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Using a Phage Display Library to Identify Basic Residues in A-Raf Required to Mediate Binding to the Src Homology 2 Domains of the p85 Subunit of Phosphatidylinositol 3′-Kinase*

Open AccessPublished:November 17, 2000DOI:https://doi.org/10.1074/jbc.M004720200
      Src homology 2 (SH2) domains are found in a variety of cytoplasmic proteins involved in mediating signals from cell surface receptors to various intracellular pathways. They fold as modular units and are capable of recognizing and binding to short linear peptide sequences containing a phosphorylated tyrosine residue. Here we show that each of the SH2 domains of the p85 subunit of phosphatidylinositol 3-kinase selects phage displayed peptide sequences containing the core (L/I)-A-(R/K)-I-R. The serine/threonine kinase A-Raf, containing the sequence LQRIRS, is associated with the p85 protein in both quiescent and growth factor stimulated cells. This suggests that p85 and A-Raf exist in a protein complex in cells and that complex formation does not require growth factor stimulation. We also show that p85 and A-Raf can bind directly to each other in vitro and that this interaction is mediated in part by the p85 SH2 domains. Further, the p85 SH2 domains require at least one of four distinct basic-X-basic sequence motifs within A-Raf for binding. This is the first description of a phosphotyrosine-independent SH2 domain interaction that requires basic residues on the SH2 ligand.
      PI 3-kinase
      phosphatidylinositol 3-kinase
      SH
      Src homology
      GST
      glutathioneS-transferase
      HA
      hemagglutinin
      PAGE
      polyacrylamide gel electrophoresis
      PDGF
      platelet-derived growth factor
      PCR
      polymerase chain reaction
      BCR
      breakpoint cluster region
      Phosphatidylinositol 3-kinase (PI 3-kinase)1 consists of an 85-kDa regulatory subunit (p85) and a 110-kDa catalytic subunit (p110), the latter of which is responsible for the phosphorylation of phosphatidylinositol lipids at the D3 position and serine phosphorylation of proteins (
      • Carpenter C.L.
      • Duckworth B.C.
      • Augers K.R.
      • Cohen B.
      • Schaffhausen B.S.
      • Cantley L.C.
      ,
      • Skolnik E.Y.
      • Margolis B.
      • Mohammadi M.
      • Lowenstein E.
      • Fischer R.
      • Drepps A.
      • Ullrich A.
      • Schlessinger J.
      ,
      • 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.
      ). The p85 subunit contains a Src homology 3 (SH3) domain capable of binding to proline-rich sequences, a region homologous to the breakpoint cluster region (BCR) gene product, a p110 binding domain (110), and two SH2 domains. PI 3-kinase activity increases in response to platelet-derived growth factor (PDGF) binding to its receptor, in large part because the p85·p110 complex is relocalized from the cytosol to the lipids at the plasma membrane, by p85 SH2 domains binding directly to tyrosine phosphorylated sites on the receptor (
      • Klippel A.
      • Escobedo J.A.
      • Fantl W.J.
      • Williams L.T.
      ,
      • Hu P.
      • Mondino A.
      • Skolnik E.Y.
      • Schlessinger J.
      ).
      The SH2 domains of p85 recognize and bind to proteins such as the PDGF receptor at sites that contain a pY-X-X-M sequence (pY = phosphotyrosine, X = any amino acid, M = methionine) in a phosphorylation-dependent manner. The residues within the p85 SH2 domain responsible for binding this sequence include a critical arginine residue that coordinates twice with the phosphate group of the phosphotyrosine residue and a hydrophobic pocket involved in methionine binding (
      • Mayer B.J.
      • Jackson P.K.
      • Etten R.A.V.
      • Baltimore D.
      ,
      • Eck M.J.
      • Shoelson S.E.
      • Harrison S.C.
      ,
      • Breeze A.L.
      • Kara B.V.
      • Barratt D.G.
      • Anderson M.
      • Smith J.C.
      • Luke R.W.
      • Best J.R.
      • Cartlidge S.A.
      ).
      Over the past several years, there have also been reports of SH2 domains binding to proteins via a phosphotyrosine-independent mechanism. These reports include several phosphoserine/phosphothreonine-dependent interactions (
      • Pendergast A.M.
      • Muller A.J.
      • Havilk M.H.
      • Maru Y.
      • Witte O.N.
      ,
      • Muller A.J.
      • Pendergast A.-M.
      • Havlik M.H.
      • Puil L.
      • Pawson T.
      • Witte O.N.
      ,
      • Cleghon V.
      • Morrison D.K.
