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Tyrosine Phosphorylation of p85 Relieves Its Inhibitory Activity on Phosphatidylinositol 3-Kinase*

Open AccessPublished:July 20, 2001DOI:https://doi.org/10.1074/jbc.M100556200
      Under resting conditions, the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K) serves to both stabilize and inactivate the p110 catalytic subunit. The inhibitory activity of p85 is relieved by occupancy of the NH2-terminal SH2 domain of p85 by phosphorylated tyrosine. Src family kinases phosphorylate tyrosine 688 in p85, a process that we have shown to be reversed by the activity of the p85-associated SH2 domain-containing phosphatase SHP1. We demonstrate that phosphorylation of the downstream PI3K target Akt is increased in cells lacking SHP1, implicating phosphorylation of p85 in the regulation of PI3K activity. Furthermore, the in vitrospecific activity of PI3K associated with tyrosine- phosphorylated p85 is higher than that associated with nonphosphorylated p85. Expression of wild-type p85 inhibits PI3K enzyme activity as indicated by PI3K- dependent Akt phosphorylation. The inhibitory activity of p85 is accentuated by mutation of tyrosine 688 to alanine and reversed by mutation of tyrosine 688 to aspartic acid, changes that block and mimic tyrosine phosphorylation, respectively Strikingly, mutation of tyrosine 688 to aspartic acid completely reverses the inhibitory activity of p85 on cell viability and activation of the downstream targets Akt and NFκB, indicative of the physiological relevance of p85 phosphorylation. Tyrosine phosphorylation of Tyr688 or mutation of tyrosine 688 to aspartic acid is sufficient to allow binding to the NH2-terminal SH2 domain of p85. Thus an intramolecular interaction between phosphorylated Tyr688 and the NH2-terminal SH2 domain of p85 can relieve the inhibitory activity of p85 on p110. Taken together, the data indicate that phosphorylation of Tyr688in p85 leads to a novel mechanism of PI3K regulation.
      PI3K
      phosphatidylinositol 3-kinase
      SH
      Src-homology
      IL
      interleukin
      HA
      hemagglutinin
      PAGE
      polyacrylamide gel electrophoresis
      MTT
      3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
      EGF
      epidermal growth factor
      GST
      glutathioneS-transferase
      The PI3K1 signaling cascade has been linked to proliferation, cell survival, differentiation, apoptosis, cytoskeletal rearrangement, and vacuolar trafficking. Growth factor-responsive Class IA PI3Ks consist of heterodimers of a 110-kDa catalytic subunit associated with an 85-kDa noncatalytic regulatory subunit designated p85. The p85 adapter subunits are encoded by at least three different genes with splice variation generating multiple proteins potentially serving many different functions (
      • Fruman D.A.
      • Snapper S.B.
      • Yballe C.M.
      • Davidson L., Yu, J.Y.
      • Alt F.W.
      • Cantley L.C.
      ). Of the known p85 adapter subunits and splice variants, nearly all contain two Src-homology 2 (SH2) domains, which enable p85 to bind phosphotyrosine in an appropriate amino acid context. The p85 SH2 domains most frequently, but not exclusively, recognize phosphotyrosine embedded in a YXXM motif (
      • Songyang Z.
      • Shoelson S.E.
      • McGlade J.
      • Olivier P.
      • Pawson T.
      • Bustelo X.R.
      • Barbacid M.
      • Sabe H.
      • Hanafusa H.
      • Yi T.
      • Ren R.
      • Baltimore D.
      • Ratnofsky S.
      • Feldman R.A.
      • Cantley L.C.
      ). Most p85 gene products also include a Src homology 3 (SH3) domain, as well as other domains involved in protein-protein interactions (
      • Wymann M.P.
      • Pirola L.
      ). All p85 family members contain a p110-binding motif located between the two SH2 domains. The diversity of protein interaction domains found among p85 family members likely contributes to the ability of multiple signaling proteins and pathways to activate PI3K. Under resting conditions, p85 serves to both stabilize p110 protein and inhibit PI3K lipid kinase activity, thereby increasing the amount of inert p110 available for activation (
      • Yu J.
      • Zhang Y.
      • McIlroy J.
      • Rordorf-Nikolic T.
      • Orr G.A.
      • Backer J.M.
      ). This inhibitory effect is alleviated by binding of the SH2 domains of p85, and in particular the NH2-terminal SH2 domain, to tyrosine-phosphorylated peptides, as well as tyrosine-phosphorylated receptors or linker molecules containing the YXXM motif (
      • Yu J.
      • Zhang Y.
      • McIlroy J.
      • Rordorf-Nikolic T.
      • Orr G.A.
      • Backer J.M.
      ). Tyrosine phosphorylation of p85 binding sites within growth factor receptor cytoplasmic domains and linker molecules thus results in the recruitment of p85 to the cell membrane with consequent release of p85-mediated inhibition of PI3K (
      • Yu J.
      • Zhang Y.
      • McIlroy J.
      • Rordorf-Nikolic T.
      • Orr G.A.
      • Backer J.M.
      ) and colocalization of PI3K with its substrate membrane phosphatidylinositols (
      • Rameh L.E.
      • Cantley L.C.
      ) and other regulatory molecules (
      • Blalock W.L.
      • Weinstein-Oppenheimer C.
      • Chang F.
      • Hoyle P.E.
      • Wang X.Y.
      • Algate P.A.
      • Franklin R.A.
      • Oberhaus S.M.
      • Steelman L.S.
      • McCubrey J.A.
      ,
      • Mayer B.J.
      • Gupta R.
      ).
      The regulatory p85 subunit of PI3K is phosphorylated at tyrosine 688 (Tyr688) by the Src family kinases Lck and Abl (
      • von Willebrand M.
      • Williams S.
      • Saxena M.
      • Gilman J.
      • Tailor P.
      • Jascur T.
      • Amarante-Mendes G.P.
      • Green D.R.
      • Mustelin T.
      ) and dephosphorylated at this site by the protein tyrosine phosphatase, SHP-1 (
      • Cuevas B.
      • Lu Y.
      • Watt S.
      • Kumar R.
      • Zhang J.
      • Siminovitch K.A.
      • Mills G.B.
      ). While p85 is known to be tyrosine-phosphorylated in response to a variety of stimuli, the role of p85 tyrosine phosphorylation in PI3K activation is unknown (
      • Blalock W.L.
      • Weinstein-Oppenheimer C.
      • Chang F.
      • Hoyle P.E.
      • Wang X.Y.
      • Algate P.A.
      • Franklin R.A.
      • Oberhaus S.M.
      • Steelman L.S.
      • McCubrey J.A.
      ,
      • Yu Z.
      • Su L.
      • Hoglinger O.
      • Jaramillo M.L.
      • Banville D.
      • Shen S.H.
      ,
      • von Willebrand M.
      • Baier G.
      • Couture C.
      • Burn P.
      • Mustelin T.
      ). Tyrosine phosphorylation of p85 does, however, correlate with proliferative rate in Jurkat cells (
      • Martinez-Lorenzo M.J.
      • Anel A.
      • Moneleon I.
      • Sierra J.J.
      • Pineiro A.
      • Naval J.
      • Alava M.A.
      ) and alters SH2 domain binding properties (
      • von Willebrand M.
      • Williams S.
      • Saxena M.
      • Gilman J.
      • Tailor P.
      • Jascur T.
      • Amarante-Mendes G.P.
      • Green D.R.
      • Mustelin T.
      ). Previous data from our group have revealed that coexpression of a constitutively active form of Lck with PI3K in COS cells results in an increase in PI3K activity (
      • Cuevas B.
      • Lu Y.
      • Watt S.
      • Kumar R.
      • Zhang J.
      • Siminovitch K.A.
      • Mills G.B.
      ). In this system, coincident expression of SHP-1 is associated with a decrease in PI3K activity, while expression of a phosphatase-inactive form of SHP-1 increases PI3K activity. These data suggest that phosphorylation/dephosphorylation of Tyr688, a residue that maps within the p85 carboxyl SH2 domain, provides a mechanism for regulating PI3K activity. The data described herein directly address this latter possibility and demonstrate that tyrosine phosphorylation of p85 and, more specifically of Tyr688, regulates PI3K activity, NFκB activation, and growth factor deprivation-induced cell death. The data also link these effects of Tyr688 phosphorylation to the formation of an intramolecular complex with the p85 NH2-terminal domain relieving the inhibitory effect of p85 on p110.

      DISCUSSION

      The biochemical events governing protein tyrosine phosphorylation are central to the regulation of cellular signaling in all eukaryotic cells. However, while a myriad of intracellular proteins undergo tyrosine phosphorylation following cell stimulation, for many proteins, the effects of phosphorylation on function are not well defined. This latter group of proteins includes PI3K, an enzyme that is inducibly tyrosine-phosphorylated in many biological contexts. It has been suggested that PI3K is negatively regulated by serine autophosphorylation of the p85 regulatory subunit (
      • Dhand R.
      • Hiles I.
      • Panayotou G.
      • Roche S.
      • Fry M.J.
      • Gout I.
      • Totty N.F.
      • Truong O.
      • Vicendo P.
      • Yonezawa K.
      • Kasuga M.
      • Courtneidge S.A.
      • Waterfield M.D.
      ). However, interaction of the p85 SH2 domains with tyrosine-phosphorylated peptides appears to alleviate this inhibition, a finding that implies a role for tyrosine phosphorylation in regulating PI3K activity (
      • Yu J.
      • Zhang Y.
      • McIlroy J.
      • Rordorf-Nikolic T.
      • Orr G.A.
      • Backer J.M.
      ). This possibility is strongly supported by the current data showing that SHP-1, an enzyme that dephosphorylates the major tyrosine phosphorylation site on p85, Tyr688, down-regulates the PI3K/Akt activation pathway. Moreover, the current data, revealing lipid kinase activity to be higher in the p85 protein present in anti-phosphotyrosine immunoprecipitates than in the p85 protein immunoprecipitated from cell lysates immunodepleted for tyrosine phosphorylated species, also indicate a direct relationship between p85 phosphorylation status and PI3K activity. Enhanced PI3K activity in this latter experiment implies that the inhibitory effect of the p85 SH2 domains on enzymatic activity has been released, a phenomenon that might relate to the tyrosine phosphorylation of p85 per seor, alternatively, to interactions of the p85 SH2 domains with tyrosine-phosphorylated proteins captured by anti-phosphotyrosine immunoprecipitation. To distinguish between these possibilities, p85 proteins mutated at the major tyrosine phosphorylation site (Tyr688) were investigated with respect to their effects on PI3K activity. The results of this analysis revealed p85 Y688A protein, which cannot be phosphorylated at Tyr688, to be associated with impaired PI3K activity as manifested by decreases in Akt phosphorylation, BAF/3 cell survival, and NFκB promoter activation. By contrast, these latter activities were all enhanced in cells expressing a mutant p85 protein, Y688D, which is predicted to mimic tyrosine-phosphorylated p85. Taken together, these data provide compelling evidence that PI3K activity is regulated by phosphorylation of p85 at position Tyr688.
      While the crystal structure of full-length p85 bound to phosphopeptide is not currently available, the predicted protein sequence of the intervening iSH2 domain (p110 binding site between the two SH2 domains) indicates a pair of antiparallel helices and thus predicts that the two SH2 domains are closely aligned (
      • Wymann M.P.
      • Pirola L.
      ). These data raise the possibility of an intramolecular association involving binding of the phosphorylated Tyr688 residue within the p85 carboxyl-terminal tail to the p85 amino-terminal SH2 domain. This model, which is illustrated in Fig. 7, is supported by the current data, which reveal the ability of full-length p85 to associate with the phosphorylated, but not nonphosphorylated, Tyr688-containing carboxyl-terminal fragment of p85 and which also suggest that this association is mediated via the p85 amino-terminal SH2 domain (Fig. 6). The data also exclude the possibility that this association depends upon Lck functioning as an intermediary “linker” protein, as the association occurs in the absence of Lck when p85 Y688D is used in the analysis. Together, these data suggest the existence of an intramolecular interaction, between phosphorylated Tyr688 and the amino-terminal SH2 domain of p85 (Fig. 7).
      Figure thumbnail gr7
      Figure 7Proposed models of the effect of phosphorylation of Tyr688 in p85 on PI3K activity. The left panel displays an intramolecular regulatory mechanism, whereas the right panel depicts an alternative intermolecular mechanism, resulting in PI3K concatamers.GFR, growth factor receptor.
      Although the amino acid sequence surrounding Tyr688 does not conform to the expected p85 SH2 target sequence (YXXM), this SH2 domain has already been shown to exhibit flexibility in terms of the target motif (
      • Ponzetto C.
      • Bardelli A.
      • Maina F.
      • Longati P.
      • Panayotou G.
      • Dhand R.
      • Waterfield M.D.
      • Comoglio P.M.
      ,
      • He T.C.
      • Zhuang H.
      • Jiang N.
      • Waterfield M.D.
      • Wojchowski D.M.
      ). Furthermore, an intramolecular association of the nature proposed here may provide a mechanism to prevent binding of the p85 SH2 domains to low affinity substrates. This possibility is supported by previous data revealing p85 association with several phosphorylated proteins to be disrupted upon Tyr688 phosphorylation (
      • von Willebrand M.
      • Williams S.
      • Saxena M.
      • Gilman J.
      • Tailor P.
      • Jascur T.
      • Amarante-Mendes G.P.
      • Green D.R.
      • Mustelin T.
      ). The current data suggest that this latter observation may reflect competitive inhibition consequent to the formation of an intramolecular association. As with SH2 occupation by other phosphopeptides, this association would serve to “relax” the p85-mediated inhibition of p110 PI3K activity. In addition to this model, the current data might also be explained by another model wherein phosphorylation of Tyr688 triggers an intermolecular interaction between individual p85 proteins, again inducing disruption of the inhibitory activity of p85 (Fig. 7). In this alternative “PI3K concatamer” model, the recruitment of multiple PI3K molecules could represent a mechanism whereby the PI3K signaling cascade is amplified. It is possible that p85 intramolecular interactions also promote PI3K signal amplification by facilitating the removal of phosphorylated PI3K and thus freeing the receptor for subsequent association with a new PI3K. The newly detached, phosphorylated PI3K could then be dephosphorylated by SHP-1 and returned to a basal state, once again available for recruitment to a phosphorylated receptor. Alternatively, an induced intramolecular interaction may represent a mechanism by which PI3K is removed from activated growth factor receptors. Recent studies have shown that an intermolecular interaction also occurs between the p85 SH3 and proline-rich domains (
      • Harpur A.G.
      • Layton M.J.
      • Das P.
      • Bottomley M.J.
      • Panayotou G.
      • Driscoll P.C.
      • Waterfield M.D.
      ), a result which suggests that concatamers of p85 may play a role in forming multimeric interaction complexes. Whichever model proves valid, the capacity of Y688D to mimic the effect of Tyr688 phosphorylation implies that the minimal requirement for this association is phosphorylation at p85 Tyr688.
      Taken together, the data indicate that phosphorylation of 688 relieves the inhibitory activity of p85 on p110 and suggest that this effect is mediated by the association of phosphorylated tyrosine 688 with the NH2-terminal SH2 domain of p85. Thus intramolecular interactions with phosphorylation sites in p85 have the potential to contribute to the outcome of ligand activation of cells.

      Acknowledgment

      We thank the DNA Core Sequencing Facility for sequencing the p85 Y688A, p85 Y688D, and HACT Y688D constructs.

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