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Regulation of the p85/p110α Phosphatidylinositol 3′-Kinase

DISTINCT ROLES FOR THE N-TERMINAL AND C-TERMINAL SH2 DOMAINS*
  • Jinghua Yu
    Affiliations
    Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461
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  • Christina Wjasow
    Footnotes
    Affiliations
    Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461
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  • Jonathan M. Backer
    Correspondence
    Established Scientist of the American Heart Association, New York Affiliate and a recipient of a Scholar Awards from the Irma T. Hirschl Trust. To whom correspondence should be addressed: Dept. of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-2153
    Affiliations
    Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461
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  • Author Footnotes
    * This work was supported by grants from the American Diabetes Association, and National Institutes of Health Grant GM55692 (to J.M.B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ‡ Supported by a fellowship from the Howard Hughes Medical Foundation.
Open AccessPublished:November 13, 1998DOI:https://doi.org/10.1074/jbc.273.46.30199
      Our previous studies on the p85/p110α phosphatidylinositol 3-kinase showed that the p85 regulatory subunit inhibits the p110α catalytic subunit, and that phosphopeptide activation of p85/p110α dimers reflects a disinhibition of p110α (Yu, J., Zhang, Y., McIlroy, J., Rordorf-Nikolic, T., Orr, G. A., and Backer, J. M. (1998) Mol. Cell. Biol. 18, 1379–1387). We now define the domains of p85 required for inhibition of p110α. The iSH2 domain of p85 is sufficient to bind p110α but does not inhibit it. Inhibition of p110α requires the presence of the nSH2 domain linked to the iSH2 domain. Phosphopeptides increase the activity of nSH2/iSH2-p110α dimers, demonstrating that the nSH2 domain mediates both inhibition of p110α and disinhibition by phosphopeptides. In contrast, phosphopeptides did not increase the activity of iSH2/cSH2-p110α dimers, or dimers composed of p110α and an nSH2/iSH2/cSH2 construct containing a mutant nSH2 domain. Phosphopeptide binding to the cSH2 domain increased p110α activity only in the context of an intact p85 containing both the nSH2 domain and residues 1–322 (the SH3, proline-rich and breakpoint cluster region-homolgy domains). These data suggest that the nSH2 domain of p85 is a direct regulator of p110α activity. Regulation of p110α by phosphopeptide binding to the cSH2 domain occurs by a mechanism that requires the additional presence of the nSH2 domain and residues 1–322 of p85.
      PI
      phosphatidylinositol
      GST
      glutathione S-transferase
      BCR
      breakpoint cluster region
      HA
      hemagglutinin.
      PI1 3′-kinases form a diverse family of lipid kinases that phosphorylate phosphatidylinositol at the D3-position (
      • Kapeller R.
      • Cantley L.C.
      ). The regulation of the p85/p110 PI 3′-kinase is particularly complex. The p85 regulatory subunit contains an N-terminal SH3 domain followed by a proline rich domain, a breakpoint cluster region-homology domain, a second proline-rich domain, and two SH2 domains linked by a putative coiled coil domain (the inter-SH2 or iSH2 domain) that binds to the N terminus of the p110α catalytic subunit (
      • 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.
      ,
      • Skolnik E.Y.
      • Margolis B.
      • Mohammadi M.
      • Lowenstein E.
      • Fischer R.
      • Drepps A.
      • Ullrich A.
      • Schlessinger J.
      ,
      • Escobedo J.A.
      • Navankasattusas S.
      • Kavanaugh W.M.
      • Milfay D.
      • Fried V.A.
      • Williams L.T.
      ). The binding of proteins such as CDC42 (to the BCR homology domain), Fyn and Lyn (to the proline-rich domains), and p21-ras (to p110α) increases the activity of p85/p110 dimers in vitro(
      • Zheng Y.
      • Bagrodia S.
      • Cerione R.A.
      ,
      • Pleiman C.M.
      • Hertz W.M.
      • Cambier J.C.
      ,
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Vanhaesebroeck B.
      • Waterfield M.D.
      • Downward J.
      ). p85/p110 activity is also increased when the two SH2 domains bind to phosphoproteins containing appropriate phosphotyrosyl motifs (
      • 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.
      ,
      • Carpenter C.L.
      • Auger K.R.
      • Chanudhuri M.
      • Yoakim M.
      • Schaffhausen B.
      • Shoelson S.
      • Cantley L.C.
      ,
      • Rordorf-Nikolic T.
      • Van Horn D.J.
      • Chen D.
      • White M.F.
      • Backer J.M.
      ).
      We previously examined the effect of p85 on p110α in mammalian cells and in vitro (
      • Yu J.
      • Zhang Y.
      • McIlroy J.
      • Rordorf-Nikolic T.
      • Orr G.A.
      • Backer J.M.
      ), and experimentally distinguished two effects of p85 on p110α: inhibition of its lipid kinase activity and stabilization against thermal inactivation. The inhibition of p110α by the p85 subunit is clearly seen during in vitroreconstitution experiments, where dimerization with p85 decreases p110 activity by 80%. The activity of p85/p110α dimers is increased when phosphotyrosyl peptides bind to the p85 SH2 domains, but to a level no greater than that of the corresponding amount of monomeric p110α. These data suggest that phosphopeptide activation of p85/p110 dimers reflects a transition between inhibited and disinhibited states.
      In addition to its inhibition of p110α, p85 stabilizes p110α against thermal denaturation. Recombinant p110α monomers lose activity rapidly when incubated at 37 °C, whereas p85/p110α dimers are stable, and coexpression of p85 with p110α in mammalian cells significantly increases the half-life of p110α (
      • Yu J.
      • Zhang Y.
      • McIlroy J.
      • Rordorf-Nikolic T.
      • Orr G.A.
      • Backer J.M.
      ). The rapid inactivation of p110α at 37 °C explains a longstanding discrepancy between mammalian and insect cells (
      • Hiles 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.J.
      • Waterfield M.D.
      ): monomeric p110α is active in insect cells but not mammalian cells because of differences in their culture temperatures (27 versus 37 °C). However, the stabilization of p110α by p85 in mammalian cells is mimicked by addition of bulky epitope tags to the N terminus of p110α (
      • Yu J.
      • Zhang Y.
      • McIlroy J.
      • Rordorf-Nikolic T.
      • Orr G.A.
      • Backer J.M.
      ). Thus, the stabilization by p85 appears to involve the overall conformation of p110α rather than the induction of a specific activated state.
      In this paper, we examine the regulation of p110α by p85 in detail. We find that the iSH2 domain of p85 is sufficient to bind p110α but does not affect its activity. Inhibition of p110α requires the presence of the nSH2 domain linked to the iSH2 domain. Phosphopeptide binding to the nSH2 domain can directly modulate p110α activity. In contrast, phosphopeptide binding to the cSH2 domain modulates p110α activity by a mechanism that requires residues 1–322 of p85 (the SH3, BCR homology and proline-rich domains) and the nSH2 domain. These data suggest that the nSH2 domain is the principle regulator of p85/p110α activity.

