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

• Published in: The Journal of biological chemistry 1987-06, Vol.262 (16), p.7504-7513
• Main Authors: Rowland, E A, Müller, T H, Goldstein, M, Greene, L A
• Format: Article
• Language: English
• Subjects: Adrenal Gland Neoplasms - enzymology; Animals; Biological and medical sciences; Cell Line; Cell physiology; Cell-Free System; Enzyme Activation; Fundamental and applied biological sciences. Psychology; Kinetics; Molecular and cellular biology; Nerve Growth Factors - pharmacology; Pheochromocytoma - enzymology; Phosphorylation; Protein Kinases - isolation & purification; Protein Kinases - metabolism; Rats; Responses to growth factors, tumor promotors, other factors; Substrate Specificity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)47595-3

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