Purification and characterization of a calmodulin-dependent kinase from rat brain cytosol able to phosphorylate tubulin and microtubule-associated proteins.
Tubulin is a major substrate for endogenous Ca2+-calmodulin-dependent phosphorylation in synaptic cytoplasm. The present study details the purification to apparent homogeneity and characterization of a brain cytosolic Ca2+-calmodulin-dependent kinase which phosphorylates tubulin and microtubule-associated proteins as major substrates. The cytosolic kinase system, purified by sequential chromatography on phosphocellulose resin, calmodulin-affinity resin, and Fractogel TSK HW-55, chromatographs as a homogeneous complex of approximately 600,000 Da on Sephacryl S-300. This calmodulin-dependent kinase possesses a group of properties which specifically characterize this enzyme system: 1) the enzyme contains two calmodulin-binding doublets, rho and sigma, of approximately 52,000 and 63,000 Da, respectively; 2) both the rho and the sigma subunits demonstrate isoelectric points between 6.7 and 7.2; 3) both the rho and sigma subunits demonstrate autophosphorylation; 4) both the rho and sigma subunits show significant homologies as assessed by tryptic peptide fingerprints; 5) in the absence of substrate, both the rho and sigma subunits manifest lower mobility autophosphorylated species; 6) the kinase phosphorylates beta-tubulin equally on threonine and serine residues. Substrate specificity, kinetic parameters, calmodulin-binding properties, subunit composition, and subunit isoelectric points clearly differentiate this enzyme from other previously reported calmodulin-dependent kinases.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002147:Calmodulin / Q000502:physiology*; D002846:Chromatography, Affinity; D003600:Cytosol / Q000201:enzymology; D005260:Female; D007700:Kinetics; D008869:Microtubule-Associated Proteins; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D014404:Tubulin / Q000378:metabolism*
D002147:Calmodulin; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; D014404:Tubulin; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases