Purification and Identification of Protein-Tyrosine Kinase-binding Proteins Using Synthetic Phosphopeptides as Affinity Reagents

Protein-tyrosine kinases are known regulators of cell division that have been implicated in the onset of a variety of malignancies. They act through cellular signaling proteins that bind to specific autophosphorylation sites. To find out whether these autophosphorylation sites can be used to identify downstream signaling proteins, synthetic peptides based on an autophosphorylation site in the colony-stimulating factor-1 (CSF-1) receptor were linked to agarose beads and incubated with lysates from macrophages. Bound proteins were analyzed by MS, leading to the identification of both known and novel CSF-1 receptor-interacting proteins. The approach presented here can be applied to phosphorylation sites in a wide variety of proteins. It will lead to the identification of novel protein-protein interactions and provide new insights into the mechanics of signal transduction. Novel protein-protein interactions may provide useful targets for the development of drugs that interfere with the activation of signaling cascades used by protein-tyrosine kinases to turn on cell division. Protein-tyrosine kinases are known regulators of cell division that have been implicated in the onset of a variety of malignancies. They act through cellular signaling proteins that bind to specific autophosphorylation sites. To find out whether these autophosphorylation sites can be used to identify downstream signaling proteins, synthetic peptides based on an autophosphorylation site in the colony-stimulating factor-1 (CSF-1) receptor were linked to agarose beads and incubated with lysates from macrophages. Bound proteins were analyzed by MS, leading to the identification of both known and novel CSF-1 receptor-interacting proteins. The approach presented here can be applied to phosphorylation sites in a wide variety of proteins. It will lead to the identification of novel protein-protein interactions and provide new insights into the mechanics of signal transduction. Novel protein-protein interactions may provide useful targets for the development of drugs that interfere with the activation of signaling cascades used by protein-tyrosine kinases to turn on cell division. The human genome encodes at least 90 different protein-tyrosine kinases (1Manning G. Whyte D.B. Martinez R. Hunter T. Sudarsanam S. The protein kinase complement of the human genome..Science. 2002; 298: 1912-1934Google Scholar , 2Robinson D.R. Wu Y.M. Lin S.F. The protein tyrosine kinase family of the human genome..Oncogene. 2000; 19: 5548-5557Google Scholar). These are involved in regulation of cell division, differentiation, adhesion, migration, metabolism, and gene transcription (1Manning G. Whyte D.B. Martinez R. Hunter T. Sudarsanam S. The protein kinase complement of the human genome..Science. 2002; 298: 1912-1934Google Scholar). Expression of constitutively active protein-tyrosine kinases results in uncontrolled cell division, and mutant protein-tyrosine kinases have been implicated as dominant oncogenes in a large variety of human cancers (3Blume-Jensen P. Hunter T. Oncogenic kinase signalling..Nature. 2001; 411: 355-365Google Scholar , 4Kolibaba K.S. Druker B.J. Protein tyrosine kinases and cancer..Biochim. Biophys. Acta. 1997; 1333: F217-F248Google Scholar). Consequently, protein-tyrosine kinases have been selected as targets for the development of anti-cancer drugs (5Fabbro D. Parkinson D. Matter A. Protein tyrosine kinase inhibitors: new treatment modalities?.Curr. Opin. Pharmacol. 2002; 2: 374-381Google Scholar, 6Al-Obeidi F.A. Lam K.S. Development of inhibitors for protein tyrosine kinases..Oncogene. 2000; 19: 5690-5701Google Scholar, 7Cohen P. Protein kinases—The major drug targets of the twenty-first century?.Nat. Rev. Drug Discov. 2002; 1: 309-315Google Scholar). Protein-tyrosine kinases are activated directly or indirectly in response to extracellular signals and translate that information into the activation of intracellular signal transduction pathways (8Pawson T. Protein modules and signalling networks..Nature. 1995; 373: 573-580Google Scholar , 9Pawson T. Gish G.D. Nash P. SH2 domains, interaction modules and cellular wiring..Trends Cell Biol. 2001; 11: 504-511Google Scholar). Most protein-tyrosine kinases do so by using autophosphorylation sites that act as binding sites for downstream signaling proteins (8Pawson T. Protein modules and signalling networks..Nature. 