| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1996: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1995: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
Phosphorylation-dependent regulation of microtubule-stabilizing activities of microtubule-associated protein 2 (MAP2) was examined using optical microscopy. MAP2, purified from mammalian brain, was phosphorylated by either cAMP-dependent protein kinase (PKA) or cyclin B-dependent cdc2 kinase. By PKA,15 mloles of phoshoryl groups was incorporated into one mole of MAP2, but about 70% was distributed to the projection region. By cdc2 kinase, 8-10 moles of phosphoryl groups was incorporated into one mole of MAP2 and more than 60% of the phosphates was distributed to the microtubule-binding region. Both phosphorylations similarly reduced binding activity of MAP2 onto microtubules. Direct observation of individual microtubules under a dark-field microscope showed that interconversion between polymerization phase and depolymerization phase was repeated in both unphosphorylated and PKA-phosphorylated MAP2. In cdc2 kinase-phosphorylated MAP2, however, phase transition from depolymerization to polymerization was difficult to take place, with the result that life-time of individual microtubules was as short as in the absence of MAP2. Examination of spontaneous nucleation of microtubules under a dark-field microscope showed that PKA-phosphorylated MAP2 redued nucleating-activity, while cdc2 kinase-phosphorylated MAP2 completely abolished it. These observations show that cdc2 kinase-dependent phosphorylation inhibited both microtubule-stabilizing activity and microtubule-nucleating activity of MAP2, while PKA-dependent phosphorylation affected only microtubule-nucleating activity of MAP2.
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