The Protein-tyrosine Kinase Fer Associates with Signaling Complexes Containing Insulin Receptor Substrate-1 and Phosphatidylinositol 3-Kinase

In a screen for 3T3-F442A adipocyte proteins that bind SH2 domains, we isolated a cDNA encoding Fer, a nonreceptor protein-tyrosine kinase of the Fes/Fps family that contains a functional SH2 domain. A truncated splicing variant, iFer, was also cloned. iFer is devoid of both the tyrosine kinase domain and a functional SH2 domain but displays a unique 42-residue C terminus and retains the ability to form oligomers with Fer. Expression of both Fer and iFer proteins are strikingly increased upon differentiation of 3T3-L1 fibroblasts to adipocytes. Platelet-derived growth factor treatment of the cultured adipocytes caused rapid tyrosine phosphorylation of Fer and its recruitment to complexes containing platelet-derived growth factor receptor and the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase. Insulin treatment of 3T3-L1 adipocytes stimulated association of Fer with complexes containing tyrosine phosphorylated IRS-1 and PI 3-kinase but did not stimulate tyrosine phosphorylation of Fer. PI 3-kinase activity in anti-Fer immunoprecipitates was also acutely activated by insulin treatment of cultured adipocytes. These data demonstrate the presence of Fer tyrosine kinase in insulin signaling complexes, suggesting a role of Fer in insulin action. In a screen for 3T3-F442A adipocyte proteins that bind SH2 domains, we isolated a cDNA encoding Fer, a nonreceptor protein-tyrosine kinase of the Fes/Fps family that contains a functional SH2 domain. A truncated splicing variant, iFer, was also cloned. iFer is devoid of both the tyrosine kinase domain and a functional SH2 domain but displays a unique 42-residue C terminus and retains the ability to form oligomers with Fer. Expression of both Fer and iFer proteins are strikingly increased upon differentiation of 3T3-L1 fibroblasts to adipocytes. Platelet-derived growth factor treatment of the cultured adipocytes caused rapid tyrosine phosphorylation of Fer and its recruitment to complexes containing platelet-derived growth factor receptor and the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase. Insulin treatment of 3T3-L1 adipocytes stimulated association of Fer with complexes containing tyrosine phosphorylated IRS-1 and PI 3-kinase but did not stimulate tyrosine phosphorylation of Fer. PI 3-kinase activity in anti-Fer immunoprecipitates was also acutely activated by insulin treatment of cultured adipocytes. These data demonstrate the presence of Fer tyrosine kinase in insulin signaling complexes, suggesting a role of Fer in insulin action. platelet-derived growth factor insulin receptor substrate 1 standard saline citrate phosphatidylinositol hemagglutinin polyacrylamide gel electrophoresis Src homology glutathione S-transferase Dulbecco's modified Eagle's medium polyvinylidene difluoride The nonreceptor tyrosine kinase Fer and the closely related protein, Fes, are the only members of a group of proteins that is unrelated to any other families of cytoplasmic tyrosine kinases (1Smithgall T.E. Rogers J.A. Peters K.L. Li J. Briggs S.D. Lionberger J.M. Cheng H. Shibata A. Scholtz B. Schreiner S. Dunham N. Crit. Rev. Oncog. 1998; 9: 43-62Crossref PubMed Scopus (61) Google Scholar). The 94-kDa isoform of Fer and Fes share the same conserved domain organization, which consists of a long N-terminal domain, a central SH2 domain, and a C-terminal kinase domain. Contained within the N-terminal domain are three coiled-coil domains, which have been shown to be required for oligomerization and have been shown to trimerize in vivo (2Craig A.W. Zirngibl R. Greer P. J. Biol. Chem. 1999; 274: 19934-19942Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 3Kim L. Wong T.W. Mol. Cell. Biol. 1995; 15: 4553-4561Crossref PubMed Scopus (154) Google Scholar). The extreme N terminus of Fer also contains a Fes/CIP4 homology domain, which has also been found in proteins involved