Purification, Characterization and Distribution of a Calmodulin-Dependent Protein Kinase from Rat Brain Cytosol (Calcium, Tubulin, Phosphorylation)
This is an Open Access Thesis
Tubulin is a major endogenous substrate for Ca('2+)- calmodulin-dependent kinase activity in rat brain cytosol. This dissertation details the purification and characterization of a calmodulin-dependent tubulin kinase from rat brain cytosol. The kinase, purified from chelated brain cytosol by sequential chromatography on phosphocellulose, calmodulin-affinity resin, and Fractogel TSK-55F, contains two calmodulin-binding, autophosphorylating subunits of 52,000 and 63,000 daltons, designated rho and sigma, respectively. The kinase holoenzyme complex has a native molecular weight of 600,000 daltons. Both kinase subunits demonstrate isoelectric points near neutrality. The kinase phosphorylated microtubule-associated protein 2, alpha tubulin and beta tubulin as major substrates. Calmodulin-dependent phosphorylation of these proteins occurred on different sites than Mg('2+)/cAMP-dependent phosphorylation. The calmodulin kinase displayed a characteristic pattern of 60% of beta tubulin phosphorylation on threonine residues.The endogenous association of this kinase with tubulin was established through the isolation of a tubulin-calmodulin kinase complex from rat brain cytosol by sequential chromatography on DEAE-cellulose, Sephacryl S-300 and Fractogel TSK-65F. The kinase complexed with tubulin was identical by all criteria including subunit composition, subunit properties, and kinase function to the purified calmodulin-dependent kinase. The association of the kinase with microtubule preparations was also demonstrated. Microtubule preparations contain calmodulin-dependent kinase activity with similar properties to the purified calmodulin kinase. These data suggest that calmodulin-dependent kinase may mediate the destabilizing effects of calmodulin on microtubules.Finally, the major protein component of the postsynaptic density was shown to be essentially identical to the rho subunit of purified calmodulin-dependent kinase. Thus, the apparent pansynaptic localization of this kinase implicates it as an important regulator of cytoskeletal dynamics.