Advertisement

Specific Association of the β Isoform of the p85 Subunit of Phosphatidylinositol-3 Kinase with the Proto-oncogene c-cbl*

  • David Hartley
    Affiliations
    Program in Molecular Medicine and Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655
    Search for articles by this author
  • Herman Meisner
    Affiliations
    Program in Molecular Medicine and Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655
    Search for articles by this author
  • Silvia Corvera
    Correspondence
    To whom all correspondence should be addressed: The Program in Molecular Medicine and Dept. of Cell Biology, University of Massachusetts Medical School, 55 Lake Ave. North, Worcester, MA 01655. Tel.: 508-856-6898; Fax: 508-856-4289;
    Affiliations
    Program in Molecular Medicine and Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655
    Search for articles by this author
  • Author Footnotes
    * This work was supported by National Institutes of Health Grant DK-40330. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:August 04, 1995DOI:https://doi.org/10.1074/jbc.270.31.18260
      Phosphatidylinositol-3 kinase (PI-3 kinase) has been implicated in cellular events such as mitogenic signaling, actin organization, and receptor sorting. The p85 subunit of PI-3 kinase contains multiple domains capable of protein-protein interactions that may contribute to mediate the multiple physiological functions of this enzyme. Here, we demonstrate that antibodies raised against the p85 subunit of PI-3 kinase immunoprecipitate a single tyrosine-phosphorylated protein of 120 kDa (pp120) from lysates of activated Jurkat T cells and A20 B cells. This protein is the only significant phosphotyrosine-containing protein in p85 immunoprecipitates from these cells, and it cannot be detected in immunoprecipitates of other signaling proteins such as PLCγ. Furthermore, antibodies specific for the β isoform of p85 but not antibodies specific for the α isoform immunoprecipitate this tyrosine-phosphorylated protein. pp120 completely comigrates with the proto-oncogene c-cbl, which is a 120 kDa protein product abundant in lymphoid cells. Furthermore, immunoblots of p85 immunoprecipitates using antibodies raised against c-cbl detect a band at exactly the position of pp120. In addition, p85 can be detected in immunoblots of c-cbl immunoprecipitates. Thus, pp120 appears to correspond to c-cbl. A direct association between c-cbl and p85 can be observed in vitro using a fusion protein comprising the Src homology 2 (SH2) domains of p85, and this binding is abolished by phenyl phosphate, suggesting that the interaction is mediated through phosphotyrosine-SH2 domain interactions. Thus, these results show important functional differences between the α and β isoforms of p85 in vivo and point to c-cbl as a potentially important mediator of some of the functions of PI-3 kinase in intact cells.

      INTRODUCTION

      Phosphatidylinositol 3-kinase is a lipid kinase that phosphorylates PIns,
      The abbreviations used are: PIns
      phosphatidylinositol
      PDGF
      platelet-derived growth factor
      PAGE
      polyacrylamide gel electrophoresis
      PI-3 kinase
      phosphatidylinositol-3 kinase
      pTyr
      phosphotyrosine
      TCR
      T cell receptor.
      PIns(4)P, and PIns(4,5)P2 on the D3 position of the inositol ring (PI-3 kinase). PI-3 kinase was first identified as a lipid kinase activity associated with the middle T antigen in polyoma virus (SV40) transformed cells. It is now known to associate with several different receptor tyrosine kinases, including the receptors for PDGF, epidermal growth factor, insulin/IRS-1, and with non-receptor tyrosine kinases, such as p60src(
      • Escobedo J.A.
      • Navankasattus S.
      • Kavanaugh W.M.
      • Milfay D.
      • Fried V.A.
      • Williams L.T.
      ,
      • 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.
      ,
      • Liu X.
      • Marengere L.E.M.
      • Koch C.A.
      • Pawson T.
      ). The association with multiple tyrosine kinases has suggested that PI-3 kinase plays an important role in signaling pathways leading to growth and proliferation. Interestingly, cloning of the catalytic subunit of PI-3 kinase revealed a high degree of homology with a yeast protein, vps34p, which plays a fundamental role in the delivery of newly synthesized proteins to the yeast vacuole, indicating an important role for PI-3 kinase activity in protein sorting(
      • Herman P.K.
      • Stack J.H.
      • Emr S.
      ). Recent studies from our laboratory strongly suggest that PI-3 kinase activity may play an important role in the intracellular sorting and down-regulation of the PDGF receptor(
      • Joly M.
      • Kazlauskas A.
      • Fay F.S.
      • Corvera S.
      ). Thus, PI-3 kinase may coordinate or regulate diverse functions of receptor tyrosine kinases in mammalian cells.
      The multiplicity of potential functions of PI-3 kinase activity is paralleled by the complexity of the PI-3 kinase itself. The mammalian kinase is composed of two subunits, an 85-kDa regulatory subunit (p85) and a 110-kDa catalytic subunit (p110), of which two different respective isoforms have been characterized, though many isoforms are likely to exist(
      • Kapeller R.
      • Cantley L.C.
      ). The p85 subunit contains two SH2 domains, one SH3 domain, a Bcr homology domain, and two different proline-rich regions, which represent potential SH3 domain binding sites(
      • Kapeller R.
      • Prasad K.V.S.
      • Janssen O.
      • Hou W.
      • Schaffhausen B.S.
      • Rudd C.E.
      • Cantley L.C.
      ). Thus, the p85 subunit has the potential for forming oligomers or heteroligomers with proteins that also contain SH3 or poly-proline domains. The signaling or sorting functions of PI-3 kinase may involve its direct interaction with these other cellular components.
      We have set out to identify proteins that stably interact with PI-3 kinase, which may constitute possible targets or effectors of PI-3 kinase function. We initiated these studies using T and B lymphoid cell lines, which, when activated through the TCR/CD3 complex or through cross-linked surface immunoglobulins, display massive increases in tyrosine phosphorylation of cellular proteins and rapid changes in cellular functions ranging from increased adhesion to receptor up-regulation. PI-3 kinase does not directly associate with the TCR/CD3 complex or with surface immunoglobulins. It only interacts directly with the membrane receptors CD28 in T cells and CD19 in B cells(
      • Truitt K.E.
      • Hicks C.M.
      • Imboden J.B.
      ,
      • Tuveson D.A.
      • Carter R.H.
      • Soltoff S.P.
      • Fearon D.T.
      ). It has also been reported to interact weakly with members of the Src family of tyrosine kinases(
      • Prasad K.V.S.
      • Janssen O.
      • Kapeller R.
      • Raab M.
      • Cantley L.C.
      • Rudd C.E.
      ,
      • Prasad K.V.S.
      • Kapeller R.
      • Janssen O.
      • Repke H.
      • Duke-Cohen J.S.
      • Cantley L.C.
      • Rudd C.E.
      ). Thus, cells stimulated through the TCR/CD3 or immunoglobulin complexes are ideally suited for investigating new potential high affinity interactions between PI-3 kinase and cellular components elicited upon activation. In this paper, we report that PI-3 kinase rapidly and stably associates with a 120-kDa protein that is tyrosine phosphorylated in cells activated through the TCR/CD3 or immunoglobulin complexes. This protein appears to correspond to the proto-oncogene c-cbl. In addition, our results suggest that, in vivo, c-cbl specifically associates with the β isoform of the p85 subunit of PI-3 kinase, suggesting important physiological differences between these kinase isoforms.