in regulating the actin cytoskeleton, and may function in microtubule binding (4Aspenstrom P. Curr. Biol. 1997; 7: 479-487Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 5Tian L. Nelson D.L. Stewart D.M. J. Biol. Chem. 2000; 275: 7854-7861Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Fer is ubiquitously expressed in a wide number of cell types and tissues (6Feldman R.A. Tam J.P. Hanafusa H. Mol. Cell. Biol. 1985; 6: 1065-1073Crossref Scopus (25) Google Scholar, 7MacDonald I. Levy J. Pawson T. Mol. Cell. Biol. 1985; 5: 2543-2551Crossref PubMed Scopus (96) Google Scholar). Through both subcellular fractionation and immunocytochemistry, Fer has been shown to be localized to the cytoplasm and the nucleus (8Hao Q.L. Ferris D.K. White G. Heisterkamp N. Groffen J. Mol. Cell. Biol. 1991; 11: 1180-1183Crossref PubMed Scopus (49) Google Scholar). Although the cellular function of Fer is not known, its tyrosine phosphorylation has been shown in A431 and Swiss 3T3 fibroblasts following activation of the epidermal growth factor receptor or the PDGF1 receptor (3Kim L. Wong T.W. Mol. Cell. Biol. 1995; 15: 4553-4561Crossref PubMed Scopus (154) Google Scholar). PDGF stimulation also results in an increase in Fer tyrosine kinase activity. These results indicate that Fer may function in signaling by growth factor receptors. One potential role Fer may have in signaling is in the regulation of cellular adhesion complexes. It has been previously demonstrated that the N-terminal region of Fer is constitutively bound to a cell adhesion complex protein p120cas , which itself is tyrosine phosphorylated upon growth factor stimulation (3Kim L. Wong T.W. Mol. Cell. Biol. 1995; 15: 4553-4561Crossref PubMed Scopus (154) Google Scholar). Fer also co-immunoprecipitates with β-catenin, another component of cell adhesion complexes. Overexpression of Fer in embryonic fibroblasts elevates tyrosine phosphorylation of both p120cas and β-catenin and decreases the amount of α-catenin and β-catenin that associates with E-cadherin. This indicates that Fer overexpression results in the dissolution of adheren junction complexes (9Rosato R. Veltmaat J.M. Groffen J. Heisterkamp N. Mol. Cell. Biol. 1998; 18: 5762-5770Crossref PubMed Scopus (109) Google Scholar). Recently the SH2 domain of Fer has been also shown to bind to the F-actin-binding protein cortactin. A dominant negative mutant in Fer inhibits the ability of macrophage colony stimulating factor to stimulate tyrosine phosphorylation of cortactin (10Kim L. Wong T.W. J. Biol. Chem. 1998; 273: 23542-23548Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Thus it is possible that Fer may also link growth factor signaling to cytoskeletal elements. During a screen of SH2-binding proteins using a 3T3-F44A adipocyte expression library, we identified a large number of clones consisting of the C-terminal kinase domain of Fer. In these differentiated adipocyte cells, insulin has the unique ability to activate a number of metabolic pathways such as glucose uptake (11Czech M.P. Covera S. J. Biol. Chem. 1999; 274: 1865-1868Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar). This led us to speculate that Fer might possibly also have a role in insulin signaling. In the present study we show that Fer expression increases upon 3T3-L1 adipocyte differentiation and that these cells express a splicing isoform that lacks most of the SH2 domain and all of the C-terminal kinase domain. We also show that insulin stimulation results in the formation of complexes between Fer with tyrosine phsophorylated IRS-1 and p85 phosphatidylinositol kinase. 