A Rho-associated Protein Kinase, ROKα, Binds Insulin Receptor Substrate-1 and Modulates Insulin Signaling*
- From the Ottawa Civic Hospital Loeb Research Institute, Ottawa Civic Hospital, and the Departments of ‡Biochemistry,§Medicine, and ¶Obstetrics and Gynecology, University of Ottawa, Ottawa K1Y 4E9, Canada
Abstract
Insulin receptor substrate-1 (IRS-1) is phosphorylated on multiple tyrosine residues by ligand-activated insulin receptors. These tyrosine phosphorylation sites serve to dock several Src homology 2-containing signaling proteins. In addition, IRS-1 contains a pleckstrin homology domain and a phosphotyrosine binding domain (PTB) implicated in protein-protein and protein-lipid interactions. In a yeast two-hybrid screening using XenopusIRS-1 (xIRS-1) pleckstrin homology-PTB domains as bait, we identified aXenopus homolog of Rho-associated kinase α (xROKα) as a potential xIRS-1-binding protein. The original clone contained the carboxyl terminus of xROKα (xROK-C) including the putative Rho binding domain but lacking the amino-terminal kinase domain. Further analyses in yeast indicated that xROK-C bound to the putative PTB domain of xIRS-1. Binding of xROK-C to xIRS-1 was confirmed inXenopus oocytes after microinjection of mRNA corresponding to xROK-C. Furthermore, microinjection of xROK-C mRNA inhibited insulin-induced mitogen-activated protein kinase activation with a concomitant inhibition of oocyte maturation. In contrast, microinjection of xROK-C mRNA did not inhibit mitogen-activated protein kinase activation or oocyte maturation induced by progesterone or by microinjection of viral Ras (v-Ras) mRNA. These results suggest that xROKα may play a role in insulin signaling via a direct interaction with xIRS-1.
Footnotes
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↵* This study was supported by operating grants from the National Cancer Institute of Canada and the Cancer Research Society of Canada (to X. J. L.).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) AF037073.
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↵‖ Scholar of the Medical Research Council of Canada. To whom correspondence should be addressed: Ottawa Civic Hospital Loeb Research Institute, Ottawa Civic Hospital, 1053 Carling Ave., Ottawa, K1Y 4E9, Canada. Tel.: 613-798-5555 (ext. 7752); Fax: 613-761-5411 or 761-5365; E-mail: johne{at}civich.ottawa.on.ca.
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↵1 The abbreviations used are: IRS-1, insulin receptor substrate-1; PH, pleckstrin homology; PTB, phosphotyrosine binding; SAIN, Shc and IRS-1 NPXpY binding domain; ROK, Rho-associated protein kinase; GVBD, germinal vesicle breakdown; PCR, polymerase chain reaction; MT, Myc tag; MBP, myelin basic protein; PAGE, polyacrylamide gel electrophoresis; MAP kinase, mitogen-activated protein kinase; CRD, cysteine-rich domain.
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↵2 R. Booth and X. J. Liu, unpublished observations.
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↵3 N. Ohan and X.-J. Liu, unpublished observations.
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- Received October 31, 1997.
- Revision received December 8, 1997.
- The American Society for Biochemistry and Molecular Biology, Inc.
