MEK Kinase 1 Interacts with Focal Adhesion Kinase and Regulates Insulin Receptor Substrate-1 Expression
2003; Elsevier BV; Volume: 278; Issue: 6 Linguagem: Inglês
10.1074/jbc.m206087200
ISSN1083-351X
AutoresToshiaki Yujiri, Ryouhei Nawata, Toru Takahashi, Yutaka Sato, Yukio Tanizawa, Toshio Kitamura, Yoshitomo Oka,
Tópico(s)Cellular Mechanics and Interactions
ResumoMEK kinase 1 (MEKK1) has been shown to contribute to the regulation of cell migration, whereas focal adhesion kinase (FAK) is a major player involved in both cell migration and integrin signaling. Here we show that MEKK1 and FAK are co-immunoprecipitated from mouse fibroblasts. Moreover, the association between MEKK1 and FAK appears to be physiologically relevant, as it is enhanced by treatment with epidermal growth factor (EGF). Targeting FAK to the membrane also enhanced its association with MEKK1, indicating that MEKK1 is localized to a membrane-related subcellular domain, perhaps focal adhesions. Interestingly, the expression of insulin receptor substrate-1 (IRS-1) was diminished in MEKK1-deficient fibroblasts, which is similar to an earlier finding in FAK-deficient fibroblasts. Insulin-like growth factor 1 (IGF-1)-induced ERK activation was diminished in MEKK1-deficient cells, but phosphatidylinositol 3-kinase/Akt activation was not. Although integrin reportedly regulates the transcription of the IRS-1 gene via FAK-mediated JNK activation, no impairment of fibronectin-stimulated activation of FAK, ERK, or JNK was observed in MEKK1-deficient cells. Reconstitution of MEKK1 expression restored IRS-1 expression as well as IGF-1-induced ERK activation. Taken together, these findings indicate that MEKK1 interacts with FAK in focal adhesions and regulates IRS-1 expression. MEK kinase 1 (MEKK1) has been shown to contribute to the regulation of cell migration, whereas focal adhesion kinase (FAK) is a major player involved in both cell migration and integrin signaling. Here we show that MEKK1 and FAK are co-immunoprecipitated from mouse fibroblasts. Moreover, the association between MEKK1 and FAK appears to be physiologically relevant, as it is enhanced by treatment with epidermal growth factor (EGF). Targeting FAK to the membrane also enhanced its association with MEKK1, indicating that MEKK1 is localized to a membrane-related subcellular domain, perhaps focal adhesions. Interestingly, the expression of insulin receptor substrate-1 (IRS-1) was diminished in MEKK1-deficient fibroblasts, which is similar to an earlier finding in FAK-deficient fibroblasts. Insulin-like growth factor 1 (IGF-1)-induced ERK activation was diminished in MEKK1-deficient cells, but phosphatidylinositol 3-kinase/Akt activation was not. Although integrin reportedly regulates the transcription of the IRS-1 gene via FAK-mediated JNK activation, no impairment of fibronectin-stimulated activation of FAK, ERK, or JNK was observed in MEKK1-deficient cells. Reconstitution of MEKK1 expression restored IRS-1 expression as well as IGF-1-induced ERK activation. Taken together, these findings indicate that MEKK1 interacts with FAK in focal adhesions and regulates IRS-1 expression. mitogen-activated protein kinase/extracellular signal regulated kinase kinase 1 mitogen-activated protein MAP kinase extracellular signal-regulated kinase MAPK/ERK kinase epidermal growth factor c-Jun N-terminal kinase transforming growth factor-α focal adhesion kinase platelet-derived growth factor insulin-like growth factor 1 insulin receptor substrate 1 phosphatidyl inositol 3-kinase mouse embryonic fibroblast human embryonic kidney 293 (cell line) Iscove's modified Eagle's medium antibody monoclonal Ab hemagglutinin phosphate-buffered saline glyceraldehyde-3-phosphate dehydrogenase Src homology glutathioneS-transferase MEKK11 is a 196-kDa serine-threonine kinase activated in response to a variety of stimuli, including EGF, lysophosphatidic acid, osmotic stress, and microtubule toxins (1Fanger G.R. Johnson N.L. Johnson G.L. EMBO J. 1997; 16: 4961-4972Crossref PubMed Scopus (253) Google Scholar, 2Yujiri T. Fanger G.R. Garrington T.P. Schlesinger T.K. Gibson S. Johnson G.L. J. Biol. Chem. 1999; 274: 12605-12610Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Upon activation, MEKK1 participates in the regulation of the JNK and ERK pathways and is involved in the activation of NF-κB (3Lee F.S. Hagler J. Chen Z.J. Maniatis T. Cell. 1997; 88: 213-222Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar, 4Meyer C.F. Wang X. Chang C. Templeton D. Tan T.H. J. Biol. Chem. 1996; 271: 8971-8976Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 5Yujiri T. Sather S. Fanger G.R. Johnson G.L. Science. 1998; 282: 1911-1914Crossref PubMed Scopus (280) Google Scholar). In addition, MEKK1 senses microtubule integrity, protects cells from committing to apoptosis, and contributes to the migration of fibroblasts and epithelial cells. The phenotype of the MEKK1-null mouse includes an eyelid closure defect that is also seen in mice lacking the EGF receptor and TGF-α (6Yujiri T. Ware M. Widmann C. Oyer R. Russell D. Chan E. Zaitsu Y. Clarke P. Tyler K. Oka Y. Fanger G.R. Henson P. Johnson G.