4G10 anti-phosphotyrosine monoclonal antibody and monoclonal p85 (N-SH2) antibody used for immunoblotting or immunoprecipitation were purchased from Upstate Biotechnology Inc. Rabbit polyclonal IRS-1 (SC559) and PDGF receptor antibody was purchased from Santa Cruz Biotechnology. Mouse monoclonal antibody HA antibody (12CAS) was purchased from BABCO. Rabbit anti-HA antibody was raised against a peptide corresponding to the HA epitope and purified on protein A-Sepharose. Mouse monoclonal Myc antibody was obtained from cell culture supernatants of hybridoma cell line 10E2. Anti-Fer serum used for both immunoprecipitation and immunoblotting (FerN) was prepared by immunizing rabbits with a GST fusion protein that contain residues 224–467 of the Fer sequence. Anti-Fer serum used for immunoblotting (FerK) that is specific for kinase domain of Fer was provided kindly by Dr. Tony Pawson. Anti-iFer serum was prepared by immunizing rabbits with a peptide containing the last 15 C-terminal amino acids of iFer. 3T3-L1 fibroblasts (2–3 days post confluent) were differentiated into adipocytes by incubating with DMEM containing 25 mm glucose, 10% fetal bovine serum, 50 μg/ml streptomycin, 50 units/ml penicillin, 0.5 mmisobutylmethylxanthine, 0.25 μm dexamethasone, and 5 μg/ml insulin for 3 days and then grown in DMEM with 10% Fetal bovine serum, 50 μg/ml streptomycin, and 50 units/ml penicillin for an additional 5–8 days. COS-1 cells were grown in DMEM containing 10% fetal bovine serum, 50 μg/ml streptomycin, and 50 units/ml penicillin. A λ-Zap mouse 3T3-F442A was screened for Grb2-binding proteins using the method described in Singh et al. (12Singh H. Clerc R.G. LeBowitz J.H. BioTechniques. 1989; 7: 252-261PubMed Google Scholar). Briefly, 40,000 plaque forming units of this library were plated on to eighteen 15-cm LB plates in LB top agar and incubated for 3 h at 42 °C. Nitrocellulose filters that were soaked in 10 mmisopropylthio-β-d-galactoside were placed on the plates and incubated for an additional 10 h at 37 °C. Plates were cooled to 4 °C, after which filters were removed and washed three times in binding buffer (10 mm Tris-HCl, pH 7.5, 50 mm NaCl, 1 mm EDTA, and 1 mmdithiothreitol) under constant agitation. Filters were then blocked with 50 mm Tris-HCl, pH 7.5, 50 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, and 5% nonfat milk and washed several times in binding buffer. GST-Grb2 fusion protein was produced from a pGEX-2TK construction (13Cherniack A.D. Klarlund J.K. Conway B.R. Czech M.P. J. Biol. Chem. 1995; 270: 1485-1488Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). GST-Grb2 fusion protein was 32P-labeled by incubating fusion protein bound to GST beads with approximately 25 units of protein kinase A (Sigma) and 1 mCi of [γ-32P]ATP in 100 μl of phosphorylation buffer (100 mm, 50 mm Tris, pH 7.5, and 1 mm dithiothreitol) for 1 h at room temperature. The beads were then washed five times with phosphate-buffered saline. Labeled Grb2 fusion protein was removed from the beads with 500 μl of 100 mm reduced glutathione and then incubated with filters in binding buffer overnight at 4 °C. The next day, filters were washed with five changes of binding buffer, dried, and exposed to film. Positive plaques were then isolated and purified by further rounds of screening. Excisions of Bluescript phagemids were preformed according to the manufacturer's protocol and dideoxy sequenced on an ABI 377 automatic sequencer. A 3T3-F44A adipocyte clone library was screened with a 32P-labeled probe made against a fragment consisting of sequences between nucleotides 669 and 1397 of the mouse Fer coding sequence by random priming (14Feinberg A.P. Vogelstein B. Anal. Biochem. 1983; 132: 6-13Crossref PubMed Scopus (16651) Google Scholar). The library was plated out according to manufacturer's instructions, and plaques were transferred to Nitrocellulose membranes (Schleicher & Schuell) for 2 min. Nitrocellulose circles were then denatured in 1.5m NaCl and 0.5 m NaOH for 5 min, neutralized in 1.5 m NaCl and 0.5 m Tris-HCl, pH 8.0, for 5 min, and then washed with 2× SSC for 2 min. DNA from plaques were fixed to nitrocellulose membranes for 2 h at 80 °C, and membranes were prehybridized in buffer 1 (50% formamide, 5× SSPE, 2% SDS, 10× Denhardt's solution, and 100 μg/ml boiled salmon sperm DNA) for 2 h at 42 °C. Denatured 32P-labeled probes were hybridized to filters in fresh buffer 1 (106 cpm/ml) overnight at 42 °C. Library filters were washed three times with 2× SSC and 0.05% SDS at room temperature for 5 min, one time in 0.1× SSC and 0.1% SDS at room temperature for 5 min, and three times with 0.1× SSC and 0.1% SDS at room temperature for 10 min. Membranes were exposed to X-Omat AR (Kodak) film overnight, and positive plaques were identified, isolated, and purified through further rounds of screening. Bluescript phagemids were excised according to manufacturer's instructions and dideoxy sequenced on an ABI 377 automatic sequencer. Six independent clones were isolated and were entirely sequenced. Three hemaglutinin epitopes were attached to the 5′ end of iFer and Fer by polymerase chain reaction and cloned into the Kpn and SmaI sites of PCMV5. Myc-HA-Fer was constructed by adding a Myc epitope to the 5′ end of 3× HA Fer. On the day before transfections, COS-1 cells were seeded at 750,000 cells/100-mm plate and transfected with 5 μg of each construction using LipofectAMINE (Life Technologies, Inc.) according to the manufacturer's protocols. Cells were lysed h after Cells were washed with phosphate-buffered saline and lysed in 1 of buffer mm pH 7.5, 100 mm NaCl, 1 mm 50 mm 1 mm 1 5 μg/ml each and 1 mm were then by at for 10 at 4 °C. The supernatants were removed and for protein using the method Anal. Biochem. PubMed Scopus Google Scholar). were incubated on an with or serum overnight and then were with protein for 2 The was by at 2 at 4 °C. were washed five times with buffer saline with 50 mm and 1 and buffer was to the after the were on and transferred to nitrocellulose or Membranes were with and were using according to the manufacturer's 3T3-L1 adipocytes were lysed with the buffer mm pH 7.5, mm NaCl, 1 mm 50 mm 0.5 mm 5 μg/ml and 5 μg/ml and Fer was overnight at 4 °C. with anti-Fer or The next day protein was to each incubated for 2 h at 4 and by at for 2 at 4 °C. complexes were washed times with buffer 1 (100 mm Tris-HCl, pH 7.5, and 500 mm one time with buffer 2 (10 mm Tris-HCl, pH 7.5, 100 mm NaCl, and 1 mm and one time with reaction buffer mm Tris-HCl, pH 7.5, 100 mm NaCl, 0.5 mm 10 mm and μm complexes were then incubated for 10 at with μl of reaction buffer with 100 μm 100 and 10 μm were with and with Labeled were and phosphatidylinositol were by Through a 3T3-F44A adipocyte expression library for proteins that bind to SH2 domain of we isolated several clones consisting of the C-terminal of Fer its tyrosine kinase. which of Fer are expressed in this cell we screened this library with 32P-labeled DNA probes consisting of a of the Fer coding sequence. Six independent clones that hybridized to this probe were of these which we as iFer, the Fer coding sequence for a within the region that for the SH2 domain. This region with the sequence which is with it the of an The iFer splicing results in a which a protein with a SH2 domain and a unique C-terminal region Fer and iFer were with HA epitopes and expressed in COS-1 cells to with previously 2 that 94-kDa Fer in COS-1 cells is constitutively and on tyrosine The protein by the iFer splicing lacks a functional tyrosine kinase domain but contains one of three Fer tyrosine phosphorylation sites (2Craig A.W. Zirngibl R. Greer P. J. Biol. Chem. 1999; 274: 19934-19942Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). in 2 iFer to tyrosine phosphorylated in cells, that a functional Fer kinase domain is required for this phosphorylation to the N-terminal region of Fer contains the domain that is required for its we that iFer is of forming complexes with Fer. we iFer, with Fer that is with both the Myc and HA and Fer oligomers with 2 that iFer with Fer. of Fer and iFer also results in the tyrosine phosphorylation of iFer, which that its tyrosine phosphorylation be phosphorylated in by an Fer kinase. Fer has been shown to be ubiquitously expressed in most tissues and cell that have been that Fer is also expressed in differentiated cell from both 3T3-L1 fibroblasts and adipocytes were on transferred to and with anti-Fer in 3 Fer is expressed in both adipocytes and but its expression is approximately by that iFer is also expressed in these cell we raised to a peptide of the last 15 amino acids of the iFer coding sequence. This sequence not have any homology to any proteins in protein sequence data a of approximately that is expressed in 3T3-L1 adipocytes but not in to 3 This is not by The in the of this to that of the of iFer on its sequence may be to and insulin stimulation not the of this on In mouse Swiss 3T3 cell PDGF tyrosine phosphorylation of Fer and complex formation of Fer with activated PDGF (3Kim L. Wong T.W. Mol. Cell. Biol. 1995; 15: 4553-4561Crossref PubMed Scopus (154) Google Scholar). 3T3-L1 adipocytes are to PDGF we were in a to this growth factor in this cell J. A. S. A. 1995; PubMed Scopus Google Scholar). we stimulated 3T3-L1 adipocytes with PDGF for 5 and cell with anti-Fer These complexes were on and with anti-Fer and anti-phosphotyrosine that to Swiss 3T3-L1 PDGF stimulation of 3T3-L1 adipocytes results in an increase of Fer tyrosine phosphorylation Fer also co-immunoprecipitates with tyrosine phosphorylated PDGF PDGF stimulation also results in the recruitment of phosphatidylinositol 3-kinase to tyrosine phosphorylated PDGF Thus PDGF stimulation PDGF receptor complexes containing Fer and the p85 subunit of phosphatidylinositol 3-kinase Anal. Biochem. PubMed Scopus Google Scholar). of anti-Fer immunoprecipitates that p85 in bind to complexes containing Fer in a a amount of p85 to Fer in the of PDGF In 3T3-L1 required for insulin signaling such as insulin receptor and expressed upon differentiation S. A. PubMed Scopus Google Scholar, A. J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar, P. B. S. A. Scopus Google Scholar). Fer is also expressed upon we that it may also have a role in insulin signaling. this we stimulated 3T3-L1 adipocytes with insulin for 5 and with anti-Fer PDGF stimulation of cultured insulin treatment results in any increase in tyrosine phosphorylation of Fer insulin stimulation in a increase in complex formation between Fer and tyrosine phosphorylated to insulin stimulation also complex formation between Fer and p85 We also these immunoprecipitates for the presence of but we were to Grb2 not This indicates that the of Fer in a screen for proteins that bind to the Grb2 SH2 domain is to the ability of the kinase domain of Fer to tyrosine phosphorylation of Grb2 binding positive The time of the formation of and complexes was by Fer from 3T3-L1 adipocytes stimulated with insulin for that within of insulin stimulation Fer is bound to both tyrosine phosphorylated IRS-1 and The binding of Fer to both these is 1 h after insulin phosphatidylinositol 3-kinase is with Fer, immunoprecipitates were for ability to phosphatidylinositol with in phosphatidylinositol 3-kinase is bound to Fer within 1 of insulin receptor activation and is not for at min. any activity is found bound to Fer PI 3-kinase activity with anti-Fer from and 3T3-L1 adipocytes were with anti-Fer or were for in PI 3-kinase activity as described under of corresponding were excised and in a PI 3-kinase activity with Fer activity with immunoprecipitates with we show that of Fer are expressed upon 3T3-L1 In 3T3-L1 we found a unique splicing variant, iFer, which displays a in the Fer The that iFer a protein a functional kinase indicates that the iFer protein has a role in regulating of Fer are in which splicing of truncated regulatory of In one splicing a in the for the growth receptor that a truncated protein that lacks most of its domain. This splicing a form of the growth which the ability of growth to activate its cellular receptor P. B. S. A. Scopus Google Scholar). a truncated splicing of protein kinase C containing was to iFer this splicing a protein that lacks a functional kinase domain, but retains the regulatory domain T. S. N. N. Biochem. Biol. 2000; Scholar). The role of this truncated protein in regulation has to be iFer retains N-terminal coiled-coil domains, one possible function of iFer be to Fer oligomerization and It has been that receptor tyrosine oligomerization Fer through the of In the of Fes, of Fes with isolated N-terminal in Fes Lionberger J.M. T.E. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). in a an N-terminal mutant of Fer, which was found to the ability to overexpression of the N-terminal region of Fer in COS-1 cells on Fer activity in in kinase (2Craig A.W. Zirngibl R. Greer P. J. Biol. Chem. 1999; 274: 19934-19942Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). we show that in COS-1 cells, iFer that immunoprecipitates with Fer is tyrosine This is with iFer not regulating Fer kinase activity. it is these results an between Fes and Fer are activated or a of the possible function of iFer is to the binding of Fer to other In we have been to a amount of iFer with both p85 and IRS-1 not but this is to an association with Fer in these complexes, as to a binding to IRS-1 or It might be possible that iFer the binding of the N-terminal domain of Fer to the cellular adhesion protein p120cas , which possibly its tyrosine phosphorylation in to growth factor are to this Fer domain might bind in 3T3-L1 adipocytes. The 94-kDa isoform of Fer is a ubiquitously expressed protein and has been found in cell line (6Feldman R.A. Tam J.P. Hanafusa H. Mol. Cell. Biol. 1985; 6: 1065-1073Crossref Scopus (25) Google Scholar, 7MacDonald I. Levy J. Pawson T. Mol. Cell. Biol. 1985; 5: 2543-2551Crossref PubMed Scopus (96) Google Scholar). are other Fer expression is Fer has been shown to be in cells but upon to antibody cells S. L. J. 1997; PubMed Scopus Google Scholar). Fer expression has also shown to be in cell from cell P. A. S. S. A. S. Mol. Cell. 2000; PubMed Scopus Google Scholar). The increased expression in differentiated 3T3-L1 cells might indicate that Fer has a role in this might indicate that Fer has a role in regulating insulin signaling pathways in adipocytes. In the we show that in 3T3-L1 adipocytes results in the association of Fer with tyrosine phosphorylated signaling complexes. In 3T3-L1 PDGF results in the binding of Fer to PDGF insulin activation results in complex formation between Fer and tyrosine We also show that Fer to complexes containing phosphatidylinositol 3-kinase. In the of PDGF it is that this complex formation is the of the binding of both Fer and p85 SH2 to the same tyrosine phosphorylated PDGF receptors. we out the that the SH2 domain of p85 to tyrosine phosphorylated Fer. is any increase of Fer tyrosine phosphorylation in to it insulin complex formation between Fer and p85 is the of this of It is most that Fer to p85 through tyrosine phosphorylated such as We out the that Fer constitutively to the amount of IRS-1 bound to Fer was the that we are to on with not It is also possible that Fer to p85 through other proteins that are tyrosine phosphorylated in to insulin such as Although it is has been that cytoplasmic tyrosine kinases such as Fer may have a role in insulin is function J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). insulin stimulation results in both the binding of the cytoplasmic tyrosine kinase to IRS-1 and and its from S. T. White J. Biol. Chem. Full Text Full Text PDF Scopus Google Scholar, Biochem. J. 1997; PubMed Scopus Google Scholar, P. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). the of these has to be data show that Fer might also have a role in insulin signaling. Although Fer is to be involved in between PDGF and cellular adhesion complexes, to be between Fer to PDGF and insulin results in the increase of tyrosine phosphorylation of Fer. This that PDGF but not insulin stimulation results in a increase of the activity of Fer kinase. it is possible that Fer may be involved in both insulin and PDGF Fer might also signaling pathways in 3T3-L1 adipocytes that are unique to to the of Fer in this cell these We Dr. Tony Pawson for us with anti-Fer antibody and for