      MATERIALS AND METHODS

      Antibodies

      Monoclonal antibodies directed to the N-terminal SH2 domain (UB 93-3, 05-217) or to the SH3 domain (05-212) of p85, as well as rabbit polyclonal antisera to rat PI-3 kinase (06-195) were obtained from Upstate Biotechnology Inc. Monoclonal antibodies were always used in combination to maximize the recovery of p85 molecules that might be complexed through SH2 or SH3 domains. Isoform-specific rabbit antisera were raised against peptides based on the extreme C-terminal 15 amino acids of each isoform as described (
      • Baltensperger K.
      • Kozma L.M.
      • Jaspers S.R.
      • Czech M.P.
      ). Unlabeled goat anti-Ig (IgM, IgG, IgA) used for stimulating A20 cells and cross-linking OKT3, was obtained from Cappel. Affinity-purified c-cbl antisera was purchased from Santa Cruz Biotechnology. The OKT3 and OKT4 hybridomas were purchased from ATCC (CRL8001, CRL8002). Antibody was concentrated from cell supernatants using ABx bakerbond (J. T. Baker). Monoclonal antibody raised against phosphotyrosine (4G10) was obtained from UBI. The fusion protein was constructed using a portion of the human cDNA for p85α, consisting of the two SH2 domains and the intervening sequences (nucleotides 986-2313). The DNA was inserted into the pGEX2T vector and expressed in Escherichia coli strain, XA90. The GST-SH2 fusion protein was adsorbed onto glutathione-agarose beads and used directly for adsorption studies.

      Cells

      Jurkat T cells and A20 murine B lymphoma cells were obtained from ATCC. All cells were grown in complete RPMI media supplemented with 10% fetal calf serum. T cells were aliquoted (3-5 × 106 cells/ml) into microfuge tubes and stimulated using 50 μg/ml ABx-purified OKT3 supplemented with excess goat anti-mouse IgG. At the time points indicated in each experiment, cells were pelleted in a microfuge for 5 s, placed on ice, and resuspended in cold lysis buffer (see below). B cells were aliquoted at 5 × 106 cells/ml and stimulated with 20 μg of purified anti-IgG. For metabolic labeling experiments, cells were incubated in serum-free, methionine-free RPMI with 200 μCi/ml [35S]methionine at 37°C for 3 h. Cells were then transferred to serum-free RPMI for an additional hour before stimulation.

      Immunoprecipitation and Immunoblotting

      Cells were lysed in 1 ml of an ice-cold buffer composed of 1% Triton X-100, 20 mM Tris, 150 mM sodium chloride, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 1 mM 1,10-phenanthroline, 1 mM sodium vanadate, and 50 mM sodium fluoride. Lysates were clarified by centrifugation at 14,000 × g for 15 min and precleared by incubation with Protein A-Sepharose prior to addition of specific antibodies. After 120 min of incubation at 5°C, protein A-Sepharose was added, and incubations continued for a further 60 min. Protein A beads were then washed three to five times in a wash buffer composed of 20 mM Tris, 150 mM sodium chloride, 0.2% Triton X-100, and 0.1% SDS. Immunoprecipitates were resolved by SDS-PAGE and transferred to nitrocellulose for immunoblotting. Primary antibodies were detected by chemiluminescence (Amersham Corp.). For adsorption to the agarose-immobilized SH2-p85 fusion protein, lysates were clarified as above and incubated with the concentrations of fusion protein indicated in Fig. 6. After 60 min of incubation at 5°C, the agarose beads were washed as described above. Beads were then boiled in SDS-sample buffer, and adsorbed proteins were resolved by SDS-PAGE.
      Figure thumbnail gr6
      Figure 6:The SH2 domain of p85 associates with cbl. Jurkat T cells were incubated at 37°C in the presence of soluble anti-CD3 and secondary cross-linking antibody for 4 min. Cells were then lysed, and extracts were incubated with the indicated concentration of a GST fusion protein comprising the two SH2 domains of p85α (A) or the indicated concentration of GST alone (B). C, p85 was immunoprecipitated from the supernatants of the lanes in B using the polyclonal antisera (UBI). Precipitates were resolved on 7.5% SDS-PAGE, transferred to nitrocellulose, and probed with anti-phosphotyrosine antibodies.