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7272-7277Crossref PubMed Scopus (207) Google Scholar). This suggests the possibility that MEKK1 is required for EGF receptor control of cell migration. Consistent with that idea, overexpression of MEKK1 induces the formation of a large lamellipodia-like structure in epithelial cells (6Yujiri T. Ware M. Widmann C. Oyer R. Russell D. Chan E. Zaitsu Y. Clarke P. Tyler K. Oka Y. Fanger G.R. Henson P. Johnson G.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7272-7277Crossref PubMed Scopus (207) Google Scholar). Still, the mechanism by which MEKK1 influences cell motility remains unclear.FAK is protein tyrosine kinase found at sites of adhesion (7Cary L.A. Guan J.L. Front. Biosci. 1999; 4: D102-D113Crossref PubMed Google Scholar). It is activated by integrin-mediated adhesion and serves as a signaling protein within cytoskeleton-associated networks (7Cary L.A. Guan J.L. Front. Biosci. 1999; 4: D102-D113Crossref PubMed Google Scholar). The Src-family protein tyrosine kinases p130 Cas, Shc, and Grb2 act in concert with FAK to transduce integrin-generated signals to the ERK/JNK MAP kinase cascades (7Cary L.A. Guan J.L. Front. Biosci. 1999; 4: D102-D113Crossref PubMed Google Scholar). Experiments using FAK-deficient cells have established that FAK is essential for integrin-stimulated cell migration and important for linking activation of the PDGF and EGF receptors to the cellular machinery that promotes directed cell migration (8Sieg D.J. Hauck C.R. Ilic D. Klingbeil C.K. Schaefer E. Damsky C.H. Schlaepfer D.D. Nat. Cell Biol. 2000; 2: 249-256Crossref PubMed Scopus (1050) Google Scholar). The fact that MEKK1 is enriched in membranes and colocalizes with α-actinin along actin stress fibers at focal adhesions (9Christerson L.B. Vanderbilt C.A. Cobb M.H. Cell Motil. Cytoskeleton. 1999; 43: 186-198Crossref PubMed Scopus (89) Google Scholar, 10Xu S. Robbins D.J. Christerson L.B. English J.M. Vanderbilt C.A. Cobb M.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5291-5295Crossref PubMed Scopus (122) Google Scholar) prompted us to investigate the possibility that MEKK1 is associated with FAK.Insulin and IGF-1 exert diverse biological effects by binding to and activating their cognate tyrosine kinase receptors. IRS-1 is a major substrate for the insulin and IGF-1 receptors, which rapidly phosphorylate it on multiple tyrosine residues upon ligand binding. Recently, it was reported that targeted disruption of FAK eliminates IRS-1 expression in MEFs and that interactions between cells and the extracellular matrix regulate the transcription of the IRS-1 gene via FAK-mediated JNK activation (11Lebrun P. Baron V. Hauck C.R. Schlaepfer D.D. Van Obberghen E. J. Biol. Chem. 2000; 275: 38371-38377Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Here, we show the following. 1) MEKK1 interacts with FAK in vivo. 2) Like FAK, MEKK1 is required for IRS-1 expression. 3) Targeted disruption of MEKK1 alters IGF-1-induced ERK activation but not PI3K/Akt activation.DISCUSSIONMEKK1 reportedly binds to a number of signaling proteins including Ras, Raf-1, Rac1, cdc42Hs, Nck-interacting kinase, SEK1, and JNK (1Fanger G.R. Johnson N.L. Johnson G.L. EMBO J. 1997; 16: 4961-4972Crossref PubMed Scopus (253) Google Scholar,17Russell M. Lange-Carter C.A. Johnson G.L. J. Biol. Chem. 1995; 270: 11757-11760Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 18Karandikar M., Xu, S. Cobb M.H. J. Biol. Chem. 2000; 275: 40120-40127Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 19Su Y.C. Han J., Xu, S. Cobb M. Skolnik E.Y. EMBO J. 1997; 16: 1279-1290Crossref PubMed Scopus (214) Google Scholar, 20Xia Y., Wu, Z., Su, B. Murray B. Karin M. Genes Dev. 1998; 12: 3369-3381Crossref PubMed Scopus (174) Google Scholar, 21Xu S. Cobb M.H. J. Biol. Chem. 1997; 272: 32056-32060Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar); consequently, MEKK1 has been proposed to function, like yeast Pbs2p, as a scaffold protein (22Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3593) Google Scholar) . In the present study, we showed that MEKK1 also interacts with FAK in vivo, suggesting that among the subcellular regions to which MEKK1 is localized are focal adhesions. The finding that a membrane-targeted form of FAK (myristoylated FAK) associated with MEKK1 more readily than did wild-type FAK supports that idea. In addition, the finding that EGF enhanced the interaction between endogenous MEKK1 and FAK suggests that the interaction is physiologically relevant. In that regard, FAK is known to associate with the activated EGF receptor signaling complex and to be required for EGF-stimulated cell motility (8Sieg D.J. Hauck C.R. Ilic D. Klingbeil C.K. Schaefer E. Damsky C.H. Schlaepfer D.D. Nat. Cell Biol. 2000; 2: 249-256Crossref PubMed Scopus (1050) Google Scholar). MEKK1 has been shown to be activated in the EGF receptor signaling pathway (1Fanger G.R. Johnson N.L. Johnson G.L. EMBO J. 1997; 16: 4961-4972Crossref PubMed Scopus (253) Google Scholar), and EGF indeed stimulated activation of ERK and JNK in MEFs. EGF might recruit MEKK1 to focal adhesion complexes containing FAK and EGF receptor. The function of MEKK1 within focal adhesions is unclear, although it is known to regulate JNK activation and survival in cells challenged with mild hyperosmolarity and microtubule toxins (2Yujiri T. Fanger G.R. Garrington T.P. Schlesinger T.K. Gibson S. Johnson G.L. J. Biol. Chem. 1999; 274: 12605-12610Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 5Yujiri T. Sather S. Fanger G.R. Johnson G.L. Science. 1998; 282: 1911-1914Crossref PubMed Scopus (280) Google Scholar). The fact that these stimuli elicit changes in cell shape suggests that MEKK1 localized in focal adhesions might be involved in sensing cytoskeletal dynamics.Our data also show that the N terminus of MEKK1 (amino acids 145–443) is important for binding FAK. This region has a proline-rich segment containing putative binding sites for proteins having SH3 domains (23Kyriakis J.M. Avruch J. Physiol. Rev. 2001; 81: 807-869Crossref PubMed Scopus (2851) Google Scholar). FAK does not contain an SH2 or SH3 domain, but it does contain SH2 domain-interacting phosphotyrosines and SH3 domain-interacting proline-rich regions. It may be that one or more other adaptor proteins (e.g. Grb2) mediates the interaction of MEKK1 with FAK. Additional studies will be necessary to precisely define the nature of the interaction between MEKK1 and FAK as well as the physiological significance of that interaction.Integrins are transmembrane proteins that mediate cell adhesion with an extracellular matrix (7Cary L.A. Guan J.L. Front. Biosci. 1999; 4: D102-D113Crossref PubMed Google Scholar). Using FAK-deficient cells, Lebrun et al. (11Lebrun P. Baron V. Hauck C.R. Schlaepfer D.D. Van Obberghen E. J. Biol. Chem. 2000; 275: 38371-38377Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) showed that integrins regulate transcription of theIRS-1 gene, in part via FAK-mediated JNK activation. That report prompted us to examine IRS-1 expression, which led to the finding that levels of IRS-1 protein and mRNA are diminished in MEKK1−/− cells and that the ectopic expression of MEKK1 in MEKK1−/− cells is sufficient to restore expression of the protein. These results demonstrate unequivocally that MEKK1 regulates expression of the IRS-1 message and protein. Interestingly, IRS-1 protein levels were increased as a function of cell density in both MEFs and HEK293T cells. In this case, IRS-1 expression may be up-regulated as the number of cell-to-cell interactions via adhesion molecules increases. Such a situation would be consistent with an earlier report that cell adhesion to the extracellular matrix up-regulates IRS-1 expression (11Lebrun P. Baron V. Hauck C.R. Schlaepfer D.D. Van Obberghen E. J. Biol. Chem. 2000; 275: 38371-38377Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The fact that cell density-dependent IRS-1 expression was also observed in MEKK1−/− MEFs indicates that a MEKK1-independent pathway to IRS-1 expression also exists.In the above mentioned study, Lebrun et al. (11Lebrun P. Baron V. Hauck C.R. Schlaepfer D.D. Van Obberghen E. J. Biol. Chem. 2000; 275: 38371-38377Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) also reported that cell adhesion to fibronectin increases IRS-1 expression and that JNK in not activated in FAK−/− MEFs when integrin engages fibronectin. They therefore concluded that FAK is essential for fibronectin-stimulated JNK activation. In the present study, by contrast, adhesion to fibronectin induced a similar activation of JNK, ERK, and FAK in wild-type and MEKK1−/− MEFs, suggesting that integrin induces IRS-1 expression via an integrin-FAK-JNK pathway that is independent of MEKK1. The signaling molecules involved in the regulation of IRS-1 expression thus remain to be determined. It is possible that, like FAK, MEKK1 is involved in organizing the cortical cytoskeleton and that disorganization due to the loss of MEKK1 participates in the down-regulation of IRS-1 expression.The results of this and our earlier study (6Yujiri T. Ware M. Widmann C. Oyer R. Russell D. Chan E. Zaitsu Y. Clarke P. Tyler K. Oka Y. Fanger G.R. Henson P. Johnson G.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7272-7277Crossref PubMed Scopus (207) Google Scholar) show that, like FAK, MEKK1 positively regulates IRS-1 expression and cell motility. On the other hand, the ectopic expression of IRS-1 in prostate cancer cells that do not express IRS-1 endogenously increased cell adhesion and decreased cell motility (24Reiss K. Wang J.Y. Romano G., Tu, X. Peruzzi F. Baserga R. Oncogene. 2001; 20: 490-500Crossref PubMed Scopus (71) Google Scholar). Although this finding is apparently opposite to ours, it is nevertheless consistent with the idea that IRS-1 plays a key role in regulating cell motility. IRS-1 also mediates various metabolic and growth-promoting actions of insulin and IGF-1. For instance, mice lacking IRS-1 display retardation of somatic growth and enhanced β-cell mass (25Araki E. Lipes M.A. Patti M.E. Bruning J.C. Haag III, B. Johnson R.S. Kahn C.R. Nature. 1994; 372: 186-190Crossref PubMed Scopus (1086) Google Scholar, 26Tamemoto H. Kadowaki T. Tobe K. Yagi T. Sakura H. Hayakawa T. Terauchi Y. Ueki K. Kaburagi Y. Satoh S. Sekihara H. Yoshioka S. Horikoshi H. Furuta Y. Ikawa Y. Kasuga M. Yazaki Y. Aizawa S. Nature. 1994; 372: 182-186Crossref PubMed Scopus (898) Google Scholar); effects on cell migration have not been reported, however.Upon insulin stimulation, IRS-1 interacts with αvβ3 integrins (27Vuori K. Ruoslahti E. Science. 1994; 266: 1576-1578Crossref PubMed Scopus (339) Google Scholar). FAK binds to both IRS-1 and integrins (7Cary L.A. Guan J.L. Front. Biosci. 1999; 4: D102-D113Crossref PubMed Google Scholar, 28Lebrun P. Mothe-Satney I. Delahaye L. Van Obberghen E. Baron V. J. Biol. Chem. 1998; 273: 32244-32253Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), suggesting that IGF-1/insulin and cell adhesion induce the formation of focal adhesion complexes that include IRS-1, FAK, and MEKK1. We found that IGF-1-induced ERK activation was significantly diminished in MEKK1−/− MEFs. The decreased activation of ERK by IGF-1 in MEKK1−/− MEFs was independent of IRS-1 expression. Although MEKK1 contributes significantly to IGF-1-stimulated ERK activation, its effect is only partial. The activation of other MAPK kinase kinases (e.g. Raf-1 and B-Raf) that also activate ERK might partially compensate for the loss of MEKK1. In contrast to ERK activation, IGF-1-induced PI3K/Akt activation was not diminished in MEKK1−/− MEFs. Although IRS-1 is a key mediator of IGF-1/insulin-induced PI3K signaling, in this case other IRS proteins (e.g. IRS-2 which is abundant in MEFs) might have compensated for the diminished levels of IRS-1. In summary, MEKK1 interacts physiologically with FAK and regulates IRS-1 expression, which in turn contributes to the regulation of IGF-1/insulin-induced signaling and cell migration. MEKK11 is a 196-kDa serine-threonine kinase activated in response to a variety of stimuli, including EGF, lysophosphatidic acid, osmotic stress, and microtubule toxins (1Fanger G.R. Johnson N.L. Johnson G.L. EMBO J. 1997; 16: 4961-4972Crossref PubMed Scopus (253) Google Scholar, 2Yujiri T. Fanger G.R. Garrington T.P. Schlesinger T.K. Gibson S. Johnson G.L. J. Biol. Chem. 1999; 274: 12605-12610Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Upon activation, MEKK1 participates in the regulation of the JNK and ERK pathways and is involved in the activation of NF-κB (3Lee F.S. Hagler J. Chen Z.J. Maniatis T. Cell. 1997; 88: 213-222Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar, 4Meyer C.F. Wang X. Chang C. Templeton D. Tan T.H. J. Biol. Chem. 1996; 271: 8971-8976Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 5Yujiri T. Sather S. Fanger G.R. Johnson G.L. Science. 1998; 282: 1911-1914Crossref PubMed Scopus (280) Google Scholar). In addition, MEKK1 senses microtubule integrity, protects cells from committing to apoptosis, and contributes to the migration of fibroblasts and epithelial cells. The phenotype of the MEKK1-null mouse includes an eyelid closure defect that is also seen in mice lacking the EGF receptor and TGF-α (6Yujiri T. Ware M. Widmann C. Oyer R. Russell D. Chan E. Zaitsu Y. Clarke P. Tyler K. Oka Y. Fanger G.R. Henson P. Johnson G.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7272-7277Crossref PubMed Scopus (207) Google Scholar). This suggests the possibility that MEKK1 is required for EGF receptor control of cell migration. Consistent with that idea, overexpression of MEKK1 induces the formation of a large lamellipodia-like structure in epithelial cells (6Yujiri T. Ware M. Widmann C. Oyer R. Russell D. Chan E. Zaitsu Y. Clarke P. Tyler K. Oka Y. Fanger G.R. Henson P. Johnson G.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7272-7277Crossref PubMed Scopus (207) Google Scholar). Still, the mechanism by which MEKK1 influences cell motility remains unclear. FAK is protein tyrosine kinase found at sites of adhesion (7Cary L.A. Guan J.L. Front. Biosci. 1999; 4: D102-D113Crossref PubMed Google Scholar). It is activated by integrin-mediated adhesion and serves as a signaling protein within cytoskeleton-associated networks (7Cary L.A. Guan J.L. Front. Biosci. 1999; 4: D102-D113Crossref PubMed Google Scholar). The Src-family protein tyrosine kinases p130 Cas, Shc, and Grb2 act in concert with FAK to transduce integrin-generated signals to the ERK/JNK MAP kinase cascades (7Cary L.A. Guan J.L. Front. Biosci. 1999; 4: D102-D113Crossref PubMed Google Scholar). Experiments using FAK-deficient cells have established that FAK is essential for integrin-stimulated cell migration and important for linking activation of the PDGF and EGF receptors to the cellular machinery that promotes directed cell migration (8Sieg D.J. Hauck C.R. Ilic D. Klingbeil C.K. Schaefer E. Damsky C.H. Schlaepfer D.D. Nat. Cell Biol. 2000; 2: 249-256Crossref PubMed Scopus (1050) Google Scholar). The fact that MEKK1 is enriched in membranes and colocalizes with α-actinin along actin stress fibers at focal adhesions (9Christerson L.B. Vanderbilt C.A. Cobb M.H. Cell Motil. Cytoskeleton. 1999; 43: 186-198Crossref PubMed Scopus (89) Google Scholar, 10Xu S. Robbins D.J. Christerson L.B. English J.M. Vanderbilt C.A. Cobb M.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5291-5295Crossref PubMed Scopus (122) Google Scholar) prompted us to investigate the possibility that MEKK1 is associated with FAK. Insulin and IGF-1 exert diverse biological effects by binding to and activating their cognate tyrosine kinase receptors. IRS-1 is a major substrate for the insulin and IGF-1 receptors, which rapidly phosphorylate it on multiple tyrosine residues upon ligand binding. Recently, it was reported that targeted disruption of FAK eliminates IRS-1 expression in MEFs and that interactions between cells and the extracellular matrix regulate the transcription of the IRS-1 gene via FAK-mediated JNK activation (11Lebrun P. Baron V. Hauck C.R. Schlaepfer D.D. Van Obberghen E. J. Biol. Chem. 2000; 275: 38371-38377Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Here, we show the following. 1) MEKK1 interacts with FAK in vivo. 2) Like FAK, MEKK1 is required for IRS-1 expression. 3) Targeted disruption of MEKK1 alters IGF-1-induced ERK activation but not PI3K/Akt activation. DISCUSSIONMEKK1 reportedly binds to a number of signaling proteins including Ras, Raf-1, Rac1, cdc42Hs, Nck-interacting kinase, SEK1, and JNK (1Fanger G.R. Johnson N.L. Johnson G.L. EMBO J. 1997; 16: 4961-4972Crossref PubMed Scopus (253) Google Scholar,17Russell M. Lange-Carter C.A. Johnson G.L. J. Biol. Chem. 1995; 270: 11757-11760Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 18Karandikar M., Xu, S. Cobb M.H. J. Biol. Chem. 2000; 275: 40120-40127Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 19Su Y.C. Han J., Xu, S. Cobb M. Skolnik E.Y. EMBO J. 1997; 16: 1279-1290Crossref PubMed Scopus (214) Google Scholar, 20Xia Y., Wu, Z., Su, B. Murray B. Karin M. Genes Dev. 1998; 12: 3369-3381Crossref PubMed Scopus (174) Google Scholar, 21Xu S. Cobb M.H. J. Biol. Chem. 1997; 272: 32056-32060Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar); consequently, MEKK1 has been proposed to function, like yeast Pbs2p, as a scaffold protein (22Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3593) Google Scholar) . In the present study, we showed that MEKK1 also interacts with FAK in vivo, suggesting that among the subcellular regions to which MEKK1 is localized are focal adhesions. The finding that a membrane-targeted form of FAK (myristoylated FAK) associated with MEKK1 more readily than did wild-type FAK supports that idea. In addition, the finding that EGF enhanced the interaction between endogenous MEKK1 and FAK suggests that the interaction is physiologically relevant. In that regard, FAK is known to associate with the activated EGF receptor signaling complex and to be required for EGF-stimulated cell motility (8Sieg D.J. Hauck C.R. Ilic D. Klingbeil C.K. Schaefer E. Damsky C.H. Schlaepfer D.D. Nat. Cell Biol. 2000; 2: 249-256Crossref PubMed Scopus (1050) Google Scholar). MEKK1 has been shown to be activated in the EGF receptor signaling pathway (1Fanger G.R. Johnson N.L. Johnson G.L. EMBO J. 1997; 16: 4961-4972Crossref PubMed Scopus (253) Google Scholar), and EGF indeed stimulated activation of ERK and JNK in MEFs. EGF might recruit MEKK1 to focal adhesion complexes containing FAK and EGF receptor. The function of MEKK1 within focal adhesions is unclear, although it is known to regulate JNK activation and survival in cells challenged with mild hyperosmolarity and microtubule toxins (2Yujiri T. Fanger G.R. Garrington T.P. Schlesinger T.K. Gibson S. Johnson G.L. J. Biol. Chem. 1999; 274: 12605-12610Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 5Yujiri T. Sather S. Fanger G.R. Johnson G.L. Science. 1998; 282: 1911-1914Crossref PubMed Scopus (280) Google Scholar). The fact that these stimuli elicit changes in cell shape suggests that MEKK1 localized in focal adhesions might be involved in sensing cytoskeletal dynamics.Our data also show that the N terminus of MEKK1 (amino acids 145–443) is important for binding FAK. This region has a proline-rich segment containing putative binding sites for proteins having SH3 domains (23Kyriakis J.M. Avruch J. Physiol. Rev. 2001; 81: 807-869Crossref PubMed Scopus (2851) Google Scholar). FAK does not contain an SH2 or SH3 domain, but it does contain SH2 domain-interacting phosphotyrosines and SH3 domain-interacting proline-rich regions. It may be that one or more other adaptor proteins (e.g. Grb2) mediates the interaction of MEKK1 with FAK. Additional studies will be necessary to precisely define the nature of the interaction between MEKK1 and FAK as well as the physiological significance of that interaction.Integrins are transmembrane proteins that mediate cell adhesion with an extracellular matrix (7Cary L.A. Guan J.L. Front. Biosci. 