Separation of endogenous calmodulin- and cAMP-dependent kinases from microtubule preparations.
Both cAMP- and calmodulin-dependent kinases are proposed regulators of microtubule function by means of their ability to phosphorylate microtubule-associated protein 2(MAP 2). A cAMP-dependent kinase/MAP 2 complex is endogenous to microtubules. In this report, we demonstrate that an endogenous calmodulin-dependent kinase that phosphorylates MAP 2 as a major substrate is also present in microtubules prepared under conditions that preserve kinase activity. This enzyme is identical to a calmodulin-dependent kinase purified previously from rat brain cytosol. A fraction containing calmodulin-dependent kinase and MAP 2 was separated from the cAMP-dependent kinase/MAP 2 complex by gel filtration chromatography of microtubule protein in high ionic strength buffer. All of the recovered calmodulin-dependent kinase activity in microtubules eluted in a single protein peak. The specific activity of the enzyme for MAP 2 was enriched 31-fold in this fraction compared to cytosol. Two-dimensional tryptic phosphopeptide mapping revealed that the endogenous cAMP- and calmodulin-dependent kinases phosphorylated distinct sites on MAP 2. These data demonstrate that both kinases are present in microtubule preparations and that they may differentially regulate MAP 2 function by phosphorylating separate sites on MAP 2.
D000818:Animals; D001921:Brain / Q000201:enzymology; D002147:Calmodulin / Q000378:metabolism*; D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D010750:Phosphoproteins / Q000032:analysis; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification*; D051381:Rats
D002147:Calmodulin; D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D011494:Protein Kinases