Binding and phosphorylation of tubulin by G protein-coupled receptor kinases

Although the beta-adrenergic receptor kinase (beta ARK) mediates agonist-dependent phosphorylation and desensitization of G protein-coupled receptors, recent studies suggest additional cellular functions. During our attempts to identify novel beta ARK interacting proteins, we found that the cytoskeletal protein tubulin could specifially bind to a beta ARR-coupled affinity column. In vitro analysis demonstrated that beta ARK and G protein-coupled receptor kinase-5 (GRK5) mere able to stoichiometrically phosphorylate purified tubulin dimers with a preference for beta-tubulin and, under certain conditions, the beta III-isotype. Examination of the GRK/tubulin binding characteristics revealed that tubulin dimers and assembled microtubules bind GRKs, whereas the catalytic domain of beta ARK contains the primary tubulin binding determinants. In vivo interaction of GRK and tubulin was suggested by the following: (i) co-purification of beta ARK with tubulin from brain tissue; (ii) co-immunoprecipitation of beta ARR and tubulin from COS-l cells; and (iii) co-localization of beta ARK and GRK5 with microtubule structures in COS-l cells. In addition, GRK-phosphorylated tubulin was found preferentially associated with the microtubule fraction during in vitro assembly assays suggesting potential functional significance. These results suggest a novel link between the cytoskeleton and GRKs that may be important for regulating GRK and/or tubulin function.