Growth Factor-dependent Phosphorylation of the Actin-binding Protein Cortactin Is Mediated by the Cytoplasmic Tyrosine Kinase FER

Previous characterization of the nonreceptor tyrosine kinase FER identified a tight physical association with the catenin pp120 and led to the suggestion that FER may be involved in cell-cell signaling. To further understand the function of FER, we have continued our analyses of the interaction of FER with pp120 and other proteins. The majority of FER is localized to the cytoplasmic fraction where it forms a complex with the actin-binding protein cortactin. The Src homology 2 sequence of FER is required for directly binding cortactin, and phosphorylation of the FER-cortactin complex is up-regulated in cells treated with peptide growth factors. Using a dominant-negative mutant of FER, we provided evidence that FER kinase activity is required for the growth factor-dependent phosphorylation of cortactin. These data suggest that cortactin is likely to be a direct substrate of FER. Our observations provide additional support for a role of FER in mediating signaling from the cell surface, via growth factor receptors, to the cytoskeleton. The nature of the FER-cortactin interaction, and their putative enzyme-substrate relationship, support the previous proposal that one of the functions of the Src homology 2 sequences of nonreceptor tyrosine kinases is to provide a binding site for their preferred substrates. Previous characterization of the nonreceptor tyrosine kinase FER identified a tight physical association with the catenin pp120 and led to the suggestion that FER may be involved in cell-cell signaling. To further understand the function of FER, we have continued our analyses of the interaction of FER with pp120 and other proteins. The majority of FER is localized to the cytoplasmic fraction where it forms a complex with the actin-binding protein cortactin. The Src homology 2 sequence of FER is required for directly binding cortactin, and phosphorylation of the FER-cortactin complex is up-regulated in cells treated with peptide growth factors. Using a dominant-negative mutant of FER, we provided evidence that FER kinase activity is required for the growth factor-dependent phosphorylation of cortactin. These data suggest that cortactin is likely to be a direct substrate of FER. Our observations provide additional support for a role of FER in mediating signaling from the cell surface, via growth factor receptors, to the cytoskeleton. The nature of the FER-cortactin interaction, and their putative enzyme-substrate relationship, support the previous proposal that one of the functions of the Src homology 2 sequences of nonreceptor tyrosine kinases is to provide a binding site for their preferred substrates. Src homology 2 platelet-derived growth factor macrophage-colony stimulating factor 100,000 × gsupernatant 100,000 × g pellet hemagglutinin glutathione S-transferase coiled coiled domain cytomegalovirus phosphate-buffered saline. The cytoplasmic tyrosine kinase FER belongs to a relatively small family of nonreceptor tyrosine kinases that consist of only one other member, FES (1Letwin K. Yee S.-P. Pawson T. Oncogene. 1988; 3: 621-627PubMed Google Scholar, 2Hao Q.L. Heisterkamp N. Groffen J. Mol. Cell. Biol. 1989; 9: 1587-1593Crossref PubMed Scopus (101) Google Scholar). The FES gene product in expressed only in cells of myeloid and endothelial lineages and has been implicated in cytokine signaling (3MacDonald I. Levy J. Pawson T. Mol. Cell. Biol. 1985; 5: 2543-2551Crossref PubMed Scopus (96) Google Scholar, 4Feldman R.A. Gabrilove J.L. Tam J.P. Moore M.A.S. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 2383-2397Google Scholar, 5Greer P. Maltby V. Rossant J. Bernstein A. Pawson T. Mol. Cell. Biol. 1990; 10: 2521-2527Crossref PubMed Google Scholar, 6Greer P. Haigh J. Mbamalu G. Khoo W. Bernstein A. Pawson T. Mol. Cell. Biol. 1994; 14: 6755-6763Crossref PubMed Scopus (93) Google Scholar, 7Hanazono Y. Chiba S. Sasaki K. Mano H. Miyajima A. Arai K. Yazaki Y. Hirai H. EMBO J. 1993; 12: 1641-1646Crossref PubMed Scopus (144) Google Scholar, 8Areces L.B. Dello Sbarba P. Jucker M. Stanley E.R. Feldman R.A. Mol. Cell. Biol. 1994; 14: 4606-4615Crossref PubMed Scopus (31) Google Scholar, 9Izuhara K. Feldman R.A. Greer P. Harada N. J. Biol. Chem. 1994; 269: 18623-18629Abstract Full Text PDF PubMed Google Scholar). In contrast, FER is expressed ubiquitously and its function remains poorly understood. FER is structurally distinct from members of the other cytoplasmic tyrosine kinase families; its amino acid sequence contains a catalytic domain adjacent to a single Src homology 2 (SH2)1 domain and an extensive coiled coil sequence in the amino terminus (2Hao Q.L. Heisterkamp N. Groffen J. Mol. Cell. Biol. 1989; 9: 1587-1593Crossref PubMed Scopus (101) Google Scholar, 10Kim L. Wong T.W. Mol. Cell. Biol. 1995; 15: 4553-4561Crossref PubMed Scopus (154) Google Scholar). We previously showed that FER is tightly associated with the novel catenin pp120 and that the FER·pp120 complex is phosphorylated on tyrosines upon treatment of cells with growth factors (10Kim L. Wong T.W. Mol. Cell. Biol. 1995; 15: 4553-4561Crossref PubMed Scopus (154) Google Scholar). However, the significance of the interaction remains to be elucidated, because the function of pp120 is also not well understood. Tyrosine phosphorylation has been demonstrated to be an important mechanism for regulating cell-cell and cell-substrate adhesions. For example, inhibition of tyrosine phosphatases in Madin-Darby canine kidney cells results in the deterioration of adherens junctions and dramatic changes in cell morphology (11Volberg T. Zick Y. Dror R. Sabanay I. Gilon C. Levitzki A. Geiger B. EMBO J. 1992; 11: 1733-1742Crossref PubMed Scopus (269) Google Scholar). Treatment of epithelial cells with epidermal growth factor leads to the tyrosine phosphorylation of β-catenin, plakoglobin and pp120, as well as changes in cell morphology (12Hoschuetzky H. Aberle H. Kemler R. J. Cell Biol. 1994; 127: 1375-1380Crossref PubMed Scopus (673) Google Scholar, 13Welsh J.B. Gill G.N. Rosenfeld M.G. Wells A. J. Cell Biol. 1991; 114: 533-543Crossref PubMed Scopus (75) Google Scholar). More importantly, the major targets of v-src kinase activity have been shown to be proteins that are directly implicated in cell adhesion or cytoskeletal function. These include focal adhesion kinase, cortactin, p130cas, and pp120 (14Scheller M.D. Bouton A.H. Flynn D.C. Parsons J.T. Prog. Nucleic Acid Res. 1993; 44: 205-227Crossref PubMed Scopus (27) Google Scholar). Results of subcellular localization and functional characterization have implicated these proteins in regulating cell motility or membrane cytoskeleton dynamics. Focal adhesion kinase and p130cas are localized to focal adhesion complexes and focal adhesion kinase has been demonstrated to be required for the turnover of the cellular structures (15Ilic D. Furuta Y. Knazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Aizawa S. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1591) Google Scholar, 16Guan J.L. Matrix Biol. 1997; 16: 195-200Crossref PubMed Scopus (133) Google Scholar, 17Schaller M.D. Borgman C.A. Cobb B.S. Vines R.R. Reynolds A.B. Parsons J.T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5192-5196Crossref PubMed Scopus (1295) Google Scholar, 18Hanks S.K. Calalb M.B. Harper M.C. Patel S.K. Proc. Natl. Acad. Sci U. S. A. 1992; 89: 8487-8491Crossref PubMed Scopus (731) Google Scholar, 19Nakamoto T. Sakai R. Honda H. Ogawa S. Ueno H. Suzuki T. Aizawa S. Yazaki Y. Hirai H. Mol. Cell. Biol. 1997; 17: 3884-3897Crossref PubMed Scopus (136) Google Scholar). Cortactin has been shown to bind F-actin in vitro and is localized to the cortical cytoskeleton (20Wu H. Reynolds A.B. Kanner S.B. Vines R.R. Parsons J.T. Mol. Cell. Biol. 1991; 11: 5113-5124Crossref PubMed Scopus (375) Google Scholar, 21Wu H. Parsons J.T. J. Cell Biol. 1993; 120: 1417-1426Crossref PubMed Scopus (452) Google Scholar). pp120 is a component of the cadherin-catenin complexes and, as such, may be involved in membrane-cytoskeleton interactions (22Reynolds A.B. Daniel J. McCrea P. Wheelock M.J. Zhang Z. Mol. Cell. Biol. 1994; 14: 8333-8342Crossref PubMed Google Scholar, 23Daniel J.M. Reynolds A.B. Bioessays. 1997; 19: 883-891Crossref PubMed Scopus (285) Google Scholar). The association of FER and pp120, and their phosphorylation in cells treated with growth factors, led us to speculate that FER may be involved in a signaling pathway linking growth factor receptors with cell adhesion or the cytoskeleton. In this report, we describe studies aimed at further characterization of the interaction of FER with other cellular components. We also report on the results of experiments, using a dominant-negative FER mutant, that seek to analyze the function of the FER kinase. NIH3T3 and human embryonic kidney 293 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% calf serum and 10% fetal bovine serum, respectively. Glutathione S-Sepharose was from Amersham Pharmacia Biotech. Monoclonal anti-phosphotyrosine and anti-pp120 antibodies were from Transduction Laboratories. Monoclonal anti-cortactin antibody (4F11) was from Upstate Biotechnology, Inc. Anti-hemagglutinin (12CA5) antibodies were from Boehringer Mannheim. Texas Red-conjugated goat anti-mouse IgG and fluorescein-conjugated goat anti-rabbit IgG were from Jackson ImmunoResearch Laboratory. Polyclonal anti-dynamin antibodies were provided by Dr. Mark McNiven of the Mayo Clinic. Polyclonal FER1 and FER5 antisera were described previously (10Kim L. Wong T.W. Mol. Cell. Biol. 1995; 15: 4553-4561Crossref PubMed Scopus (154) Google Scholar). They were generated by immunizing rabbits with fusion proteins that contain codons 502–675 and codons 451–564 of FER, respectively. Antiserum FER2 was prepared also using a TrpE fusion protein containing codons 502–675. Full-length FER cDNA was isolated from a human foreskin fibroblast cDNA library (CLONTECH) using as probe a polymerase chain reaction fragment that encompasses codons 149–332. A single lysine to arginine mutation was introduced at codon 591, using the SculptorTM in vitro mutagenesis kit (Amersham Pharmacia Biotech). Both the wild-type and K591R mutant cDNA sequences were subcloned in a CMV-based eukaryotic expression vector to yield pCMV-HA-FER(WT) and pCMV-HA-FER(K591R). The subcloning was designed to introduce an influenza virus hemagglutinin (HA) epitope at the amino terminus of the FER sequences. The human CSF-1 receptor cDNA was kindly provided by Dr. C. Sherr of the Howard Hughes Medical Institute at St. Jude's Medical Center and was subcloned into the CMV expression vector to yield pCMV-CSF1R. Details of the subcloning procedures will be provided upon request. Confluent cultures of NIH3T3 cells were serum-starved and stimulated with platelet-derived growth factor (PDGF) as described before (10Kim L. Wong T.W. Mol. Cell. Biol. 1995; 15: 4553-4561Crossref PubMed Scopus (154) Google Scholar). Cells were rinsed twice with cold PBS and scraped into ice-cold hypotonic buffer (20 mmTris·HCl, pH 7.7, 15 mm NaCl, 2 mmMgCl2, 1 mm sodium orthovanadate, and 40 μm ammonium molybdate) (1 ml/10-cm plate). The cell suspension was allowed to swell on ice for 15 min, and cell lysis was achieved by homogenization in a tight-fitting glass homogenizer. The cell lysates were centrifuged for 10 min at 2000 rpm in a tabletop centrifuge to remove nuclei and unbroken cells. The postnuclear supernatants were centrifuged for 90 min at 100,000 ×g to yield an S100 supernatant. The pelleted material was resuspended by homogenization in 3 ml of buffer that contained 5% glycerol, 20 mm Tris·HCl, pH 7.7, 1 mm EDTA, 150 mm NaCl, 1 mm sodium orthovanadate, and 40 mm ammonium molybdate. To that suspension was added 10% Triton X-100 to a final concentration of 1%. The mixture was clarified by centrifugation (10,000 ×g, 10 min) to yield a solubilized membrane (P100) fraction. Protein concentrations were determined using the microBCA reagents (Pierce). Whole-cell lysates were prepared as described previously (10Kim L. Wong T.W. Mol. Cell. Biol. 1995; 15: 4553-4561Crossref PubMed Scopus (154) Google Scholar). Immunoprecipitations were performed as described before, using typically 300 μg of S100 and 30 μg of P100 fractions and 5 μg of purified antibodies or 5 μl of antisera (10Kim L. Wong T.W. Mol. Cell. Biol. 1995; 15: 4553-4561Crossref PubMed Scopus (154) Google Scholar). Immunoblotting was performed using standard techniques, and binding was with reagents (Amersham Pharmacia Biotech). For complex kinase lysates were with FER5 and kinase were performed as described before (10Kim L. Wong T.W. Mol. Cell. Biol. 1995; 15: 4553-4561Crossref PubMed Scopus (154) Google Scholar). of fusion proteins and in vitro binding were performed as described before (10Kim L. Wong T.W. Mol. Cell. Biol. 1995; 15: 4553-4561Crossref PubMed Scopus (154) Google Scholar). In binding that involved the of cortactin required an because the with cortactin on and the of cortactin. In experiments, proteins to fusion proteins were by in The were with Triton X-100 to the final concentration to The were with anti-cortactin antibody before analyses by in were performed by S100 fractions with 5 μg of anti-phosphotyrosine The were by in and with Triton X-100 as The proteins were with fusion proteins on and proteins were by NIH3T3 cells were on glass and were serum-starved before treatment with for 5 Cells were with in PBS for 5 min) and were with Triton X-100 in PBS with bovine serum in cells were with FER5 and anti-cortactin was by with antibodies and of the Cells were using an and 293 cells were in and were using the modified C. H. Mol. Cell. Biol. PubMed Scopus Google Scholar). 2 μg of in the of μg of or pCMV-HA-FER(K591R). The and of cDNA were in to expression of and the FER cells were rinsed and with the medium was with medium modified Eagle's medium fetal bovine serum and 10 mm The cultures were and stimulated with for 5 Cells were and as described We previously an to the association of FER with pp120 (10Kim L. Wong T.W. Mol. Cell. Biol. 1995; 15: 4553-4561Crossref PubMed Scopus (154) Google Scholar). To further other that may with FER, we prepared additional antisera and their to complexes from lysates of NIH3T3 cells. shown FER1 a complex of FER and pp120 3 and treatment in an in the phosphorylation of FER and other FER2 and also FER, as well as a small of the receptor However, or pp120 was by of these FER5 additional of and The of these has to be These antisera may distinct or of FER. proteins that cell-cell or cell-substrate adhesion are in cytoskeletal the of lysates may the of FER We prepared subcellular fractions by hypotonic lysis and the of subcellular by The membrane (P100) fraction showed the of the is the major tyrosine phosphorylated protein in cells treated with 2 3 and shown pp120 is in the P100 fraction S.B. Reynolds A.B. Parsons J.T. Mol. Cell. Biol. 1991; 11: PubMed Scopus Google Scholar). FER is localized in the a is also in the membrane signaling as cortactin and showed localization with of membrane association as the S100 FER1 and FER2 antisera a of and 2 of only the with FER antibodies in 2 Antiserum FER2 also an phosphorylated with antibodies to the actin-binding protein cortactin and treatment in an in tyrosine phosphorylation of FER, the as well as cortactin. In the of cortactin, is and the of association with FER to be to growth factor The of the that with FER was by with antibodies to signaling of this to with a that was by the FER not by antibodies 2 However, the of the in the FER not with the to the anti-phosphotyrosine antibodies of with in from the localization of pp120, the FER·pp120 complex was in the P100 fraction and was by FER1 2 prepared with FER2 contained a of cortactin, only a of pp120 In to the prepared with the S100 the P100 fraction not contain of the To further the FER-cortactin the subcellular localization of the proteins were by In cortactin was previously shown to with cortical (20Wu H. Reynolds A.B. Kanner S.B. Vines R.R. Parsons J.T. Mol. Cell. Biol. 1991; 11: 