Binding and phosphorylation of tubulin by G protein-coupled receptor kinases.
Although the beta-adrenergic receptor kinase (betaARK) mediates agonist-dependent phosphorylation and desensitization of G protein-coupled receptors, recent studies suggest additional cellular functions. During our attempts to identify novel betaARK interacting proteins, we found that the cytoskeletal protein tubulin could specifically bind to a betaARK-coupled affinity column. In vitro analysis demonstrated that betaARK and G protein-coupled receptor kinase-5 (GRK5) were able to stoichiometrically phosphorylate purified tubulin dimers with a preference for beta-tubulin and, under certain conditions, the betaIII-isotype. Examination of the GRK/tubulin binding characteristics revealed that tubulin dimers and assembled microtubules bind GRKs, whereas the catalytic domain of betaARK contains the primary tubulin binding determinants. In vivo interaction of GRK and tubulin was suggested by the following: (i) co-purification of betaARK with tubulin from brain tissue; (ii) co-immunoprecipitation of betaARK and tubulin from COS-1 cells; and (iii) co-localization of betaARK and GRK5 with microtubule structures in COS-1 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.
Carman
C V
CV
Departments of Biochemistry & Molecular Pharmacology and Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Som
T
T
Kim
C M
CM
Benovic
J L
JL
D000818:Animals; D001921:Brain / Q000201:enzymology; D019556:COS Cells; D002417:Cattle; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003599:Cytoskeleton / Q000502:physiology; D019281:Dimerization; D005455:Fluorescent Antibody Technique; D054774:G-Protein-Coupled Receptor Kinase 5; D019204:GTP-Binding Proteins / Q000502:physiology; D007700:Kinetics; D008870:Microtubules / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding / Q000502:physiology; D017346:Protein Serine-Threonine Kinases; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D014162:Transfection / Q000235:genetics; D014404:Tubulin / Q000378:metabolism*; D051552:beta-Adrenergic Receptor Kinases
D014404:Tubulin; D020794:Receptor Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D054774:G-Protein-Coupled Receptor Kinase 5; D019204:GTP-Binding Proteins