      ,
      • Malek S.N.
      • Desiderio S.
      ,
      • Nantel A.
      • Mohammad-Ali K.
      • Sherk J.
      • Posner B.I.
      • Thomas D.Y.
      ,
      • Dutartre H.
      • Harris M.
      • Olive D.
      • Collette Y.
      ). In addition, there have been a few reports that concluded that the SH2-mediated interaction was phosphotyrosine-independent but did not determine whether or not the interaction was instead dependent upon phosphoserine or phosphothreonine (
      • Park I.
      • Chung J.
      • Walsh C.T.
      • Yun Y.
      • Strominger J.L.
      • Shin J.
      ,
      • Raffel G.D.
      • Parmar K.
      • Rosenberg N.
      ,
      • Anderson S.M.
      • Burton E.A.
      • Koch B.L.
      ,
      • Schmandt R.
      • Liu S.K.
      • McGlade C.J.
      ). In each instance, the precise amino acid residues involved in mediating these SH2 domain interactions both within the SH2 domain and on the SH2-bound ligand have yet to be identified. There are also two reports of SH2 domains that can bind to unphosphorylated forms of the preferred tyrosine-containing peptides, albeit more weakly than their phosphorylated counterparts (
      • Bibbins K.B.
      • Boeuf H.
      • Varmus H.E.
      ,
      • Li S.-C.
      • Gish G.
      • Yang D.
      • Coffey A.J.
      • Forman-kay J.D.
      • Ernberg I.
      • Kay L.E.
      • Pawson T.
      ). The physiological significance of these low affinity interactions has yet to be demonstrated.
      In this report, we describe a unique SH2 domain interaction involving the SH2 domains of the p85 subunit of PI 3-kinase and the serine/threonine kinase A-Raf. This is the first description of an SH2 domain interaction that requires positively charged basic residues within the SH2 ligand (i.e. the A-Raf protein) to mediate binding to the p85 SH2 domains. Our results indicate that there are four distinct sites on the A-Raf protein, each of which is sufficient for p85 SH2 domain binding. Further, we also find that the p85 SH3 domain can bind A-Raf, suggesting that the p85·A-Raf complex is mediated by multiple domain interactions. We observe the presence of this p85·A-Raf complex in both quiescent and PDGF-stimulated cells, but do not find a complex between p85 and the more extensively characterized c-Raf kinase. These results suggest the possibility that p85 may act as an adapter protein for A-Raf, as it has been shown to do for the p110 catalytic subunit of PI 3-kinase (
      • Hu P.
      • Mondino A.
      • Skolnik E.Y.
      • Schlessinger J.
      ).

      DISCUSSION

      These results demonstrate that a phage display library can be used to provide target peptide sequences devoid of phosphotyrosine residues, that are capable of binding to bait SH2 domains. Similar peptide sequences to those selected from the phage display library by the p85 SH2 domains were present in the serine/threonine kinase A-Raf and were required for p85 SH2 binding. The p85 and A-Raf proteins were found in the same protein complex within cells, indicating that they interact together in a biological setting, as well as in vitro. Therefore, this approach has facilitated the identification of a previously uncharacterized protein:protein interaction between p85 and A-Raf. The function of the p85·A-Raf complex is not known; however, the fact that it is present in both quiescent and PDGF-stimulated cells suggests that it is a constitutive association. A similar constitutive interaction has been described previously between p85 and p110 (
      • Hu P.
      • Mondino A.
      • Skolnik E.Y.
      • Schlessinger J.
      ). This raises the possibility that p85 may act as an adapter protein for A-Raf, relocalizing it to activated growth factor receptors at the membrane, as p85 has been suggested to do for p110 (
      • Hu P.
      • Mondino A.
      • Skolnik E.Y.
      • Schlessinger J.
      ). Since A-Raf has been shown to phosphorylate and activate MEK1 (
      • Wu X.
      • Noh S.J.
      • Zhou G.
      • Dixon J.E.
      • Guan K.L.
      ), this could provide a Ras-independent mechanism to activate the mitogen-activated protein kinase pathway.
      The fact that c-Raf is not found in a similar complex with p85 suggests that different Raf family members may also play unique roles in signal transduction pathways. In support of this hypothesis, it has recently been reported that A-Raf but not c-Raf was detected in highly purified rat liver mitochondria (
      • Yuryev A.
      • Ono M.
      • Goff S.A.
      • Macaluso F.
      • Wennogle L.P.
