The G Protein-coupled Receptor Kinase 2 Is a Microtubule-associated Protein Kinase That Phosphorylates Tubulin*
- Julie A. Pitcher,
- Randy A. Hall,
- Yehia Daaka,
- Jie Zhang‡,
- Stephen S. G. Ferguson‡§,
- Susan Hester¶,
- Sara Miller¶,
- Marc G. Caron‡,
- Robert J. Lefkowitz‖ and
- Larry S. Barak‡
- From the Howard Hughes Medical Institute Laboratories and Departments of Medicine and Biochemistry, ‡Cell Biology, and ¶Microbiology, Duke University Medical Center, Durham, North Carolina 27710
Abstract
The G protein-coupled receptor kinase 2 (GRK2) is a serine/threonine kinase that phosphorylates and desensitizes agonist-occupied G protein-coupled receptors (GPCRs). Here we demonstrate that GRK2 is a microtubule-associated protein and identify tubulin as a novel GRK2 substrate. GRK2 is associated with microtubules purified from bovine brain, forms a complex with tubulin in cell extracts, and colocalizes with tubulin in living cells. Furthermore, an endogenous tubulin kinase activity that copurifies with microtubules has properties similar to GRK2 and is inhibited by anti-GRK2 monoclonal antibodies. Indeed, GRK2 phosphorylates tubulinin vitro with kinetic parameters very similar to those for phosphorylation of the agonist-occupied β2-adrenergic receptor, suggesting a functionally relevant role for this phosphorylation event. In a cellular environment, agonist occupancy of GPCRs, which leads to recruitment of GRK2 to the plasma membrane and its subsequent activation, promotes GRK2-tubulin complex formation and tubulin phosphorylation. These findings suggest a novel role for GRK2 as a GPCR signal transducer mediating the effects of GPCR activation on the cytoskeleton.
Footnotes
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↵* This work was supported by National Institutes of Health Grants HL16037 (to R. J. L), NS 19576 (to M. G. C.), and HL 03422 (to L. S. B.) and a Bristol Myers Squibb unrestricted grant award (to M. G. 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.
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↵§ Recipient of a Michael Smith Postdoctoral Fellowship from the Medical Research Council Canada. Present address: The John P. Robarts Research Institute, P.O. Box 5015, 100 Perth Dr., London, Ontario N6A 5K8, Canada.
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↵‖ To whom correspondence should be addressed: Duke University Medical Center, Box 3281, Durham, NC 27710. Tel.: 919-684-2974; Fax: 919-684-8875.
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↵1 The abbreviations used are: GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; β-AR, β2-adrenergic receptor; HEK, human embryonic kidney; PBS, phosphate-buffered saline; Gβγ, the βγ subunits of heterotrimeric G proteins; GFP, green fluorescent protein; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Pipes, piperazine-N,N′-bis(2-ethanesulfonic acid).
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- Received October 8, 1997.
- Revision received February 9, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
