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Identification of Endogenous Calmodulin‐Dependent Kinase and Calmodulin‐Binding Proteins in Cold‐Stable Microtubule Preparations from Rat Brain

1985; Wiley; Volume: 44; Issue: 5 Linguagem: Inglês

10.1111/j.1471-4159.1985.tb08796.x

1471-4159

Roy Edward Larson, James R. Goldenring, Mary Lou Vallano, Robert J. DeLorenzo,

Bioinformatics and Genomic Networks

Abstract: Calmodulin‐dependent kinase activity was investigated in cold‐stable microtubule fractions. Calmodulin‐dependent kinase activity was enriched approximately 20‐fold over cytosol in cold‐stable microtubule preparations. Calmodulin‐dependent kinase activity in cold‐stable microtubule preparations phosphorylated microtubule‐associated protein‐2, α‐ and β‐tubulin, an 80,000‐dalton doublet, and several minor phosphoproteins. The endogenous calmodulin‐dependent kinase in cold‐stable microtubule fractions was identical to a previously purified calmodulin‐dependent kinase from rat brain by several criteria including (1) subunit molecular weights, (2) subunit isoelectric points, (3) calmodulin‐binding properties, (4) subunit autophosphorylation, (5) calmodulin‐binding subunit composition on high‐resolution sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, (6) isolation of kinase on calmodulin affinity resin, (7) kinetic parameters, (8) phosphoamino acid phosphorylation sites on β‐tubulin, and (9) phosphopeptide mapping. Endogenous cold‐stable calmodulin‐dependent kinase activity was isolated from the microtubule fraction by calmodulin affinity resin column chromatography and specifically eluted with EGTA. This kinase fraction contained the calmodulin‐binding, autophosphorylating p and σ subunits of the previously purified kinase. The p and σ subunits of this kinase represented the major calmodulin‐binding proteins in the cold‐stable microtubule fractions as assessed by denaturing and nondenaturing procedures. These results indicate that calmodulin‐dependent kinase is a major calmodulin‐binding enzyme system in cold‐stable microtubule fractions and may play an important role in mediating some of the effects of calcium on microtubule and cytoskeletal dynamics.