1999; 4: D102-D113Crossref PubMed Google Scholar). Using FAK-deficient cells, Lebrun et al. (11Lebrun P. Baron V. Hauck C.R. Schlaepfer D.D. Van Obberghen E. J. Biol. Chem. 2000; 275: 38371-38377Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) showed that integrins regulate transcription of theIRS-1 gene, in part via FAK-mediated JNK activation. That report prompted us to examine IRS-1 expression, which led to the finding that levels of IRS-1 protein and mRNA are diminished in MEKK1−/− cells and that the ectopic expression of MEKK1 in MEKK1−/− cells is sufficient to restore expression of the protein. These results demonstrate unequivocally that MEKK1 regulates expression of the IRS-1 message and protein. Interestingly, IRS-1 protein levels were increased as a function of cell density in both MEFs and HEK293T cells. In this case, IRS-1 expression may be up-regulated as the number of cell-to-cell interactions via adhesion molecules increases. Such a situation would be consistent with an earlier report that cell adhesion to the extracellular matrix up-regulates IRS-1 expression (11Lebrun P. Baron V. Hauck C.R. Schlaepfer D.D. Van Obberghen E. J. Biol. Chem. 2000; 275: 38371-38377Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The fact that cell density-dependent IRS-1 expression was also observed in MEKK1−/− MEFs indicates that a MEKK1-independent pathway to IRS-1 expression also exists.In the above mentioned study, Lebrun et al. (11Lebrun P. Baron V. Hauck C.R. Schlaepfer D.D. Van Obberghen E. J. Biol. Chem. 2000; 275: 38371-38377Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) also reported that cell adhesion to fibronectin increases IRS-1 expression and that JNK in not activated in FAK−/− MEFs when integrin engages fibronectin. They therefore concluded that FAK is essential for fibronectin-stimulated JNK activation. In the present study, by contrast, adhesion to fibronectin induced a similar activation of JNK, ERK, and FAK in wild-type and MEKK1−/− MEFs, suggesting that integrin induces IRS-1 expression via an integrin-FAK-JNK pathway that is independent of MEKK1. The signaling molecules involved in the regulation of IRS-1 expression thus remain to be determined. It is possible that, like FAK, MEKK1 is involved in organizing the cortical cytoskeleton and that disorganization due to the loss of MEKK1 participates in the down-regulation of IRS-1 expression.The results of this and our earlier study (6Yujiri T. Ware M. Widmann C. Oyer R. Russell D. Chan E. Zaitsu Y. Clarke P. Tyler K. Oka Y. Fanger G.R. Henson P. Johnson G.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7272-7277Crossref PubMed Scopus (207) Google Scholar) show that, like FAK, MEKK1 positively regulates IRS-1 expression and cell motility. On the other hand, the ectopic expression of IRS-1 in prostate cancer cells that do not express IRS-1 endogenously increased cell adhesion and decreased cell motility (24Reiss K. Wang J.Y. Romano G., Tu, X. Peruzzi F. Baserga R. Oncogene. 2001; 20: 490-500Crossref PubMed Scopus (71) Google Scholar). Although this finding is apparently opposite to ours, it is nevertheless consistent with the idea that IRS-1 plays a key role in regulating cell motility. IRS-1 also mediates various metabolic and growth-promoting actions of insulin and IGF-1. For instance, mice lacking IRS-1 display retardation of somatic growth and enhanced β-cell mass (25Araki E. Lipes M.A. Patti M.E. Bruning J.C. Haag III, B. Johnson R.S. Kahn C.R. Nature. 1994; 372: 186-190Crossref PubMed Scopus (1086) Google Scholar, 26Tamemoto H. Kadowaki T. Tobe K. Yagi T. Sakura H. Hayakawa T. Terauchi Y. Ueki K. Kaburagi Y. Satoh S. Sekihara H. Yoshioka S. Horikoshi H. Furuta Y. Ikawa Y. Kasuga M. Yazaki Y. Aizawa S. Nature. 1994; 372: 182-186Crossref PubMed Scopus (898) Google Scholar); effects on cell migration have not been reported, however.Upon insulin stimulation, IRS-1 interacts with αvβ3 integrins (27Vuori K. Ruoslahti E. Science. 1994; 266: 1576-1578Crossref PubMed Scopus (339) Google Scholar). FAK binds to both IRS-1 and integrins (7Cary L.A. Guan J.L. Front. Biosci. 1999; 4: D102-D113Crossref PubMed Google Scholar, 28Lebrun P. Mothe-Satney I. Delahaye L. Van Obberghen E. Baron V. J. Biol. Chem. 1998; 273: 32244-32253Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), suggesting that IGF-1/insulin and cell adhesion induce the formation of focal adhesion complexes that include IRS-1, FAK, and MEKK1. We found that IGF-1-induced ERK activation was significantly diminished in MEKK1−/− MEFs. The decreased activation of ERK by IGF-1 in MEKK1−/− MEFs was independent of IRS-1 expression. Although MEKK1 contributes significantly to IGF-1-stimulated ERK activation, its effect is only partial. The activation of other MAPK kinase kinases (e.g. Raf-1 and B-Raf) that also activate ERK might partially compensate for the loss of MEKK1. In contrast to ERK activation, IGF-1-induced PI3K/Akt activation was not diminished in MEKK1−/− MEFs. Although IRS-1 is a key mediator of IGF-1/insulin-induced PI3K signaling, in this case other IRS proteins (e.g. IRS-2 which is abundant in MEFs) might have compensated for the diminished levels of IRS-1. In summary, MEKK1 interacts physiologically with FAK and regulates IRS-1 expression, which in turn contributes to the regulation of IGF-1/insulin-induced signaling and cell migration. MEKK1 reportedly binds to a number of signaling proteins including Ras, Raf-1, Rac1, cdc42Hs, Nck-interacting kinase, SEK1, and JNK (1Fanger G.R. Johnson N.L. Johnson G.L. EMBO J. 1997; 16: 4961-4972Crossref PubMed Scopus (253) Google Scholar,17Russell M. Lange-Carter C.A. Johnson G.L. J. Biol. Chem. 1995; 270: 11757-11760Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 18Karandikar M., Xu, S. Cobb M.H. J. Biol. Chem. 2000; 275: 40120-40127Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 19Su Y.C. Han J., Xu, S. Cobb M. Skolnik E.Y. EMBO J. 1997; 16: 1279-1290Crossref PubMed Scopus (214) Google Scholar, 20Xia Y., Wu, Z., Su, B. Murray B. Karin M. Genes Dev. 1998; 12: 3369-3381Crossref PubMed Scopus (174) Google Scholar, 21Xu S. Cobb M.H. J. Biol. Chem. 1997; 272: 32056-32060Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar); consequently, MEKK1 has been proposed to function, like yeast Pbs2p, as a scaffold protein (22Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3593) Google Scholar) . In the present study, we showed that MEKK1 also interacts with FAK in vivo, suggesting that among the subcellular regions to which MEKK1 is localized are focal adhesions. The finding that a membrane-targeted form of FAK (myristoylated FAK) associated with MEKK1 more readily than did wild-type FAK supports that idea. In addition, the finding that EGF enhanced the interaction between endogenous MEKK1 and FAK suggests that the interaction is physiologically relevant. In that regard, FAK is known to associate with the activated EGF receptor signaling complex and to be required for EGF-stimulated cell motility (8Sieg D.J. Hauck C.R. Ilic D. Klingbeil C.K. Schaefer E. Damsky C.H. Schlaepfer D.D. Nat. Cell Biol. 2000; 2: 249-256Crossref PubMed Scopus (1050) Google Scholar). MEKK1 has been shown to be activated in the EGF receptor signaling pathway (1Fanger G.R. Johnson N.L. Johnson G.L. EMBO J. 1997; 16: 4961-4972Crossref PubMed Scopus (253) Google Scholar), and EGF indeed stimulated activation of ERK and JNK in MEFs. EGF might recruit MEKK1 to focal adhesion complexes containing FAK and EGF receptor. The function of MEKK1 within focal adhesions is unclear, although it is known to regulate JNK activation and survival in cells challenged with mild hyperosmolarity and microtubule toxins (2Yujiri T. Fanger G.R. Garrington T.P. Schlesinger T.K. Gibson S. Johnson G.L. J. Biol. Chem. 1999; 274: 12605-12610Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 5Yujiri T. Sather S. Fanger G.R. Johnson G.L. Science. 1998; 282: 1911-1914Crossref PubMed Scopus (280) Google Scholar). The fact that these stimuli elicit changes in cell shape suggests that MEKK1 localized in focal adhesions might be involved in sensing cytoskeletal dynamics. Our data also show that the N terminus of MEKK1 (amino acids 145–443) is important for binding FAK. This region has a proline-rich segment containing putative binding sites for proteins having SH3 domains (23Kyriakis J.M. Avruch J. Physiol. Rev. 2001; 81: 807-869Crossref PubMed Scopus (2851) Google Scholar). FAK does not contain an SH2 or SH3 domain, but it does contain SH2 domain-interacting phosphotyrosines and SH3 domain-interacting proline-rich regions. It may be that one or more other adaptor proteins (e.g. Grb2) mediates the interaction of MEKK1 with FAK. Additional studies will be necessary to precisely define the nature of the interaction between MEKK1 and FAK as well as the physiological significance of that interaction. Integrins are transmembrane proteins that mediate cell adhesion with an extracellular matrix (7Cary L.A. Guan J.L. Front. Biosci. 1999; 4: D102-D113Crossref PubMed Google Scholar). Using FAK-deficient cells, Lebrun et al. (11Lebrun P. Baron V. Hauck C.R. Schlaepfer D.D. Van Obberghen E. J. Biol. Chem. 2000; 275: 38371-38377Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) showed that integrins regulate transcription of theIRS-1 gene, in part via FAK-mediated JNK activation. That report prompted us to examine IRS-1 expression, which led to the finding that levels of IRS-1 protein and mRNA are diminished in MEKK1−/− cells and that the ectopic expression of MEKK1 in MEKK1−/− cells is sufficient to restore expression of the protein. These results demonstrate unequivocally that MEKK1 regulates expression of the IRS-1 message and protein. Interestingly, IRS-1 protein levels were increased as a function of cell density in both MEFs and HEK293T cells. In this case, IRS-1 expression may be up-regulated as the number of cell-to-cell interactions via adhesion molecules increases. Such a situation would be consistent with an earlier report that cell adhesion to the extracellular matrix up-regulates IRS-1 expression (11Lebrun P. Baron V. Hauck C.R. Schlaepfer D.D. Van Obberghen E. J. Biol. Chem. 2000; 275: 38371-38377Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The fact that cell density-dependent IRS-1 expression was also observed in MEKK1−/− MEFs indicates that a MEKK1-independent pathway to IRS-1 expression also exists. In the above mentioned study, Lebrun et al. (11Lebrun P. Baron V. Hauck C.R. Schlaepfer D.D. Van Obberghen E. J. Biol. Chem. 2000; 275: 38371-38377Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) also reported that cell adhesion to fibronectin increases IRS-1 expression and that JNK in not activated in FAK−/− MEFs when integrin engages fibronectin. They therefore concluded that FAK is essential for fibronectin-stimulated JNK activation. In the present study, by contrast, adhesion to fibronectin induced a similar activation of JNK, ERK, and FAK in wild-type and MEKK1−/− MEFs, suggesting that integrin induces IRS-1 expression via an integrin-FAK-JNK pathway that is independent of MEKK1. The signaling molecules involved in the regulation of IRS-1 expression thus remain to be determined. It is possible that, like FAK, MEKK1 is involved in organizing the cortical cytoskeleton and that disorganization due to the loss of MEKK1 participates in the down-regulation of IRS-1 expression. The results of this and our earlier study (6Yujiri T. Ware M. Widmann C. Oyer R. Russell D. Chan E. Zaitsu Y. Clarke P. Tyler K. Oka Y. Fanger G.R. Henson P. Johnson G.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7272-7277Crossref PubMed Scopus (207) Google Scholar) show that, like FAK, MEKK1 positively regulates IRS-1 expression and cell motility. On the other hand, the ectopic expression of IRS-1 in prostate cancer cells that do not express IRS-1 endogenously increased cell adhesion and decreased cell motility (24Reiss K. Wang J.Y. Romano G., Tu, X. Peruzzi F. Baserga R. Oncogene. 2001; 20: 490-500Crossref PubMed Scopus (71) Google Scholar). Although this finding is apparently opposite to ours, it is nevertheless consistent with the idea that IRS-1 plays a key role in regulating cell motility. IRS-1 also mediates various metabolic and growth-promoting actions of insulin and IGF-1. For instance, mice lacking IRS-1 display retardation of somatic growth and enhanced β-cell mass (25Araki E. Lipes M.A. Patti M.E. Bruning J.C. Haag III, B. Johnson R.S. Kahn C.R. Nature. 1994; 372: 186-190Crossref PubMed Scopus (1086) Google Scholar, 26Tamemoto H. Kadowaki T. Tobe K. Yagi T. Sakura H. Hayakawa T. Terauchi Y. Ueki K. Kaburagi Y. Satoh S. Sekihara H. Yoshioka S. Horikoshi H. Furuta Y. Ikawa Y. Kasuga M. Yazaki Y. Aizawa S. Nature. 1994; 372: 182-186Crossref PubMed Scopus (898) Google Scholar); effects on cell migration have not been reported, however. Upon insulin stimulation, IRS-1 interacts with αvβ3 integrins (27Vuori K. Ruoslahti E. Science. 1994; 266: 1576-1578Crossref PubMed Scopus (339) Google Scholar). FAK binds to both IRS-1 and integrins (7Cary L.A. Guan J.L. Front. Biosci. 1999; 4: D102-D113Crossref PubMed Google Scholar, 28Lebrun P. Mothe-Satney I. Delahaye L. Van Obberghen E. Baron V. J. Biol. Chem. 1998; 273: 32244-32253Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), suggesting that IGF-1/insulin and cell adhesion induce the formation of focal adhesion complexes that include IRS-1, FAK, and MEKK1. We found that IGF-1-induced ERK activation was significantly diminished in MEKK1−/− MEFs. The decreased activation of ERK by IGF-1 in MEKK1−/− MEFs was independent of IRS-1 expression. Although MEKK1 contributes significantly to IGF-1-stimulated ERK activation, its effect is only partial. The activation of other MAPK kinase kinases (e.g. Raf-1 and B-Raf) that also activate ERK might partially compensate for the loss of MEKK1. In contrast to ERK activation, IGF-1-induced PI3K/Akt activation was not diminished in MEKK1−/− MEFs. Although IRS-1 is a key mediator of IGF-1/insulin-induced PI3K signaling, in this case other IRS proteins (e.g. IRS-2 which is abundant in MEFs) might have compensated for the diminished levels of IRS-1. In summary, MEKK1 interacts physiologically with FAK and regulates IRS-1 expression, which in turn contributes to the regulation of IGF-1/insulin-induced signaling and cell migration. We thank Drs. S. Hanks and S. Gutkind for the generous gift of plasmids. We also thank Dr. G. L. Johnson for valuable discussions and Yukari Kora and Atsuko Tanimura for technical assistance.
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