Correspondence to K. Haga, Department of Neurochemistry, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Tokyo 1130033, Japan
GTP-binding-protein-coupled receptor kinase 2 (GRK2) binds and phosphorylates tubulin
Article first published online: 25 DEC 2001
European Journal of Biochemistry
Volume 255, Issue 2, pages 363–368, July (II) 1998
How to Cite
Haga, K., Ogawa, H., Haga, T. and Murofushi, H. (1998), GTP-binding-protein-coupled receptor kinase 2 (GRK2) binds and phosphorylates tubulin. European Journal of Biochemistry, 255: 363–368. doi: 10.1046/j.1432-1327.1998.2550363.x
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Abbreviations. m2 receptor, muscarinic acetylcholine receptor m2 subtype; I3-del m2 receptor, m2 receptor mutant which lacks the central part of the third intracellular loop; G protein, GTP-binding regulatory protein; GRK2, G-protein-coupled receptor kinase 2 : GST, glutathione S-transferase; GRK2C-GST, GST fusion protein containing the carboxyterminal peptide of GRK2; GraP-DH, glyceraldehyde-3-phosphate dehydrogenase; I3-GST, GST fusion protein containing a peptide corresponding to the central part of the third intracellular loop of m2 receptor; MAP, microtubule-associated protein; PH, pleckstrin homology.
- Issue published online: 25 DEC 2001
- Article first published online: 25 DEC 2001
- (Received 9 February/14 April 1998)
- Cited By
- G-protein-coupled receptor;
- G-protein-coupled receptor kinase;
- muscarinic acetylcholine receptor;
- protein phosphorylation;
Tubulin was found to bind to a glutathione S-transferase fusion protein containing the carboxy-terminal domain of GTP-binding-protein-coupled receptor kinase 2 (GRK2) (residues 467−689), which is known to contain a pleckstrin homology site and to bind GTP-binding protein βγ subunits. The binding of tubulin to the fusion protein was not affected by GTP-binding protein βγ subunits, indicating that tubulin and βγ subunits bind GRK2 independently. Western-blotting analysis with anti-GRK2 Ig indicated that GRK2 was copurified with tubulin through the polymerization-depolymerization procedure. Tubulin was phosphorylated by GRK2, in contrast with the facts that the known substrates of GRK2 are restricted to activated forms of GTP-binding-protein-coupled receptors and that tubulin is a poor substrate for most kinases. GRK2 did not phosphorylate microtubule-associated proteins (MAPs), under conditions where MAPs were well phosphorylated by endogenous kinases copurified with tubulin. The Km for tubulin was estimated to be 3 μM, and 1.3 mol phosphate/tubulin dimer was incorporated. The phosphorylation of tubulin was stimulated by βγ subunits and agonist-bound muscarinic receptors. Phosphorylated tubulin could be polymerized into microtubules, and polymerized tubulin was also phosphorylated by GRK2.