      ). This report also demonstrates that A-Raf interacts specifically with hTOM and hTIM, human proteins with sequence similarity to mitochondrial outer and inner membrane protein-import receptors from lower organisms. The authors suggest that hTOM and hTIM may be involved in the mitochondrial transport of A-Raf. Interestingly, the p110 catalytic subunit of PI 3-kinase has sequence homology to Vps34p, a yeast protein involved in the sorting of proteins to the vacuole (
      • 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.
      ). This fact, coupled with our identification of the p85 subunit of PI 3-kinase as an A-Raf-binding protein, raises the possibility that p85 may play a role in the subcellular localization of A-Raf.
      Both the SH3 domain and each of the SH2 domains of p85 were found to be capable of binding independently to the A-Raf protein, suggesting that several distinct regions of each protein are involved in mediating binding. In addition, we find that a p85 fusion protein lacking only its SH3 domain still binds the C/R/D/E HA-A-Raf mutant (unable to bind to p85 SH2 domains) (Fig. 5 B). This raises the distinct possibility that region(s) of p85 in addition to the SH3 domain, the N-SH2 domain, and the C-SH2 domain are involved in mediating binding between p85 and A-Raf. The abilities of isolated SH3 and SH2 domains to fold as modular domains that retain there binding activities has been demonstrated using nuclear magnetic resonance, x-ray crystallography, and functional binding assays (
      • Anderson D.
      • Koch C.A.
      • Grey L.
      • Ellis C.
      • Moran M.F.
      • Pawson T.
      ,
      • Moran M.F.
      • Koch C.A.
      • Anderson D.
      • Ellis C.
      • England L.
      • Martin G.S.
      • Pawson T.
      ,
      • Mayer B.J.
      • Jackson P.K.
      • Baltimore D.
      ,
      • Pawson T.
      • Gish G.D.
      ,
      • Ren R.
      • Mayer B.J.
      • Cicchetti P.
      • Baltimore D.
      ,
      • Yu H.
      • Chen J.K.
      • Feng S.
      • Dalgarno D.C.
      • Brauer A.W.
      • Schreiber S.L.
      ,
      • Cohen G.B.
      • Ren R.
      • Baltimore D.
      ). Whether or not the BCR homology and p110 binding regions of p85 are similarly able to fold appropriately when expressed in isolation is less clear. Therefore, although the BCR homology and p110 binding regions of p85 did not bind HA-A-Raf when expressed as isolated fragments of p85 fused to GST, they may contribute to A-Raf binding, when present in the context of the full-length p85 protein. X-ray crystallography of the p85·A-Raf complex will be required to resolve this question.
      We have characterized a novel interaction for the p85 SH2 domains that requires basic residues on A-Raf within the sequence motif basic-X-basic. Our results suggest that A-Raf contains four separate basic-X-basic sequences (designated C, R, D, E, and containing the sequences LIKGRK, LQRIRS, EQRERK, and DKKKVKNL respectively), each of which is capable of binding to both the N-SH2 and C-SH2 of p85. This is the first report of a p85 SH2 ligand that lacks phosphotyrosine residues.
      There have been several reports of phosphotyrosine-independent interactions for other SH2 domains. These reports include several phosphoserine/phosphothreonine-dependent interactions, such as those between: the BCR protein, and the SH2 domains of Abl (
      • Pendergast A.M.
      • Muller A.J.
      • Havilk M.H.
      • Maru Y.
      • Witte O.N.
      ) and other proteins (
      • Muller A.J.
      • Pendergast A.-M.
      • Havlik M.H.
      • Puil L.
      • Pawson T.
      • Witte O.N.
      ), the c-Raf kinase and the Fyn SH2 domain (
      • Cleghon V.
      • Morrison D.K.
      ), the cyclin-dependent kinase homologue p130PITSLRE and the Blk SH2 domain (
      • Malek S.N.
      • Desiderio S.
      ), both the c-Raf and MEK1 kinases with the Grb10 SH2 domain (
      • Nantel A.
      • Mohammad-Ali K.
      • Sherk J.
      • Posner B.I.
      • Thomas D.Y.
      ), as well as the human immunodeficiency virus type 1 Nef protein and the Lck SH2 domain (
      • Dutartre H.
      • Harris M.
      • Olive D.
      • Collette Y.
      ). In addition, there have been a few reports that concluded that the SH2-mediated interaction was phosphotyrosine-independent but did not determine whether or not the interaction was instead dependent upon phosphoserine or phosphothreonine. These interactions include: the ubiquitin-binding protein p62 and the Lck SH2 domain (
      • Park I.
      • Chung J.
      • Walsh C.T.
      • Yun Y.
      • Strominger J.L.
      • Shin J.
      ), the SHC adapter protein and the Abl SH2 domain (
      • Raffel G.D.
      • Parmar K.
      • Rosenberg N.
      ), and the Cbl adapter protein and the Fyn SH2 domain (
      • Anderson S.M.
      • Burton E.A.
      • Koch B.L.
      ). One report of a phosphotyrosine-independent SH2-mediated interaction involved a protein expressed inactivated lymphocytes, PAL, binding to the SH2 domain of SHC (
      • Schmandt R.
      • Liu S.K.
      • McGlade C.J.
      ). Since the authors were unable to detect any phosphorylation of the PAL protein, they concluded that the interaction must be phosphotyrosine-independent, if not phosphorylation-independent. Curiously, mutation of the conserved arginine required for phosphotyrosine-dependent binding, within the SHC SH2 domain, prevented PAL interaction (
      • Schmandt R.
      • Liu S.K.
      • McGlade C.J.
      ), suggesting that more experiments will be required to establish the basis for this interaction.
      In each of these reports, the precise sequence of the ligand binding to the SH2 domain in a phosphotyrosine-independent manner was not determined. Identification of such sequences may be facilitated using the approach we describe here, i.e. by screening a phage display library with each of the SH2 domains involved and then searching the ligand for similar sequences. It is important to note that both of the SH2 domains of GTPase-activating protein and phospholipase Cγ1 also selected distinct sequences from the phage displayed hexapeptide library. Many of these sequences were specific for a particular SH2 domain, but each of these four SH2 domains selected a common sequence, GDYTLF. We therefore suggest that phage display libraries may be used to characterize the binding specificities of other SH2 domains, in addition to those of the p85 protein. This approach should facilitate the identification of novel SH2-binding proteins that may serve important functions in cell signaling pathways.
      A-Raf is a very different p85 SH2 ligand compared with the typical pY-X-X-M-containing protein/peptide. SH2 domains in general are best known for their ability to bind proteins or peptides in a phosphotyrosine-dependent manner. The molecular details of many of these interactions have been elucidated using nuclear magnetic resonance and x-ray crystallography (
      • Eck M.J.
      • Shoelson S.E.
      • Harrison S.C.
      ,
      • Waksman G.
      • Shoelson S.E.
      • Pant N.
      • Cowburn D.
      • Kuriyan J.
      ,
      • Pascal S.M.
      • Singer A.U.
      • Gish G.
      • Yamazaki T.
      • Shoelson S.E.
      • Pawson T.
      • Kay L.E.
      • Forman-Kay J.D.
      ,
      • Waksman G.
      • Kaminos D.
      • Robertson S.C.
      • Pant N.
      • Baltimore D.
      • Birge R.B.
      • Cowburn D.
      • Hanafusa H.
      • Mayer B.J.
      • Overduin M.
      • Resh M.D.
      • Rios C.B.
      • Silverman L.
      • Kuriyan J.
      ,
      • Zhou M.-M.
      • Meadows R.P.
      • Logan T.M.
      • Yoon H.S.
      • Wade W.S.
      • Ravichandran K.S.
      • Burakoff S.J.
      • Fesik S.W.
      ,
      • Siegal G.
      • Davis B.
      • Kristensen S.M.
      • Sankar A.
      • Linacre J.
      • Stein R.C.
      • Panayotou G.
      • Waterfield M.D.
      • Driscoll P.C.
      ). SH2 domains have numerous highly conserved residues important for maintaining a common modular structure and for interaction with the phosphotyrosine portion of the ligand. The specificity of SH2 domain interactions is provided by the unique residues within the SH2 domain that contact residues on the ligand, usually C-terminal to the phosphotyrosine residue (
      • 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.
      ,
      • 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.
      ). For example, the unique regions within the p85 SH2 domains are responsible for its binding specificity for pY-X-X-M ligands.
      A-Raf, on the other hand, requires one of several basic-X-basic motifs for binding to p85 SH2 domains. Given the fact that this newly identified p85 SH2 ligand is positively charged, while the previously characterized phosphotyrosine-containing p85 SH2 ligand is negatively charged and hydrophobic, it is unlikely that A-Raf binds to the same site on the p85 SH2 domains. Our results, therefore, raise the interesting possibility that p85 SH2 domains may have a second binding site: a phosphorylation-independent binding site distinct from the phosphotyrosine-dependent binding pocket.

      Acknowledgments

      We thank C. McGlade, U. Rapp and M. Waterfield for generously supplying DNAs. Expert technical assistance was provided by T. Taylor.

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