      RESULTS AND DISCUSSION

      A Single Tyrosine-phosphorylated Protein Associates with PI-3 Kinase in Activated Lymphocytes

      The complex structure of the p85 subunit of PI-3 kinase can potentially give rise to numerous protein-protein interactions in cells stimulated by mitogenic factors. At least a subset of interacting proteins might be expected to interact stably and to coimmunoprecipitate with kinase isolated from cell extracts. To identify such components, we analyzed the polypeptide composition of p85 immunoprecipitates from stimulated Jurkat T cells labeled to equilibrium with [35S]methionine. Polypeptides in such immunoprecipitates were separated by polyacrylamide gel electrophoresis and transferred onto nitrocellulose paper for immunoblotting.
      Immunoprecipitates obtained using monoclonal antibodies to p85 contained two major bands that migrated with molecular weights of approximately 85 and 110 kDa (Fig. 1A). These two bands probably represent the p85 and catalytic subunits of PI-3 kinase, respectively. Minor polypeptides were also observed, the most prominent of which migrated slightly above the 110-kDa band (Fig. 1, left lane). In contrast, immunoprecipitates of activated cell extracts obtained with monoclonal antibodies to phosphotyrosine contained a large number of bands, many of which were clustered around 100-120 kDa. Immunoblotting of the p85 immunoprecipitates with antibodies against phosphotyrosine (Fig. 1B) revealed only a single band of 120 kDa. This phosphoprotein comigrates with the methionine-labeled band detected above the p110 subunit of PI-3 kinase. It was striking in these experiments that pp120 was the only major tyrosine phosphoprotein detected in p85 immunoprecipitates, despite the massive increase in the number of tyrosine-containing proteins in cell extracts after activation. The coprecipitation of a single phosphotyrosine protein with p85 in T lymphocytes is in marked contrast to that observed in non-lymphoid cells. For example, immunoprecipitation of p85 from fibroblastic cells stimulated with PDGF coprecipitates many phosphotyrosine-containing proteins including the PDGF receptor and other proteins associated with the receptor complex, such as PLCγ1, GAP, and Syp. These results suggest that pp120 may be a major target or effector of PI-3 kinase function in lymphoid cells.
      Figure thumbnail gr1
      Figure 1:A tyrosine-phosphorylated protein is coprecipitated with PI-3 kinase. Jurkat T cells were labeled with [35S]methionine as described under “Materials and Methods” and then stimulated with soluble anti-CD3 for 4 min at 37°C. Total cell lysates were immunoprecipitated with a combination of two monoclonal antibodies raised against the SH2 and SH3 domains of p85 or with a monoclonal antibody raised against pTyr. Immunoprecipitates were then resolved on 7.5% SDS-PAGE and transferred to nitrocellulose. A, an autoradiogram of the nitrocellulose blot. Arrows indicate major bands immunoprecipitated specifically by the antibodies. B, phosphotyrosine immunoblot of lanes shown in A. The primary antibody was detected using horseradish peroxidase-coupled secondary antibodies and ECL. An arrow points to the single phosphotyrosine band, pp120. The intense lower band at approximately 40 kDa corresponds to the precipitating immunoglobulin, detected by the anti-mouse secondary antibodies.
      Immunoprecipitation of p85 at various time points after activation revealed some pp120 in immunoprecipitates from non-stimulated Jurkat T cells. However, the level of pp120 phosphorylation significantly increased over this constitutive basal level after 1 min of stimulation (Fig. 2A). In addition, a decrease in mobility of pp120 could be observed. Phosphorlyation of pp120 decreased subsequently, approximating background levels after 15 min of stimulation. This increase in the phosphotyrosine signal is not due to changes in the amount of p85 present in the immunoprecipitates (Fig. 2B).
      Figure thumbnail gr2
      Figure 2:Time course of tyrosine phosphorylation of the PI-3 kinase-associated pp120. Jurkat T cells were incubated at 37°C in the presence of soluble anti-CD3 and secondary cross-linking antibody for the times indicated above each lane. Cells were then lysed, and extracts were immunoprecipitated with polyclonal antisera to p85. Immunoprecipitates were then resolved on 7.5% SDS-PAGE and transferred to nitrocellulose. A, phosphotyrosine immunoblot; B, P85 immunoblot. Arrows point to pp120 (A) and p85 (B). The intense band at 105 kDa is a contaminant in the polyclonal anti-p85 antiserum, which is detected by the secondary antibody.
      We next investigated whether the association of p85 with pp120 was restricted to T lymphocytes. For these experiments, we employed the murine B cell lymphoma, A20, which expresses IgG on the cell surface and is efficient at presenting antigen to T cells. Resting A20 B cells display very low levels of tyrosine phosphorylation, and stimulation results in a massive increase in tyrosine phosphorylation of numerous bands (not shown). Immunoprecipitates of p85 from resting A20 B cells were devoid any coprecipitating tyrosine phosphoprotein, but p85 immunoprecipitates from activated A20 cells contained three prominent tyrosine-phosphorylated bands at 95-100, 120, and 150 kDa (Fig. 3). Thus, the association of a tyrosine-phosphorylated, 120-kDa protein with p85 also appears to occur in B cell lines. A potential candidate for the 95-100-kDa band is the surface molecule CD19, known to be tyrosine phosphorylated and associate with PI-3 kinase after stimulation(
      • Tuveson D.A.
      • Carter R.H.
      • Soltoff S.P.
      • Fearon D.T.
      ). The phosphotyrosine band at 150 kDa was not consistently observed in p85 precipitates and was occasionally observed in the preimmune precipitates.
      Figure thumbnail gr3
      Figure 3:Tyrosine-phosphorylated bands associated with p85 in A20 B cells. A20 B cells were incubated at 37°C with anti-Ig for the times indicated above each lane. Cells were then lysed, and extracts were immunoprecipitated with polyclonal antisera to p85. Immunoprecipitates were then resolved on 7.5% SDS-PAGE, transferred to nitrocellulose, and immunoblotted for phosphotyrosine.

