Microtubule assembly using the microtubule-associated protein MAP-2 prepared in defined states of phosphorylation with protein kinase and phosphatase.
A microtubule-associated protein (the 270-kDa MAP-2) was prepared in two defined states of phosphorylation by (a) phosphorylation by associated kinase to the extent of 11-14 mol/mol, and (b) removal of 70-80% of this phosphate with a protein phosphatase purified from brain. The newly introduced phosphate was in addition to about 10 mol/mol already present in MAP-2 as isolated; these phosphates were not appreciably released by the phosphatase and did not exchange with ATP. In microtubules assembled with phosphorylated (24 mol/mol) MAP-2 the assembly rate was decreased, microtubule length and critical concentration for assembly were unaffected, and rates of loss of subunits were increased from both microtubule ends. Phosphorylation also reduced the binding of MAP-2 to taxol-stabilized microtubules. These changes were unequivocally due to phosphorylation, since phosphatase treatment reversed all of them. The brain phosphatase used in these experiments was purified 3000-fold towards histone, but only 100-fold towards MAP-2, suggesting brain may contain another enzyme more specific for MAP-2. Calcineurin, however, had only a low activity for MAP-2.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D001704:Biopolymers / Q000378:metabolism; D001921:Brain / Q000201:enzymology; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D066298:In Vitro Techniques; D008869:Microtubule-Associated Proteins; D008870:Microtubules / Q000378:metabolism*; D017239:Paclitaxel; D010710:Phosphates / Q000032:analysis; D010749:Phosphoprotein Phosphatases; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases; D011506:Proteins / Q000378:metabolism*; D012756:Sheep; D014404:Tubulin / Q000378:metabolism
D000470:Alkaloids; D001704:Biopolymers; D008869:Microtubule-Associated Proteins; D010710:Phosphates; D011506:Proteins; D014404:Tubulin; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases; D017239:Paclitaxel