5113-5124Crossref PubMed Scopus (375) Google H. Parsons J.T. J. Cell Biol. 1993; 120: 1417-1426Crossref PubMed Scopus (452) Google Scholar). We in cortactin is in the a is also in the In these FER is localized in the a of with cortactin in the cell also be and The with the FER was because it was not with serum or the was with the In cortactin in the cell and with FER is in the membrane and The of the FER-cortactin association was further in in vitro binding A fusion protein containing the sequence of FER cortactin from a fraction A and The of FER, was previously shown to interaction with pp120, not with cortactin. Cortactin is one of the major proteins that are to tyrosine phosphorylation and bind the FER sequence in vitro and We also the of the FER sequence to directly with cortactin. proteins were with anti-phosphotyrosine antibodies and Cortactin was the component and the to bind the FER sequence and that the FER sequence has the to directly with cortactin. In to cortactin, a was to with FER from the fraction. be in the FER its in the complexes that it is distinct from the component in the anti-phosphotyrosine The of the was further by the interactions the of the The complex was isolated using FER2 and was The were to a of using we to be to FER 5 with anti-phosphotyrosine antibodies that the was by with FER5 antibodies 5 was by was not in the FER5 was in a with anti-phosphotyrosine antibodies 5 and was in the FER2 was not in of the the that was by the FER2 antibodies was not These results the that is the with FER. They also suggest that the is to FER or that its association with FER is by a novel interaction, is to To analyze the functional of the FER-cortactin interaction, we prepared an FER by a mutation in the lysine of the binding site of the kinase. expression of the K591R FER mutant cDNA showed that it a that is the FER kinase, as a of the of an epitope to the amino terminus A and In an complex kinase the of cells with the K591R mutant showed of the mutant FER protein and of the kinase In contrast, with an wild-type FER cDNA in a of the kinase These results suggest that the K591R FER protein may function as a To the of the K591R FER mutant on pp120 and cortactin we 293 cells with cDNA CSF-1 receptor and mutant or wild-type FER. was shown previously that the tyrosine phosphorylation of pp120 and cortactin is also by the CSF-1 receptor Reynolds A.B. Oncogene. 1991; Google Scholar). S100 fractions of cells were with FER Antiserum FER1 a complex mixture of and FER as well as a small of pp120 the anti-phosphotyrosine the of of in the wild-type FER was in the of cells with the K591R mutant and 3 and the contained a complex mixture of FER proteins and cortactin The anti-phosphotyrosine that expression of the K591R FER mutant in a in the tyrosine phosphorylation of cortactin and The of the FER mutant on cortactin phosphorylation was further by with anti-cortactin shown with the FER2 with anti-cortactin antibodies demonstrated the association of FER and cortactin In cells the CSF-1 receptor and wild-type FER, treatment in an in phosphorylation of FER and cortactin 1 and In contrast, expression of the K591R FER mutant in a of and factor-dependent phosphorylation of cortactin 3 and is that the mutant FER not in its association with cortactin. of the FER in the anti-cortactin the of the wild-type and mutant FER We also further the of the FER mutant on pp120 phosphorylation by P100 fractions using anti-pp120 shown epithelial cells as 293 cells a mixture of pp120 (22Reynolds A.B. Daniel J. McCrea P. Wheelock M.J. Zhang Z. Mol. Cell. Biol. 1994; 14: 8333-8342Crossref PubMed Google Scholar). was in the of pp120 phosphorylation in cells wild-type or K591R mutant FER. We previously demonstrated the association of FER with the catenin pp120 and that of the FER protein was involved in that complex (10Kim L. Wong T.W. Mol. Cell. Biol. 1995; 15: 4553-4561Crossref PubMed Scopus (154) Google Scholar). shown in the subcellular studies that previous is with the of FER that is associated with the membrane