      DISCUSSION

      We have previously shown that p85 is an inhibitor of p110α activity, and that binding of tyrosyl phosphopeptides to the p85 SH2 domains relieves this inhibition (
      • Yu J.
      • Zhang Y.
      • McIlroy J.
      • Rordorf-Nikolic T.
      • Orr G.A.
      • Backer J.M.
      ). This study demonstrates that the SH2 domains of p85 are critical for the inhibitory effects of p85 on p110α. Consistent with previous reports, we find that the iSH2 domain of p85 is sufficient to bind to p110α (
      • Dhand R.
      • Hara K.
      • Hiles I.
      • Bax B.
      • Gout I.
      • Panayotou G.
      • Fry M.J.
      • Yonezawa K.
      • Kasuga M.
      • Waterfield M.D.
      ,
      • Klippel A.
      • Escobedo J.A.
      • Hu Q.
      • Williams L.T.
      ,
      • Holt K.H.
      • Olson A.L.
      • Moye-Rowley W.S.
      • Pessin J.E.
      ,
      • Hu P.
      • Schlessinger J.
      ). However, iSH2 domain binding alone does not affect p110α activity. Instead, the presence of an SH2 domain linked to the N terminus of the iSH2 domain is required for inhibition of p110α.
      Two mechanisms could explain the inhibition of p110α by the nSH2/iSH2versus iSH2 fragments. The iSH2 domain is predicted to form a coiled-coil domain (
      • Dhand R.
      • Hara K.
      • Hiles I.
      • Bax B.
      • Gout I.
      • Panayotou G.
      • Fry M.J.
      • Yonezawa K.
      • Kasuga M.
      • Waterfield M.D.
      ). The presence of an nSH2 domain at the N-terminal end of the iSH2 domain could exert a conformational strain on the coiled-coil and alter its interactions with p110α. Alternatively, the nSH2 domain may directly contact p110α, inhibiting its activity. In both mechanisms, conformational changes induced by phosphoprotein binding to the nSH2 domain would relieve the inhibition of p110α. We have no direct experimental evidence to distinguish these hypotheses at this time. However, we have noticed that unlike the iSH2 domain itself, a GST-iSH2 fusion protein binds p110α and inhibits its activity by 50%. Inhibition by attachment of a bulky GST moiety to the N terminus of the iSH2 domain is consistent with the first mechanism. Also consistent with this model is a recent paper by Jimenez et al. (19a) describing an oncogenic truncated p85 molecule. Expression of this mutant with p110α caused an increase in activity as compared with wild-type p85, which we would interpret as the loss of p85-induced inhibition of p110α. Since the nSH2 domain is present in the truncation mutant, the loss of inhibition would seem to be because of a conformational change in the iSH2 domain caused by the removal of its extreme C terminus. On the other hand, Cooper and Kashishian reported a direct interaction between p110α and the p85 nSH2 domain in transfected cells (
      • Cooper J.A.
      • Kashishian A.
      ), which would be consistent with the second mechanism.
      Importantly, the iSH2 domain itself neither activates nor inhibits p110α. In contrast, others have suggested that iSH2 domain binding to p110α provides critical activating interactions that are required for p110α activity in mammalian cells (
      • Klippel A.
      • Escobedo J.A.
      • Hirano M.
      • Williams L.T.
      ), and attachment of the iSH2 domain to p110 has been used to produce a constitutively active enzyme (
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.T.
      ). However, attachment of bulky moieties such as GST or a tris-HA tag to the N terminus of p110α increases the activity of monomeric p110α activity in mammalian cells by stabilizing the protein (
      • Yu J.
      • Zhang Y.
      • McIlroy J.
