Ca2+, Calmodulin‐Dependent Regulation of Microtubule Formation via Phosphorylation of Microtubule‐Associated Protein 2, τ Factor, and Tubulin, and Comparison with the Cyclic AMP‐Dependent Phosphorylation

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Abstract
Isolated microtubule-associated protein 2 (MAP2), .tau. factor and tubulin were phosphorylated by a purified Ca2+, calmodulin-dependent protein kinase (640K enzyme) from rat brain. The phosphorylation of MAP2 and .tau. factor separately induced the inhibition of microtubule assembly, in accordance with the degree. Tubulin phosphorylation by the 640K [kilodalton] enzyme induced the inhibition of microtubule assembly, whereas the effect of tubulin phosphorylation by the catalytic subunit was undetectable. The effects of tubulin and MAP phosphorylation on microtubule assembly were greater than that of either tubulin or MAP phosphorylation. Because MAP2, .tau. factor and tubulin were also phosphorylated by the catalytic subunit of type-II cAMP-dependent protein kinase from rat brain, the kinetic properties and phosphorylation sites were compared. The amount of phosphate incorporated into each microtubule protein was 3-5 times higher by the 640K enzyme than by the catalytic subunit. The Km values of the 640K enzyme for microtubule proteins were 4-24 times lower than those of the catalytic subunit. The peptide mapping analysis showed that the 640K enzyme and the catalytic subunit incorporated phosphate into different sites on MAP2, .tau. factor and tubulin. Investigation of phosphoamino acids revealed that only the seryl residue was phosphorylated by the catalytic subunit, whereas both seryl and threonyl residues wre phosphorylated by the 640K enzyme. The Ca2+, calmodulin system via phosphorylation of MAP2, .tau. factor and tubulin by the 640K enzyme is more effective than the cAMP system on the regulation of microtubule assembly.