fraction. The of subcellular and the FER2 the of a novel interaction FER and the cytoskeletal protein cortactin. of FER in the of cortactin or pp120, not FER to with pp120 and cortactin in a binding to the distinct FER sequences. These observations suggest that the FER·pp120 and FER-cortactin complexes may be involved in signaling function. The FER-cortactin complex is in a not in a prepared using The association was by functional and analyses using and is to be an of the lysis The FER-cortactin association is demonstrated by in the membrane of cells. the of the FER K591R mutant on cortactin phosphorylation a direct physical interaction the proteins. We that the interaction cytoskeletal structures that are Our analyses suggest that 10 and of the cellular cortactin is associated with FER. In to cortactin, we identified also a novel that with FER. We showed that cortactin with has an of L. and T. W. in Our analyses showed that be in prepared with by of the interaction of FER with cortactin. However, the results also showed that the is distinct from The of the FER5 to the FER and the that the is a modified of FER or a is in the expression is a in the of wild-type FER The that the is in the of the FER mutant that it is on FER kinase that the may be a phosphorylated of FER that is in as to be by with The functional significance of this and its localization in the to be In vitro binding demonstrated that the sequence of FER is of directly binding cortactin. with our previous that the coiled coil sequence at the amino terminus of FER is required for binding pp120 (10Kim L. Wong T.W. Mol. Cell. Biol. 1995; 15: 4553-4561Crossref PubMed Scopus (154) Google Scholar). of the FER K591R mutant was to in an inhibition of the of the FER kinase, that the mutant was of in a The FER mutation has on its to with cortactin and pp120, and expression of the FER mutant has on the expression of the cytoskeletal proteins not The mutation in an inhibition of the tyrosine phosphorylation of cortactin, not of pp120, in to growth factor receptor signaling. These data that cortactin, not pp120, is a direct substrate of FER kinase. However, we the that a of FER activity is to the phosphorylation of be that we have been to a direct binding FER and peptide were to the substrate of protein tyrosine kinases Z. M.J. Feldman R.A. M. J. C. M.J. Nature. 1995; PubMed Scopus Google Scholar). analyses led to the that nonreceptor tyrosine kinases to peptide sequences that are by their Our observations on the FER-cortactin interaction and their putative enzyme-substrate be with the on the of the peptide substrate our data suggest that the of cortactin phosphorylation by growth factor receptors may be in by FER. cortactin was identified as a of tyrosine phosphorylation by a role for in cortactin phosphorylation has not been The subcellular localization of to that and cortactin are localized to cellular J. Cell Biol. 1992; PubMed Scopus Google Scholar). In to and FER, the cytoplasmic kinase has also been implicated in the of cortactin phosphorylation C. 1995; 9: PubMed Scopus Google Scholar). is that cortactin may be a substrate of tyrosine kinases that to The phosphorylation of cortactin may be by an tyrosine kinase. The phosphorylation cortactin to bind FER, and the FER-cortactin complex a of by growth factor signaling. The localization of cortactin to the cortical and its for led to the suggestion that cortactin may be involved in from the membrane to the cytoskeleton. proposal is further by the that cortactin phosphorylation is upon signaling in cell adhesion and in the cytoskeletal that of epithelial cells K. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus (269) Google Scholar, C. EMBO J. 1995; 14: PubMed Scopus Google Scholar). cortactin was shown to F-actin in and the activity was by the tyrosine phosphorylation of cortactin C. Y. T. Y. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). These observations have a role for and FER and cortactin in the of for of the cytoskeleton. We Dr. M. McNiven for the anti-dynamin