as it is translated, as p110α has been shown to be prone to inactivation and degradation at 37 °C without the bound regulatory subunit (
      • Yu J.
      • Zhang Y.
      • McIlroy J.
      • Rordorf Nikolic T.
      • Orr G.A.
      • Backer J.M.
      ).
      The existence of forms of the regulatory subunit of PI3K, such as p49α or p55γ, that do not have the potential for self-association may represent a mechanism for divergent signaling. PI3K-mediated signaling through the insulin receptor and insulin receptor substrate 1 has been shown to preferentially utilize forms of the PI3K regulatory subunit that do not have the potential for self-association (
      • Inukai K.
      • Anai M.
      • Van Breda E.
      • Hosaka T.
      • Katagiri H.
      • Funaki M.
      • Fukushima Y.
      • Ogihara T.
      • Yazaki Y.
      • Kikuchi
      • Oka Y.
      • Asano T.
      ,
      • Shepherd P.R.
      • Nave B.T.
      • Rincon J.
      • Nolte L.A.
      • Bevan A.P.
      • Siddle K.
      • Zierath J.R.
      • Wallberg Henriksson H.
      ). As yet it is unclear what implication a lack of dimerization of some regulatory subunit isoforms has for PI3K signaling. The use of protein-protein interactions of SH3 and BH domains and proline-rich motifs to regulate the nature and localization of PI3K activity would not be an option for the lower molecular mass isoforms. The regulatory subunit of PI3K may therefore represent both an adaptor for the catalytic subunit and a mediator of other protein-protein interactions that utilize these domains. This study has highlighted that these same domains are also implicated in self-association of the p85 protein, which adds a further layer to the potential complexity of the regulation of PI3K activity in cell signaling events.

      Acknowledgments

      We thank Akunna Akpan and Krishna Pitrola for assistance with Sf9 cell culture, Professor I. D. Campbell and Dr. K. Drickamer (University of Oxford) for access to the analytical ultracentrifuge, and Dr. R. Wallis for kind assistance in the implementation and interpretation of the SE-AUC experiments.

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