GTP-binding-protein-coupled receptor kinase 2 (GRK2) binds and phosphorylates tubulin.
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 betagamma subunits. The binding of tubulin to the fusion protein was not affected by GTP-binding protein betagamma subunits, indicating that tubulin and betagamma 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 microM, and 1.3 mol phosphate/tubulin dimer was incorporated. The phosphorylation of tubulin was stimulated by betagamma subunits and agonist-bound muscarinic receptors. Phosphorylated tubulin could be polymerized into microtubules, and polymerized tubulin was also phosphorylated by GRK2.
Haga
K
K
Department of Neurochemistry, Faculty of Medicine, University of Tokyo, Japan. haga@m.u-tokyo.ac.jp
Ogawa
H
H
Haga
T
T
Murofushi
H
H
D000818:Animals; D001285:Atropine / Q000494:pharmacology; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005982:Glutathione Transferase; D006493:Heparin / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D010766:Phosphorylation; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013552:Swine; D014404:Tubulin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D051552:beta-Adrenergic Receptor Kinases
D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D001285:Atropine; D002217:Carbachol; D006493:Heparin; D005982:Glutathione Transferase; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D019204:GTP-Binding Proteins