Identification and characterization of the ATP·Mg-dependent protein phosphatase activator (F_A) as a microtubule protein kinase in the brain

The activating factor of ATP·Mg-dependent protein phosphatase (F_A) has been identified in brain microtubules. When using purified MAP-2 (microtubule associated protein 2) and tau proteins as substrates, F_A could phosphorylate MAP-2 to 16 moles of phosphates per mole of protein with a K_m value of 0.4 μM, and tau proteins to 4 moles of phosphates per mole of proteins with a K_m value of about 3 μM. When using microtubules as substrates, F_A could enhance many-fold the endogenous phosphorylation of many microtubule-associated proteins including MAP-2, tau proteins, and several low-molecular-weight MAPs. In contrast to other reported MAP kinases, such as cAMP-dependent protein kinase and Ca⁺²/phospholipid-dependent protein kinase, the F_A-catalyzed phosphorylation of tau proteins could cause an electrophoretic mobility shift on sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that a dramatic conformational change of tau proteins was produced by F_A. Peptide mapping analysis of the phosphopeptides derived from SV8 protease digestion revealed that F_A could phosphorylate MAP-2 and tau proteins on at least four specific sites distinctly different from those phosphorylated by cAMP-dependent and Ca⁺²/phospholipid-dependent MAP kinases. Quantitative analysis further indicated that approximately 19% of the total endogenous kinase activity in brain microtubules was due to F_A. Taken together, the results provide initial evidence that the ATP·Mg-dependent protein phosphatase activating factor (F_A) is a potent and unique MAP kinase, and may represent one of the major factors involved in phosphorylation of brain microtubules.