Characterization of Graf, the GTPase-activating Protein for Rho Associated with Focal Adhesion Kinase
PHOSPHORYLATION AND POSSIBLE REGULATION BY MITOGEN-ACTIVATED PROTEIN KINASE*
- Joan M. Taylor‡§,
- Jeffrey D. Hildebrand‡¶,
- Christopher P. Mack‖,
- Michael E. Cox‡ and
- J. Thomas Parsons‡**
- From the ‡Department of Microbiology and‖Department of Molecular and Cellular Physiology, Health Sciences Center, University of Virginia, Charlottesville, Virginia 22908
Abstract
Graf is a GTPase-activating protein for Rho that interacts with focal adhesion kinase and co-localizes with the actin cytoskeleton (Hildebrand, J. D., Taylor, J. M. and Parsons, J. T. (1996) Mol. Cell. Biol. 16, 3169–3178). We examined the expression and regulation of Graf as a prelude to understanding the role of Graf in mediating signal transductionin vivo. We demonstrated that Graf is a ubiquitously expressed 95-kDa protein with high levels observed in heart and brain and cells derived from these tissues. Stimulation of PC12 cells with epidermal growth factor or nerve growth factor induced a phosphatase-reversible mobility shift upon gel electrophoresis, indicative of phosphorylation. In vitro, purified mitogen-activated protein (MAP) kinase catalyzed the phosphorylation of Graf on serine 510, suggesting that Graf phosphorylation may be mediated through MAP kinase signaling. In addition, the mutation of serine 510 to alanine inhibited the epidermal growth factor-induced mobility shift of mutant Graf protein in vivo, consistent with serine 510 being the site of in vivo phosphorylation. Based on these data we suggest that phosphorylation of Graf by MAP kinase or related kinases may be a mechanism by which growth factor signaling modulates Rho-mediated cytoskeletal changes in PC12 and perhaps other cells.
Footnotes
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↵* This work was supported in part by Grants CA29243 and CA40042 from the DHHS-NCI and Grant 4491 from the Council for Tobacco Research, Inc.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.
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↵§ Supported by National Research Service Award H1-F32- GM18297-01.
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↵¶ Present address: Dept. of Molecular Medicine, Fred Hutchinson Cancer Research Center, Seattle, WA 98104.
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↵** To whom correspondence should be addressed: Dept. of Microbiology, Box 441, Health Sciences Center, University of Virginia, Charlottesville, VA 22908. Tel.: 804-924-5395; Fax: 804-982-1071; E-mail: jtp{at}virginia.edu.
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↵1 The abbreviations used are: smw, small molecular weight; MAP, mitogen-activated protein; MEK, MAP kinase kinase or Erk kinase; NGF, nerve growth factor; EGF, epidermal growth factor; GEF, guanine nucleotide exchange factors; GAP, GTPase-activating protein; SH3, Src homology 3 domain; PCR, polymerase chain reaction; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; TBS, tris-buffered saline; RIPA, radioimmunoprecipitation assay buffer; Ab, antibody; mAb, monoclonal antibody; p42MAPK, 42-kDa isoform of MAP kinase; kb, kilobase(s).
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↵2 J. M. Taylor and J. T. Parsons, unpublished observation.
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↵3 W. Xiong and J. T. Parsons, manuscript in preparation.
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- Received November 12, 1997.
- Revision received January 21, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
