Autophosphorylation of brain microtubule protein: evidence for endogenous protein kinase/phosphoprotein phosphatase cycling and multiple phosphorylation of a microtubule associated protein |
| |
Authors: | B A Coughlin H D White D L Purich |
| |
Institution: | Department of Chemistry, University of California Santa Barbara, California 93106 USA |
| |
Abstract: | Brain microtubule protein, prepared by two types of recycling methods, undergoes “flash” phosphorylation in the presence of γ-32p]ATP through sequential action of protein kinase and phosphoprotein phosphatase present in microtubule protein. SDS electrophoretic analysis indicates that MAP1, tau protein, and tubulin are poorly phosphorylated, and MAP2 is the major site of phosphorylation. To improve 32P]phosphoprotein stability in the presence of the kinase/phosphatase cycle, 3′,5′-cyclicAMP, orthophosphate, or fluoride ion may be added. After separation from tubulin by phosphocellulose chromatography, the MAP fraction exhibits autophosphorylation. Finally, the maximal extent of autophosphorylation is observed with an ATP regenerating system using ADP, 32P]acetyl-P, and bacterial acetate kinase; this results in the incorporation of 3–4 phosphoryl groups per MAP2 subunit. |
| |
Keywords: | SDS sodium dodecyl sulfate MTP microtubule protein |
本文献已被 ScienceDirect 等数据库收录! |
|