Identification of endogenous calmodulin-dependent kinase and calmodulin-binding proteins in cold-stable microtubule preparations from rat brain.
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.
Larson
R E
RE
Goldenring
J R
JR
Vallano
M L
ML
DeLorenzo
R J
RJ
D000818:Animals; D001923:Brain Chemistry; D002118:Calcium / Q000502:physiology; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins; D003080:Cold Temperature; D007526:Isoelectric Point; D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000032:analysis; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010750:Phosphoproteins / Q000032:analysis; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D014404:Tubulin / Q000378:metabolism*
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D014404:Tubulin; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases; D002118:Calcium