      pp120 Is the Proto-oncogene c-cbl

      The identity of some of the numerous proteins that are tyrosine phosphorylated in response to stimulation through the T cell receptor has been determined. Two of the proteins in the molecular range of 100-120 kDa have been shown to correspond to the mammalian protein VCP (
      • Egerton M.
      • Ashe O.R.
      • Chen D.
      • Druker B.J.
      • Burgess W.H.
      • Samelson L.E.
      ) and to the proto-oncogene c-cbl(
      • Donovan J.A.
      • Wnage R.L.
      • Langdon W.Y.
      • Samelson L.E.
      ). Antibodies to VCP and to c-cbl were used to determine whether these polypeptides corresponded to the 120-kDa tyrosine-phosphorylated polypeptide associated with p85. Antibodies against c-cbl but not antibodies against VCP immunoprecipitated a tyrosine-phosphorylated protein that comigrated exactly with the p120 band associated with p85 in lysates of A20 B cells (Fig. 4A). In addition, immunoblotting with anti-p85 antibodies revealed the presence of p85 in c-cbl immunoprecipitates of extracts from stimulated but not resting B cells (Fig. 4B). Although the amount of p85 in c-cbl immunoprecipitates represented a small fraction of the total cellular p85 pool, it represented a significant amount (approximately 50%) of the p85 protein associated with phosphotyrosine immunoprecipitates (not shown). These results suggest that c-cbl is the major tyrosine phosphoprotein associated with p85 in these cells.
      Figure thumbnail gr4
      Figure 4:c-cbl is associated with p85. A20 B cells were incubated at 37°C with anti-Ig for the times indicated above each lane. Cells were then lysed, and extracts were divided into four aliquots. Aliquots were immunoprecipitated with polyclonal antisera to p85, non-immune rabbit serum (P.I.), polyclonal antisera against c-cbl (cbl), or boiled in sample buffer (TCL). Immunoprecipitates and total cell lysate were resolved on 7.5% SDS-PAGE and transferred to nitrocellulose. The nitrocellulose blot was cut, and the regions above and below the 100-kDa marker were blotted with anti-phosphotyrosine (A) or anti-p85 antibodies (B), respectively. The position of prestained molecular weight markers is indicated.
      The significance of the p85-c-cbl interaction cannot be determined from these studies because the functional cellular role of c-cbl is unknown. The v-cbl oncogene was originally identified as expressed by the transforming retrovirus Cas NS-1 (
      • Donovan J.A.
      • Wnage R.L.
      • Langdon W.Y.
      • Samelson L.E.
      ,
      • Blake T.J.
      • Shapiro M.
      • Morse III, H.C.
      • Langdon W.Y.
      ,
      • Mushinski J.F.
      • Goodnight J.
      • Rudikoff E.
      • Morse H.C.
      • Langdon W.Y.
      ) and was later found to be a truncated form of an endogenous cellular protein. The truncated form, v-cbl, can be found in the nucleus and has been shown to have the ability to bind DNA. In contrast, the endogenous c-cbl exists as a 120-kDa protein that does not appear to reside in the nucleus and does not bind DNA. It has not been possible to detect truncated forms of c-cbl, though a smaller mRNA transcript has been shown to be expressed by some cells. The mechanism whereby v-cbl is transforming and the physiological function of the larger c-cbl have yet to be determined.

