Phosphorylation states of microtubule-associated protein 2 (MAP2) determine the regulatory role of MAP2 in microtubule dynamics.
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. Using PKA, 15 mol of phosphoryl groups was incorporated per mole of MAP2, but about 70% of the phosphates was distributed to the projection region. Using cdc2 kinase, 7-10 mol of phosphoryl groups was incorporated per mole of MAP2, and more than 60% of the phosphates was distributed to the microtubule-binding region. Both types of phosphorylation similarly reduced binding activity of MAP2 onto microtubules. Direct observation of individual microtubules using dark-field microscopy showed that interconversion between the polymerization phase and the depolymerization phase was repeated in both unphosphorylated and PKA-phosphorylated MAP2. In cdc2 kinase-phosphorylated MAP2, however, the phase transition from depolymerization to polymerization occurred with difficulty, with the result being that the half-life of individual microtubules was as short as in the absence of MAP2. Examination of spontaneous polymerization of microtubules using dark-field microscopy showed that the microtubule-nucleating activity of MAP2 was reduced by PKA-dependent phosphorylation and was completely abolished by cdc2 kinase-dependent phosphorylation. These observations show that cdc2 kinase-dependent phosphorylation inhibits both the microtubule-stabilizing activity and the microtubule-nucleating activity of MAP2, while PKA-dependent phosphorylation affects only the microtubule-nucleating activity of MAP2.
Itoh
T J
TJ
Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-01, Japan.
Hisanaga
S
S
Hosoi
T
T
Kishimoto
T
T
Hotani
H
H
D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002417:Cattle; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D010766:Phosphorylation; D013552:Swine
D008869:Microtubule-Associated Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D016203:CDC2 Protein Kinase