1995; 373: 573-580Google Scholar , 10van der Geer P. Hunter T. Lindberg R.A. Receptor protein-tyrosine kinases and their signal transduction pathways..Annu. Rev. Cell Biol. 1994; 10: 251-337Google Scholar). Signaling proteins, in turn, contain protein-interaction modules, such as Src homology 2 (SH2) 1The abbreviations used are: SH2, Src homology 2; CSF-1, colony-stimulating factor 1; EGF, epidermal growth factor; PTB, phosphotyrosine binding; P.Tyr, phosphotyrosine; SH3, Src homology 3; TKB, tyrosine kinase binding; PLC, phospholipase C. 1The abbreviations used are: SH2, Src homology 2; CSF-1, colony-stimulating factor 1; EGF, epidermal growth factor; PTB, phosphotyrosine binding; P.Tyr, phosphotyrosine; SH3, Src homology 3; TKB, tyrosine kinase binding; PLC, phospholipase C. and phosphotyrosine-binding (PTB) domains, that bind to phosphorylated tyrosine residues (8Pawson T. Protein modules and signalling networks..Nature. 1995; 373: 573-580Google Scholar, 9Pawson T. Gish G.D. Nash P. SH2 domains, interaction modules and cellular wiring..Trends Cell Biol. 2001; 11: 504-511Google Scholar, 10van der Geer P. Hunter T. Lindberg R.A. Receptor protein-tyrosine kinases and their signal transduction pathways..Annu. Rev. Cell Biol. 1994; 10: 251-337Google Scholar). The specificity of these interactions depends on the protein-interaction domain as well as on the amino acid sequence surrounding the phosphorylation site. Signaling proteins that are activated downstream of protein-tyrosine kinases may provide useful targets for the development of anti-cancer drugs. In theory, these targets can be identified using the binding sites that are present on their upstream activators. Most signaling proteins identified thus far use SH2 or PTB domains to bind to upstream protein-tyrosine kinases. The specificity of many of these domains has been characterized in detail and generally involves only residues present immediately upstream or downstream of the phosphorylation site (11Margolis B. Borg J.P. Straight S. Meyer D. The function of PTB domain proteins..Kidney Int. 1999; 56: 1230-1237Google Scholar, 12Songyang Z. Margolis B. Chaudhuri M. Shoelson S.E. Cantley L.C. The phosphotyrosine interaction domain of SHC recognizes tyrosine-phosphorylated NPXY motif..J. Biol. Chem. 1995; 270: 14863-14866Google Scholar, 13Songyang 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. Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav..Mol. Cell. Biol. 1994; 14: 2777-2785Google Scholar, 14Songyang 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. SH2 domains recognize specific phosphopeptide sequences..Cell. 1993; 72: 767-778Google Scholar). To identify potential binding proteins for newly identified tyrosine phosphorylation sites, a common bioinformatics approach has been established. This approach is based on comparison of the amino acid sequence surrounding a tyrosine phosphorylation site with the specificity of known SH2 and PTB domains. In vitro binding and co-immunoprecipitation experiments are subsequently carried out to test the prediction. The disadvantage of this approach is that it considers only known signaling proteins containing well-characterized protein-protein interaction domains. Here we have demonstrated the use of peptides based on tyrosine phosphorylation sites as affinity regents to identify protein-tyrosine kinase-interacting proteins and substrates. This approach has yielded both known signaling proteins and proteins with no previously known signaling function. Murine P388D1 macrophages were grown in Iscove’s-modified Dulbecco’s medium supplemented with 10% fetal bovine serum at 37°C in 10% CO2 until confluent and then starved for 12 h in serum-free medium. Control or colony-stimulating factor-1 (CSF-1)-stimulated cells (200 ng/ml) were lysed in 1 ml of phospholipase C (PLC) lysis buffer per 10-cm dish (15Wilhelmsen K. Burkhalter S. van der Geer P. C-Cbl binds the CSF-1 receptor at tyrosine 973, a novel phosphorylation site in the receptor’s carboxy-terminus..Oncogene. 2002; 21: 1079-1089Google Scholar). Polyclonal antisera against Cbl, the CSF-1 receptor, NonO, and PSF were generated as previously described (15Wilhelmsen K. Burkhalter S. van der Geer P. C-Cbl binds the CSF-1 receptor at tyrosine 973, a novel phosphorylation site in the receptor’s carboxy-terminus..Oncogene. 2002; 21: 1079-1089Google Scholar , 16Mathur M. Tucker P.W. Samuels H.H. PSF is a novel corepressor that mediates its effect through Sin3A and the DNA binding domain of nuclear hormone receptors..Mol. Cell. Biol. 2001; 21: 2298-2311Google Scholar), a polyclonal anti-Shp2 serum was provided by G.-S. Feng (Burnham Institute, La Jolla, CA), a polyclonal anti-Grb2 serum was provided by T. Hunter (Salk Institute, La Jolla, CA), polyclonal sera against Sos and SHIP were purchased from Upstate Biotechnology Inc. (Waltham, MA), a polyclonal serum against Shp1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and a monoclonal antibody against clathrin heavy chain was purchased from BD Biosciences Pharmingen (San Diego, CA). Immunoprecipitation and immunoblotting were carried out exactly as described previously (15Wilhelmsen K. Burkhalter S. van der Geer P. C-Cbl binds the CSF-1 receptor at tyrosine 973, a novel phosphorylation site in the receptor’s carboxy-terminus..Oncogene. 2002; 21: 1079-1089Google Scholar). GST-Cbl tyrosine kinase binding (TKB) was generated as previously described (15Wilhelmsen K. Burkhalter S. van der Geer P. C-Cbl binds the CSF-1 receptor at tyrosine 973, a novel phosphorylation site in the receptor’s carboxy-terminus..Oncogene. 2002; 21: 1079-1089Google Scholar), GST-Grb2 SH2 was provided by T. Pawson (Samuel Lunenfeld Research Institute, Toronto, Canada), and GST-Shp2 SH2(N+C) was provided by G.-S. Feng (Burnham Institute). GST fusion proteins were produced and purified exactly as previously described (15Wilhelmsen K. Burkhalter S. van der Geer P. C-Cbl binds the CSF-1 receptor at tyrosine 973, a novel phosphorylation site in the receptor’s carboxy-terminus..Oncogene. 2002; 21: 1079-1089Google Scholar). Phosphorylated and peptides based on surrounding and of the CSF-1 receptor were by CA). The and peptides were with a by an acid to the amino The was used for to the acid was to provide the and the The peptides were with acid to the amino and to lysates were incubated with peptides at on a The beads were with lysis buffer and proteins were analyzed by or Protein were from a with and protein were and using and acid The were then and at 37°C with were in acid and on and into of the was into a K. S. D. of from for protein in 2000; 11: Scholar, J. J. P. of proteins for sequence or Scholar, C. J. of an for the of protein for amino acid 1995; Scholar, A. M. M. of proteins Chem. Scholar). of the from to were on an with a and were selected for and were and sequence were used to both the and for proteins that the and sequence and were carried out using and of on the The CSF-1 receptor is a protein-tyrosine kinase that is on the of macrophages and van C. D. A. of of receptor at Scholar, R. The is to the receptor for the growth Scholar, R. Expression of the human factor-1 on cells and cell Scholar, J. of the human gene and its in cells of the Cell. Biol. Scholar). of CSF-1 to the extracellular domain results in activation of the kinase domain and autophosphorylation on a of tyrosine (15Wilhelmsen K. Burkhalter S. van der Geer P. C-Cbl binds the CSF-1 receptor at tyrosine 973, a novel phosphorylation site in the receptor’s carboxy-terminus..Oncogene. 2002; 21: 1079-1089Google Scholar , in receptor 2000; Scholar). In to of the to the kinase residues immediately upstream and downstream of have been This that the an function that is to protein-protein To test this phosphorylated and peptides based on the sequence were used as affinity to CSF-1 receptor-interacting proteins. were on agarose beads and incubated with lysates of and macrophages. Bound proteins were analyzed by The results that are proteins that bind to the bind to the phosphorylated can be used to specific binding lysates of P388D1 macrophages were incubated with phosphorylated and and Bound proteins were by and by lysates of and macrophages were incubated with and phosphorylated on agarose Bound proteins were analyzed in with lysates by To find out whether these interactions are specific for the sequence the of proteins that to the peptides was the of proteins to peptides based on the and phosphorylation sites. the and phosphotyrosine peptides to a of proteins, binding with a of in binding the phosphorylated and were based on a of proteins were proteins with a to that only to the phosphorylated and to the peptides proteins to the These results that peptides can be used to protein-tyrosine proteins. The of proteins that bind to a is by its and both and interactions can be To their phosphorylation proteins were analyzed by lysates were analyzed in The results that are tyrosine-phosphorylated proteins that bind to the phosphorylated of these proteins were phosphorylated in as well as were tyrosine-phosphorylated only in cells This that proteins that are tyrosine-phosphorylated in response to CSF-1 can be using the phosphorylated from P388D1 macrophages were incubated with agarose beads containing the phosphorylated or Bound proteins were by and by protein were from the with and analyzed by and sequence were used to were identified based on and sequence proteins and peptides that were identified are identified proteins that bind to the clathrin heavy a protein involved in a transcription and factor; a and NonO, a transcription factor C. and function of Rev. Cell Biol. 2001; Scholar, J. P. B. and of a novel 1993; Scholar, and of a gene the major protein in J. Scholar, Tucker P.W. 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Cbl, and have been implicated previously in CSF-1 receptor signaling To that bind and in a to lysates of P388D1 macrophages were incubated with or agarose beads and proteins were analyzed by The results that Cbl, and bind to the phosphorylated to the a protein-tyrosine that is to has been implicated in CSF-1 receptor signal transduction To test whether can bind to the phosphorylation P388D1 lysates were incubated with peptides and proteins were analyzed by anti-Shp2 immunoblotting The results that binds to the phosphorylated Cbl, and bind to the phosphorylated proteins known to be involved in CSF-1 receptor signaling that bind to a phosphopeptide based on the autophosphorylation with the CSF-1 in and in vitro to and is tyrosine-phosphorylated in response to K. Burkhalter S. van der Geer P. 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D. colony-stimulating factor binding of to in human Biol. Chem. 1995; 270: Scholar, H. B. H. S. B. CSF-1 the of a CSF-1 receptor, and 1997; 14: in response to CSF-1, can with the receptor, a binding site has been S. K. R. a signal transduction with 10: Scholar , K. D. and a tyrosine-phosphorylated protein with in Cell. Biol. 1994; 14: Scholar , S. J. G. are to of 2002; Scholar , K. of a novel tyrosine phosphorylated protein that binds to and in Biol. Chem. 1997; in response to CSF-1, a binding site on the receptor has been Protein tyrosine is phosphorylated in tyrosine in macrophages in response to Biol. Chem. Scholar , S. T. Neel B.G. of colony-stimulating factor 1 receptor signaling by the SH2 tyrosine Cell. Biol. in response to CSF-1, with the receptor in a binding site has been K. of a novel tyrosine phosphorylated protein that binds to and in Biol. Chem. 1997; Scholar , McGlade The of in cell 2001; Scholar , B.J. M. P. a new family Cell. Biol. 2002; with the CSF-1 receptor through CSF-1 of the CSF-1 receptor and of their tyrosine phosphorylation and with signaling Cell. 1999; 72: Scholar , K. D. and a tyrosine-phosphorylated protein with in Cell. Biol. 1994; 14: Scholar , A. S. D. colony-stimulating factor binding of to in human Biol. Chem. 1995; 270: Scholar in a new To find out whether binding was purified GST fusion proteins containing the the SH2 domains, or the SH2 domain were for their to bind to the and peptides fusion proteins to the phosphorylated binding to the or the peptides was These that Cbl, and bind directly and to in a PSF has been implicated previously in CSF-1 receptor To that binds to lysates of and P388D1 macrophages were incubated with peptides and proteins were analyzed by The results are with the and that binds to both the phosphorylated and bind to phosphorylated and peptides based on the phosphorylation site To these interactions and were analyzed by receptor The results that PSF and with CSF-1 from P388D1 macrophages. To that clathrin with lysates of P388D1 macrophages were incubated with based on the sequence were as Bound proteins were analyzed by immunoblotting The results that the clathrin heavy chain with the and downstream signaling proteins are to activated protein-tyrosine kinases through binding to specific autophosphorylation sites (8Pawson T. Protein modules and signalling networks..Nature. 1995; 373: 573-580Google Scholar, 9Pawson T. Gish G.D. Nash P. SH2 domains, interaction modules and cellular wiring..Trends Cell Biol. 2001; 11: 504-511Google Scholar, 10van der Geer P. Hunter T. Lindberg R.A. Receptor protein-tyrosine kinases and their signal transduction pathways..Annu. Rev. Cell Biol. 1994; 10: 251-337Google Scholar , by receptor tyrosine Rev. 1993; Scholar). To identify binding for tyrosine phosphorylation sites, have been used in the Margolis and the use of the of the epidermal growth factor receptor to R.J. Margolis B. R. A. J. The SH2 and protein receptor tyrosine kinases to Scholar , B. F. C. A. J. of epidermal growth proteins with Src homology 2 Scholar). In this the receptor domain was by autophosphorylation in the of the is from the of the protein and used to a This approach has been used to identify a large of growth factor proteins R.J. Margolis B. R. A. J. The SH2 and protein receptor tyrosine kinases to Scholar , B. F. C. A. J. of epidermal growth proteins with Src homology 2 Scholar , Margolis B. M. R. A. A. J. of a novel for of proteins for receptor tyrosine Scholar). it is and has been used to identify binding for specific phosphorylation sites. In it is whether this approach can be used to identify binding for protein-tyrosine kinases. approach is based on the In this the kinase of is to the domain of a transcription factor A. interaction of SHC and receptor 1 with the of the receptor a novel Cell. Biol. 1995; Scholar). This fusion protein is in with an in the are to a activation domain A. interaction of SHC and receptor 1 with the of the receptor a novel Cell. Biol. 1995; Scholar). Expression of proteins that bind to the kinase of results in of a transcription factor that the of a have been used to identify of protein-tyrosine kinase signaling S. K. R. a signal transduction with 10: Scholar , A. interaction of SHC and receptor 1 with the of the receptor a novel Cell. Biol. 1995; Scholar). have with the of specific protein-tyrosine kinases and with identification of The may be the to identify binding for specific phosphorylation sites. a complement to these we have used as affinity to binding for specific tyrosine phosphorylation sites. this is based on synthetic peptides that are phosphorylated at a specific this can be applied to phosphorylation site of can be at on agarose beads using a this can be used to of proteins for identification by have used peptides based on the sequence surrounding in the of the CSF-1 This to the identification of proteins that bind to the and proteins that bind to the phosphorylated In a peptides based on the sequence surrounding in the receptor were leading to the identification of M. novel for Biol. Chem. Scholar). These experiments that protein with can be used as a to identify binding for receptor protein-tyrosine kinases. of the proteins that bind to the phosphorylated Cbl, and are to act as regulators of cell division. The is a of an a a and a containing tyrosine phosphorylation sites P. signaling in the regulation of cell Scholar). is to of activated CSF-1 It was that binds directly to the of the activated CSF-1 of that the approach used here proteins. that in to Cbl, the SHIP can bind to the phosphorylated CSF-1 receptor SHIP is a protein that is of an SH2 a and a containing tyrosine phosphorylation sites and of SHIP 2000; 14: Scholar , K. D. and a tyrosine-phosphorylated protein with in Cell. Biol. 1994; 14: Scholar). SHIP is tyrosine-phosphorylated in response to CSF-1, and is that that SHIP is a of CSF-1 receptor signaling S. K. R. a signal transduction with 10: Scholar , K. D. and a tyrosine-phosphorylated protein with in Cell. Biol. 1994; 14: Scholar , S. J. G. are to of 2002; Scholar). results that SHIP with the CSF-1 receptor directly using its SH2 domain or indirectly through we that both the SH2 protein Shp1 and can bind to the phosphorylation site. Shp1 is known to be tyrosine-phosphorylated of macrophages with CSF-1 T. of cell with with Cell. Biol. 1993; Scholar , Protein tyrosine is phosphorylated in tyrosine in macrophages in response to Biol. Chem. Scholar). have of and macrophages an in the of with CSF-1 D.R. cell function in 11: Scholar, Yi T. R.J. D.R. at the are the cell protein-tyrosine 1993; Scholar, S. T. Neel B.G. of colony-stimulating factor 1 receptor signaling by the SH2 tyrosine Cell. Biol. Scholar). These that Shp1 is a of CSF-1 receptor is a protein-tyrosine that is to Shp1 J. Neel B.G. of a human homology Scholar). is known to act in with to cell division and in response to CSF-1 K. of a novel tyrosine phosphorylated protein that binds to and in Biol. 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Cell Biol. 2001; Scholar , for of proteins to and Rev. 72: Scholar). is present a sequence that to the to has been in many proteins, this is present at the of the CSF-1 receptor for of proteins to and Rev. 72: Scholar). These that clathrin with the CSF-1 receptor indirectly through an clathrin binds to the This that this interaction is involved in the of activated in of the This is with large of CSF-1 with in macrophages K. van der Geer P. of the colony-stimulating factor 1 receptor domain into the involves Cell. Biol. Scholar). and PSF are proteins that in a and that have been implicated in and the activation of gene transcription J. H. and of a of and from J. 1993; Scholar , D. PSF and nuclear 2002; Scholar). of and PSF is of is that receptor protein-tyrosine kinases can be to by of their domain into the and into the K. van der Geer P. of the colony-stimulating factor 1 receptor domain into the involves Cell. Biol. Scholar , G. and nuclear of receptor tyrosine 2001; Scholar , G. and of receptor tyrosine Opin. Cell Biol. Scholar). we have identified a variety of proteins that to with the of the CSF-1 The that the of the CSF-1 receptor as a site for signaling proteins with is with the that it is have that peptides based on tyrosine phosphorylation sites are affinity that can be used for the and identification of signaling of these proteins will lead to new insights in signal transduction. In may provide targets for the development of drugs that interfere with protein-tyrosine kinase we that this approach may be used to the of and that it can be used for the identification of binding for and phosphorylation sites.

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