      c-cbl Specifically Interacts with p85β

      Jurkat T cells have been shown to contain two of the known isoforms of p85 (α and β), which display high sequence similarity. The functional differences between the two p85 isoforms are not known, though there is some evidence that p85 isoforms regulate the catalytic activity of PI-3 kinase differentially (
      • Egerton M.
      • Ashe O.R.
      • Chen D.
      • Druker B.J.
      • Burgess W.H.
      • Samelson L.E.
      ) and that in lymphocytes they are differentially phosphorylated in response to stimulation(
      • Reif K.
      • Gout I.
      • Waterfield M.D.
      • Cantrell D.A.
      ). We sought to determine the specificity of the interaction of c-cbl with the α or β isoforms of p85. These two characterized isoforms of p85 display slightly different mobilities on SDS-PAGE and can be separated by immunoprecipitation with isoform-specific antisera (Fig. 5A, top panel). Despite the presence of large amounts of p85α in immunoprecipitates obtained with α-isoform-specific antiserum, c-cbl could not be detected by immunoblotting these precipitates with c-cbl antiserum (Fig. 5A, middle panel). Furthermore, p85α immunoprecipitates from activated Jurkat T cells were devoid of coprecipitating tyrosine phosphoproteins. These results are consistent with the findings of Ward et al.(
      • Ward S.G.
      • Reif K.
      • Ley S.
      • Fry M.J.
      • Waterfield M.D.
      • Cantrell D.A.
      ), which concluded that PI-3 kinase is not a substrate for tyrosine kinases in T cells and does not interact with any tyrosine-phosphorylated proteins. These investigators employed anti-p85 antibodies specific for p85α. In contrast, p85β immunoprecipitates contained a single phosphotyrosine-containing protein at 120 kDa (Fig. 5A, bottom), which was also detected with anti-c-cbl antibodies (Fig. 5A, middle). A time course shows that this isoform-specific interaction occurs rapidly after activation and declines over 15 min (Fig. 5B). This time course is identical to that observed using polyclonal antisera against p85 which does not distinguish among different isoforms (Fig. 2).
      Figure thumbnail gr5
      Figure 5:c-cbl is specifically associated with p85β. A, Jurkat T cells (2.5 × 107) were stimulated for 2 min at 37°C by cross-linking CD3 and CD4, lysed, and immunoprecipitated with isoform-specific antisera raised against p85α and p85β. Immunoprecipitates were resolved on 7.5% SDS-PAGE, blotted onto nitrocellulose, and probed with anti-p85 antiserum, anti-phosphotyrosine, and anti-c-cbl. Top panel illustrates the region of the blot containing p85, and middle and bottom panels are the region above the 105-kDa marker, which contained the only tyrosine phosphoprotein in p85 immunoprecipitates. B, Jurkat T cells were incubated at 37°C in the presence of soluble anti-CD3 and secondary cross-linking antibody for the times indicated above each lane. Cells were then lysed, and extracts were immunoprecipitated with isoform-specific anti-p85 antisera as indicated. Immunoprecipitates were then resolved on 7.5% SDS-PAGE, transferred to nitrocellulose, and probed with anti-phosphotyrosine antibodies.

