Identification of sites of phosphorylation by G-protein-coupled receptor kinase 2 in beta-tubulin.
G-protein-coupled receptor kinase 2 (GRK2) is known to specifically phosphorylate the agonist-bound forms of G-protein-coupled receptors (GPCRs). This strict specificity is due at least partly to activation of GRK2 by agonist-bound GPCRs, in which basic residues in intracellular regions adjacent to transmembrane segments are thought to be involved. Tubulin was found to be phosphorylated by GRK2, but it remains unknown if tubulin can also serve as both a substrate and an activator for GRK2. Purified tubulin, phosphorylated by GRK2, was subjected to biochemical analysis, and the phosphorylation sites in beta-tubulin were determined to be Thr409 and Ser420. In addition, the Ser444 in beta III-tubulin was also indicated to be phosphorylated by GRK2. The phosphorylation sites in tubulin for GRK2 reside in the C-terminal domain of beta-tubulin, which is on the outer surface of microtubules. Pretreatment of tubulin with protein phosphatase type-2A (PP2A) resulted in a twofold increase in the phosphorylation of tubulin by GRK2. These results suggest that tubulin is phosphorylated in situ probably by GRK2 and that the phosphorylation may affect the interaction of microtubules with microtubule-associated proteins. A GST fusion protein of a C-terminal region of beta I-tubulin (393-445 residues), containing 19 acidic residues but only one basic residue, was found to be a good substrate for GRK2, like full-length beta-tubulin. These results, together with the finding that GRK2 may phosphorylate synuclein and phosducin in their acidic domains, indicate that some proteins with very acidic regions but without basic activation domains could serve as substrates for GRK2.
Yoshida
Norihiro
N
Department of Neurochemistry, Faculty of Medicine, University of Tokyo, Tokyo, Japan.
Haga
Kazuko
K
Haga
Tatsuya
T
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D014404:Tubulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051552:beta-Adrenergic Receptor Kinases
D011993:Recombinant Fusion Proteins; D014404:Tubulin; D013912:Threonine; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases