Cell-free detection and characterization of a novel nerve growth factor-activated protein kinase in PC12 cells.

摘要: We have developed a cell-free assay to detect and characterize nerve growth factor (NGF)-activated protein kinase activity. Cultured PC12 cells were briefly exposed NGF, extracts of these assayed for phosphorylating activity using exogenously added tyrosine hydroxylase as substrate. Tyrosine was employed since it is an endogenous substrate NGF-regulated activated by phosphorylation. In the assay, prepared from NGF-treated yielded 2-3-fold greater incorporation phosphate into compared with control, NGF-untreated cells. Activation did not occur, however, if NGF directly cell extracts. The NGF-stimulated appeared be due regulation rather than phosphoprotein phosphatase. Characterization (designated N) showed that soluble, detectably within 1-3 min after are maximally 10 min, half-maximally 0.5 nM 1 detectable in presence either Mg2+ or Mn2+ but does require Ca2+, nonmacromolecular cofactors, can use histone H1 substrate, exhibits 2-fold increase apparent Vmax response undergo significant change Km ATP GTP. A number characteristics N assessed including susceptibility inhibitors, specificity, cofactor requirements, dependence, lack down-regulation prolonged expose phorbol ester. These studies indicated lacks distinct variety well-characterized kinases cAMP-dependent kinase, C (Ca2+/phospholipid-dependent enzyme), Ca2+/calmodulin-dependent casein II. Preliminary purification data show has basic pI Mr 22,000-25,000. only amino acid found phosphorylated semipurified serine.