      p85 Can Associate with c-cbl through Its SH2 Domains

      An analysis of the human c-cbl sequence shows a C-terminal proline-rich region as well as multiple tyrosine residues(
      • Blake T.J.
      • Shapiro M.
      • Morse III, H.C.
      • Langdon W.Y.
      ), including one within the context of a consensus p85 binding site (YEXM, tyrosine 731), as determined by Songyang et al.(
      • Songyang Z.
      • Shoelson S.E.
      • McGlage 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.
      ). The proline-rich region has recently been reported to bind the SH3 domains of the adaptor molecule Grb-2 in vitro(
      • Donovan J.A.
      • Wnage R.L.
      • Langdon W.Y.
      • Samelson L.E.
      ) and was previously shown to bind p47 Nck(
      • Rivero-Lezcano O.
      • Sameshima J.H.
      • Marcilla A.
      • Robbins K.C.
      ). To determine the nature of the interactions between p85 and c-cbl, we tested the ability of a p85 fusion protein to adsorb to c-cbl from cell extracts. A GST-fusion protein comprising the two SH2 domains of p85α readily adsorbed a tyrosine-phosphorylated protein of 120 kDa from Jurkat cells, as well as many other bands not seen in polyclonal or isoform-specific p85 precipitates (Fig. 6). Both the fusion protein and polyclonal p85 antisera fail to coprecipitate this band when 40 mM phenyl phosphate was added to the lysis buffer (data not shown). These results suggest that the interaction between p85 and c-cbl occurs through SH2 domains of p85 and phosphotyrosine residues in c-cbl.
      Work by Songyang and co-workers (
      • Songyang Z.
      • Shoelson S.E.
      • McGlage 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.
      ) to determine the consensus binding sites for p85 SH2 domains failed to show any differences in selectivity between the SH2 domains of the α or β isoforms(
      • Songyang Z.
      • Shoelson S.E.
      • McGlage 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.
      ). Thus, it is unlikely that different affinities for c-cbl can explain the observed differential interaction between the α and β isoforms of p85 and c-cbl that appear to occur in intact cells. Other potential factors that may influence in vivo interactions and that may not be apparent from in vitro peptide affinity studies are additional protein-protein interactions or cellular localization. These factors may also explain our failure to detect an interaction between PLCγ1 and c-cbl in immunoprecipitates of c-cbl or PLCγ1 obtained from cell extracts (not shown and (
      • Gilliland L.K.
      • Schieven G.L.
      • Norris N.A.
      • Kanner S.B.
      • Aruffo A.
      • Ledbetter J.A.
      )), despite the fact that an interaction between the SH2 domains of PLCγ1 and c-cbl can be readily observed in vitro(
      • Donovan J.A.
      • Wnage R.L.
      • Langdon W.Y.
      • Samelson L.E.
      ). We believe that the observed in vitro interaction between the p85 fusion protein and c-cbl probably reflects the mechanism of interaction between p85β and c-cbl in intact cells. The basis for the isoform-specific interaction in vivo between p85β and c-cbl cannot be determined from the data presented here, but understanding the nature and significance of this selective interaction is an important goal for future studies.

      Acknowledgments

      We acknowledge Drs. L. Samelson and A. B. Reynolds for the generous gifts of reagents.

      REFERENCES

        • Escobedo J.A.
        • Navankasattus S.
        • Kavanaugh W.M.
        • Milfay D.
        • Fried V.A.
        • Williams L.T.
        Cell. 1991; 65: 75-82
        • 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.
        Cell. 1991; 65: 91-104
        • Liu X.
        • Marengere L.E.M.
        • Koch C.A.
        • Pawson T.
        Mol. Cell Biol. 1993; 13: 5225-5232
        • Herman P.K.
        • Stack J.H.
        • Emr S.
        Trends Cell Biol. 1992; 2: 363-368
        • Joly M.
        • Kazlauskas A.
        • Fay F.S.
        • Corvera S.
        Science. 1994; 263: 684-687
        • Kapeller R.
        • Cantley L.C.
        BioEssays. 1994; 16: 565-576
        • Kapeller R.
        • Prasad K.V.S.
        • Janssen O.
        • Hou W.
        • Schaffhausen B.S.
        • Rudd C.E.
        • Cantley L.C.
        J. Biol. Chem. 1994; 269: 1927-1933
        • Prasad K.V.S.
        • Janssen O.
        • Kapeller R.
        • Raab M.
        • Cantley L.C.
        • Rudd C.E.
        Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7366-7370
        • Prasad K.V.S.
        • Kapeller R.
        • Janssen O.
        • Repke H.
        • Duke-Cohen J.S.
        • Cantley L.C.
        • Rudd C.E.
        Mol. Cell Biol. 1993; 13: 7708-7717
        • Truitt K.E.
        • Hicks C.M.
        • Imboden J.B.
        J. Exp. Med. 1994; 179: 1071-1076
        • Tuveson D.A.
        • Carter R.H.
        • Soltoff S.P.
        • Fearon D.T.
        Science. 1993; 260: 986-989
        • Baltensperger K.
        • Kozma L.M.
        • Jaspers S.R.
        • Czech M.P.
        J. Biol. Chem. 1994; 269: 28937-28946
        • Egerton M.
        • Ashe O.R.
        • Chen D.
        • Druker B.J.
        • Burgess W.H.
        • Samelson L.E.
        EMBO J. 1992; 11: 3533-3540
        • Donovan J.A.
        • Wnage R.L.
        • Langdon W.Y.
        • Samelson L.E.
        J. Biol. Chem. 1994; 269: 22921-22924
        • Blake T.J.
        • Shapiro M.
        • Morse III, H.C.
        • Langdon W.Y.
        Oncogene. 1991; 6: 653-657
        • Mushinski J.F.
        • Goodnight J.
        • Rudikoff E.
        • Morse H.C.
        • Langdon W.Y.
        Oncogene. 1994; 9: 2489-2497
        • Reif K.
        • Gout I.
        • Waterfield M.D.
        • Cantrell D.A.
        J. Biol. Chem. 1993; 268: 10780-10788
        • Ward S.G.
        • Reif K.
        • Ley S.
        • Fry M.J.
        • Waterfield M.D.
        • Cantrell D.A.
        J. Biol. Chem. 1992; 267: 23862-23869
        • Songyang Z.
        • Shoelson S.E.
        • McGlage 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.
        Mol. Cell. Biol. 1994; 14: 2777-2785
        • Rivero-Lezcano O.
        • Sameshima J.H.
        • Marcilla A.
        • Robbins K.C.
        J. Biol. Chem. 1994; 269: 17363-17366
        • Gilliland L.K.
        • Schieven G.L.
        • Norris N.A.
        • Kanner S.B.
        • Aruffo A.
        • Ledbetter J.A.
        J. Biol. Chem. 1992; 267: 13610-13616