PHOSPHORYLATION STATES OF MICROTUBULE-ASSOCIATED PROTEIN-2 (MAP2) DETERMINE THE REGULATORY ROLE OF MAP2 IN MICROTUBULE DYNAMICS

Phosphorylation-dependent regulation of microtubule-stabilizing activi ties of microtubule-associated protein 2 (MAP2) was examined using opt ical microscopy. MAP2, purified from mammalian brain, was phosphorylat ed by either cAMP-dependent protein kinase (PKA) or cyclin B-dependent cdc2 kinase. Using PKA, 15 mol of phosphoryl groups was incorporated per mole of MAP2, but about 70% of the phosphates was distributed to t he projection region. Using cdc2 kinase, 7-10 mol of phosphoryl groups was incorporated per mole of MAP2, and more than 60% of the phosphate s was distributed to the microtubule-binding region, Both types of pho sphorylation similarly reduced binding activity of MAP2 onto microtubu les. Direct observation of individual microtubules using dark-field mi croscopy showed that interconversion between the polymerization phase and the depolymerization phase was repeated in both unphosphorylated a nd PKA-phosphorylated MAP2. In cdc2 kinase-phosphorylated MAP2, howeve r, the phase transition from depolymerization to polymerization occurr ed with difficulty, with the result being that the half-life of indivi dual microtubules was as short as in the absence of MAP2. Examination of spontaneous polymerization of microtubules using dark-field microsc opy showed that the microtubule-nucleating activity of MAP2 was reduce d by PKA-dependent phosphorylation and was completely abolished by cdc 2 kinase-dependent phosphorylation. These observations show that cdc2 kinase-dependent phosphorylation inhibits both the microtubule-stabili zing activity and the microtubule-nucleating activity of MAP2, while P KA-dependent phosphorylation affects only the microtubule-nucleating a ctivity of MAP2.