Microheterogeneity of microtubule-associated proteins, MAP-1 and MAP-2, and differential phosphorylation of individual subcomponents.
High molecular weight microtubule-associated proteins 1 and 2 (MAP-1 and MAP-2), prepared by copolymerization with tubulin, were electrophorectically separated into three and two major subcomponents, respectively, using 5% sodium dodecyl sulfate-polyacrylamide gels. By two-dimensional gel electrophoresis, all five MAP components were shown to possess a pI of around 5. Four of these proteins, MAP-1A, MAP-1C, MAP-2A, and MAP-2B, present in comparable amounts, were iodinated after electrophoretic separation and analyzed by two-dimensional peptide mapping. With both trypsin and V8 protease, almost identical patterns were obtained from MAP-2A and MAP-2B. MAP-1A and MAP-1C, too, gave similar digestion patterns, although some differences were noted. Incubation with [gamma-32P]ATP demonstrated that endogeneous protein kinase activities phosphorylated individual subcomponents at different rates. MAP-2A, the highest labeled component, was phosphorylated 2.5-fold compared to MAP-2B both in the presence and the absence of cAMP. Labeling of MAP-1 subcomponents was 4 times less than that of MAP-2A in the absence and 16 times less in the presence of cAMP. 32P-labeled MAP-2A and MAP-2B bands were indistinguishable by one-dimensional peptide mapping, as were the three MAP-1 bands. For both MAP-1 and MAP-2 subcomponents, cAMP induced phosphorylation at new molecular sites. Incubation of radiolabeled microtubule proteins with 1 mM ATP effected, upon electrophoresis, a clear shift of MAP-2A and MAP-2B bands to positions of higher apparent molecular weights, while only slightly affecting MAP-1 bands.
Herrmann
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Dalton
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Wiche
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D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001923:Brain Chemistry; D000242:Cyclic AMP / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins / Q000032:analysis*; D010766:Phosphorylation; D012697:Serine Endopeptidases; D013552:Swine; D014357:Trypsin / Q000378:metabolism
D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D010450:Endopeptidases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin