Identification of Endogenous Calmodulin-Dependent Kinase and Calmodulin-Binding Proteins in Cold-Stable Microtubule Preparations from Rat Brain |
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Authors: | Roy E Larson James R Goldenring Mary Lou Vallano Robert J DeLorenzo |
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Institution: | Department of Biochemistry, Faculdade de Medicina de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, Sao Paulo, Brazil;;Department of Neurology, Yale University School of Medicine, New Haven, Connecticut, U.S.A. |
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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, alpha- and beta-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 beta-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 rho and sigma subunits of the previously purified kinase. The rho and sigma subunits of this kinase represented the major calmodulin-binding proteins in the cold-stable microtubule fractions as assessed by denaturing and non-denaturing 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. |
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Keywords: | Calmodulin Kinase activity Tubulin Cytosol Binding Microtubule |
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