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

  • Masanori Iwanishi
    Footnotes
    Affiliations
    Program in Molecular Medicine and Department of Biochemistry and Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605
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  • Michael P. Czech
    Affiliations
    Program in Molecular Medicine and Department of Biochemistry and Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605
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  • Andrew D. Cherniack
    Correspondence
    To whom correspondence should be addressed: Program in Molecular Medicine , 373 Plantation St., Worcester, MA 01605. Fax: 508-856-1617
    Affiliations
    Program in Molecular Medicine and Department of Biochemistry and Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605
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  • Author Footnotes
    * This work is supported in part by National Institutes of Health Grant DK30648 (to M. P. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) AF286537.
    ‡ Present address: Third Department of Medicine, Shiga University of Medical Science, Seta, Ohtsu, Shiga 520-21, Japan.
Open AccessPublished:December 15, 2000DOI:https://doi.org/10.1074/jbc.M006665200
      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.
      PDGF
      platelet-derived growth factor
      IRS-1
      insulin receptor substrate 1
      SSC
      standard saline citrate
      PI
      phosphatidylinositol
      HA
      hemagglutinin
      PAGE
      polyacrylamide gel electrophoresis
      SH
      Src homology
      GST
      glutathione S-transferase
      DMEM
      Dulbecco's modified Eagle's medium
      PVDF
      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 (
      • Smithgall 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.
      ). 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 (
      • Craig A.W.
      • Zirngibl R.
      • Greer P.
      ,
      • Kim L.
      • Wong T.W.
      ). 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 (
      • Aspenstrom P.
      ,
      • Tian L.
      • Nelson D.L.
      • Stewart D.M.
      ). Fer is ubiquitously expressed in a wide number of cell types and tissues (
      • Feldman R.A.
      • Tam J.P.
      • Hanafusa H.
      ,
      • MacDonald I.
      • Levy J.
      • Pawson T.
      ). Through both subcellular fractionation and immunocytochemistry, Fer has been shown to be localized to the cytoplasm and the nucleus (
      • Hao Q.L.
      • Ferris D.K.
      • White G.
      • Heisterkamp N.
      • Groffen J.
      ).
      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 (
      • Kim L.
      • Wong T.W.
      ). 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 (
      • Kim L.
      • Wong T.W.
      ). 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 (
      • Rosato R.
      • Veltmaat J.M.
      • Groffen J.
      • Heisterkamp N.
      ).
      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 (
      • Kim L.
      • Wong T.W.
      ). 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 (
      • Czech M.P.
      • Covera S.
      ). 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.

      DISCUSSION

      Here we show that two isoforms of Fer are highly expressed upon 3T3-L1 differentiation. In 3T3-L1 adipocytes, we found a unique splicing variant, iFer, which displays a 65-base pair deletion in the Fer open reading frame. The finding that iFer deletion encodes a protein without a functional kinase indicates that the iFer protein probably has a role in regulating some aspect of Fer function. There are many examples in which alternative splicing of genes produce truncated regulatory forms of proteins. In one case, alternate splicing produces a 26-base pair deletion in the message for the growth hormone receptor that encodes a truncated protein that lacks most of its intracellular domain. This splicing variant produces a soluble circulating form of the growth hormone receptor, which regulates the ability of growth hormone to activate its cellular receptor (
      • Dastot F.
      • Sobrier M.L.
      • Duquesnoy P.
      • Duriez B.
      • Goossens M.
      • Amselem S.
      ). More recently a truncated splicing variant of protein kinase C δ containing 83-base pair insertion variant was identified. Similar to iFer this splicing variant PKC δIII produces a protein that lacks a functional kinase domain, but retains the regulatory domain (
      • Ueyama T.
      • Ren Y.
      • Ohmori S.
      • Sakai K.
      • Tamaki N.
      • Saito N.
      ). The role of this truncated protein in PKC regulation has yet to be determined.
      Because iFer retains N-terminal coiled-coil domains, one possible function of iFer could be to regulate Fer oligomerization and activation. It has been hypothesized that like receptor tyrosine kinases, oligomerization activates Fer through the trans-phosphorylation of subunits. In the case of Fes, incubation of wild type full-length Fes with isolated N-terminal domains does in fact suppress Fes autophosphorylation (
      • Read R.D.
      • Lionberger J.M.
      • Smithgall T.E.
      ). However, in a different study, an N-terminal deletion mutant of Fer, which cannot oligomerize, was found to retain the ability to autophosphorylate itself. Furthermore, overexpression of the N-terminal region of Fer in COS-1 cells had no effect on Fer activity in in vitro kinase assays (
      • Craig A.W.
      • Zirngibl R.
      • Greer P.
      ). Here we show that in COS-1 cells, overexpressed iFer that immunoprecipitates with Fer is tyrosine phosphorylated. This result is consistent with iFer not regulating Fer kinase activity. Nevertheless, it is unclear whether these results reflect an actual biochemical difference between how Fes and Fer are activated or whether they actually reflect a difference because of the experimental design. Another possible function of iFer is to regulate the binding of Fer to other proteins. In initial experiments, we have been able to detect a small amount of iFer associated with both p85 and IRS-1 (data not shown), but this is probably due to an association with full-length Fer in these complexes, as opposed to a direct binding to either IRS-1 or p85. It might be possible that iFer regulates the binding of the N-terminal domain of Fer to the cellular adhesion protein p120cas , which could possibly prevent its tyrosine phosphorylation in response to growth factor stimulation. Further studies are needed to determine exactly what targets this Fer domain might bind in 3T3-L1 adipocytes.
      The 94-kDa full-length isoform of Fer is a ubiquitously expressed protein and has been found in almost every cell line studied (
      • Feldman R.A.
      • Tam J.P.
      • Hanafusa H.
      ,
      • MacDonald I.
      • Levy J.
      • Pawson T.
      ). However, there are other cases where Fer expression is increased. Fer has been shown to be absent in pre-B cells but accumulates upon their induction to antibody producing cells (
      • Halachmy S.
      • Bern O.
      • Schreiber L.
      • Carmel M.
      • Sharabi Y.
      • Shoham J.
      • Nir U.
      ). Fer expression has also shown to be enhanced in cell extracts from human prostate cancer cell lines (
      • Allard P.
      • Zoubeidi A.
      • Nguyen L.T.
      • Tessier S.
      • Tanguay S.
      • Chevrette M.
      • Aprikian A.
      • Chevalier S.
      ). The increased expression in differentiated 3T3-L1 cells might indicate that Fer has a role in promoting adipogenesis. Alternatively, this might indicate that Fer has a role in regulating insulin signaling pathways in adipocytes.
      In the current work, we show that in 3T3-L1 adipocytes insulin, like PDGF, results in the association of Fer with tyrosine phosphorylated signaling complexes. In 3T3-L1 adipocytes, PDGF results in the binding of Fer to PDGF receptors, whereas insulin activation results in complex formation between Fer and tyrosine phosphosphorylated IRS molecules. We also show that Fer binds to complexes containing phosphatidylinositol 3-kinase. In the case of PDGF stimulation, it is likely that this complex formation is the result of the binding of both Fer and p85 SH2 domains to the same tyrosine phosphorylated PDGF receptors. However, we cannot rule out the possibility that the SH2 domain of p85 binds directly to tyrosine phosphorylated Fer. Because there is little if any increase of Fer tyrosine phosphorylation in response to insulin, it seem unlikely insulin sensitive complex formation between Fer and p85 is the result of this sort of direct interaction. It is most likely that Fer binds to p85 through tyrosine phosphorylated adaptors such as IRS-1. We cannot rule out the possibility that Fer binds constitutively to IRS-1, because the amount of IRS-1 bound to Fer was below the sensitivity that we are able to detect on immunoblots probed with anti-IRS-1 antibodies (data not shown). It is also possible that Fer binds to p85 through other adaptor proteins that are tyrosine phosphorylated in response to insulin such as IRS-2.
      Although it is has been hypothesized that cytoplasmic tyrosine kinases such as Fer may have a role in insulin signaling, very little is actually known about their function (
      • Kanzaki M.
      • Watson R.T.
      • Artemyev N.O.
      • Pessin J.E.
      ). For example, insulin stimulation results in both the binding of the cytoplasmic tyrosine kinase Fyn to IRS-1 and c-Cbl and its dissociation from Sirm (
      • Sun X.J.
      • Pons S.
      • Asano T.
      • Myers M.G.
      • Glasheen E.
      • White M.F.
      ,
      • Ribon V.
      • Saltiel A.R.
      ,
      • Salvatore P.
      • Hanash C.R.
      • Kido Y.
      • Imai Y.
      • Accili D.
      ). Yet the significance of these associations has yet to be determined. Our data show that Fer might also have a role in insulin signaling. Although Fer is believed to be involved in cross-talk between PDGF receptors and cellular adhesion complexes, there appears to be differences between how Fer responds to PDGF and insulin stimulation. PDGF, unlike insulin, results in the increase of tyrosine phosphorylation of Fer. This suggests that PDGF but not insulin stimulation results in a increase of the catalytic activity of Fer kinase. Thus, although it is possible that Fer may be involved in both insulin and PDGF signaling, Fer might also mediate signaling pathways in 3T3-L1 adipocytes that are unique to PDGF. Further studies to determine the substrates of Fer in this cell type will help unravel these pathways.

      ACKNOWLEDGEMENTS

      We thank Dr. Tony Pawson for providing us with anti-Fer antibody and Sarah Nicoloro for helpful discussions.

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