      • Rordorf-Nikolic T.
      • Orr G.A.
      • Backer J.M.
      ). Given that the iSH2 domain has no effect on p110α activity in vitro, we think it likely that the iSH2-p110α chimera is active because of the attachment of a bulky group, rather than the provision of specific activating interactions.
      A surprising finding in this study is the marked difference in the roles of the nSH2 and cSH2 domains. The nSH2/iSH2 fragment inhibits p110α, and nSH2/iSH2-p110α complexes are activated by phosphotyrosine peptides. In contrast, iSH2/cSH2 fragments bind p110α but have little effect on its activity. Phosphopeptide binding to the cSH2 domain does contribute to p110α activation, but only in the context of the entire p85 protein. Thus, phosphopeptide modulation of p110α via the cSH2 domain appears to be distinct from modulation via the nSH2 domain.
      Previous studies have suggested that intramolecular interactions may occur between the SH3 domain and the proline-rich domain of p85 (
      • Kapeller R.
      • Prasad K.V.S.
      • Janssen O.
      • Hou W.
      • Schaffhausen B.S.
      • Rudd C.E.
      • Cantley L.C.
      ). In this case, the cSH2 domain, SH3-PRD domains, and the nSH2 domain may form a compact structure (Fig. 5). Our data would suggest that the nSH2 domain is the major regulator of p110α activity, and that occupancy of the nSH2 domain induces a conformational change (
      • Shoelson S.E.
      • Sivaraja M.
      • Williams K.P.
      • Hu P.
      • Schlessinger J.
      • Weiss M.A.
      ,
      • Panayotou G.
      • Bax B.
      • Gout I.
      • Federswisch M.
      • Wrobolowski B., R.
      • Dhand
      • Fry M.J.
      • Blundell T.L.
      • Wollmer A.
      • Waterfield M.D.
      ) that is transmitted to the iSH2 domain and/or p110α (Fig. 5 A). In contrast, phosphopeptide occupancy of the cSH2 domain may induce a conformational change that is transmitted to the regulatory nSH2 domain by way of residues 1–322 of p85 (the SH2, Bcr and proline-rich domains) (Fig. 5 B). This model would predict that disruption of intramolecular interactions within the SH3 and proline-rich domains of p85 would minimize phosphopeptide-induced activation via the cSH2 domain, but would not affect activation via the nSH2 domain. Experiments to test this hypothesis are in progress.
      Figure thumbnail gr5
      Figure 5Effects of phosphopeptide binding to the nSH2 and cSH2 domains of p85. A, phosphopeptide binding to the nSH2 induces a conformational change that is transmitted to the iSH2 domains and/or p110α. B, phosphopeptide binding to the cSH2 domain induces a conformational change that is transmitted to the nSH2 domain via residues 1–322 of p85 (the SH3, Bcr and proline-rich domains). The secondary conformational change at the nSH2 domain is transmitted to the iSH2 domain and/or p110α.
      In summary, we have shown that the iSH2 domain of p85 mediates binding to p110α, whereas the inhibitory effects of p85 on p110α are largely mediated by an additional constraint imposed by the nSH2 domain. Phosphopeptide occupancy of the nSH2 domain can directly modulate p110α activity. In contrast, modulation of p110α activity by the cSH2 domain occurs by a mechanism that requires residues 1–234 of p85 as well as the nSH2 domain. These studies highlight the complexities of p110α regulation by p85.

      ACKNOWLEDGEMENTS

      We thank Dr. Steve Almo for helpful discussions, and Dr. George Orr for discussion and critical reading of the manuscript. We thank Ms. Jenny Yip for excellent technical assistance. We thank Dr. Michael Waterfield for the p110α cDNA.

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