Tubulin-associated calmodulin-dependent kinase: evidence for an endogenous complex of tubulin with a calcium-calmodulin-dependent kinase.
A Ca2+ -calmodulin kinase that phosphorylates tubulin and microtubule-associated proteins as major substrates has been purified and characterized from brain cytoplasm. It is important to determine if cytoskeletal proteins are major natural substrates for this kinase system. This report demonstrates that a significant fraction of brain cytosolic calmodulin-dependent kinase activity exists in tight association with tubulin in the form of a stable complex. The tubulin-calmodulin kinase complex displayed an apparent molecular weight on gel filtration of approximately 1.8 X 10(6) daltons. The specific activity of tubulin kinase in the complex was enriched over 20-fold in comparison with brain cytosol. Although purified tubulin alone did not adhere to a calmodulin column, the tubulin associated with the calmodulin kinase complex did bind specifically to the calmodulin affinity resin. The kinase activity was shown to be tightly associated in complex with tubulin by (1) copurification, (2) isolation on gel filtration chromatography, (3) isolation on ion-exchange chromatography, and (4) binding to calmodulin. The kinase complexed with tubulin was identical to the previously purified kinase as judged by several criteria including (1) subunit molecular weights, (2) isoelectric points, (3) autophosphorylation characteristics, (4) calmodulin binding properties, (5) kinetic parameters of tubulin phosphorylation, (6) phosphoamino acid phosphorylation sites on alpha- and beta-tubulin, and (7) identical subunit 125I-tryptic peptide maps. The results indicate that a significant fraction of this previously purified calmodulin kinase is endogenously associated with tubulin in brain cytoplasm and may play a role in mediating some of the effects of calcium on neuronal function.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002147:Calmodulin / Q000378:metabolism; D002148:Calmodulin-Binding Proteins; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D046911:Macromolecular Substances; D008970:Molecular Weight; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D014404:Tubulin / Q000378:metabolism*
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D046911:Macromolecular Substances; D014404:Tubulin; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases