Partial purification and characterization of a nerve growth factor-sensitive kinase and its substrate from PC12 cells.
The cell-free, nerve growth factor-sensitive incorporation of radioactive phosphate into a 100,000-dalton protein (Nsp100), observed in a previous study (End,D., Tolson, N., Hashimoto, S., and Guroff, G. (1983) J. Biol. Chem. 258, 6549-6555), has been characterized and the system fractionated. It is shown here that the decrease in incorporation due to treatment of the cells with nerve growth factor is transient, even in the continued presence of nerve growth factor. The decrease in radioactive phosphate incorporation is due to an inhibition of phosphorylation, not to a stimulation of a dephosphorylation. Evidence is presented to suggest that no soluble cofactors are needed for the phosphorylation and no soluble second messengers are responsible for the inhibition. It is demonstrated that the phosphorylation requires divalent cations; both Mg2+ and Mn2+ are effective in this regard. ATP is the preferred phosphate donor, the phosphorylation is maximal at pH values between 5 and 6, and Na+, K+, and Zn2+ are rather specific inhibitors. The system has been partially purified and the resolved components have been used to show that the kinase and the substrate are separate molecules, that the kinase, not the substrate, is the heat-labile portion, and that the kinase has a molecular weight of 110,000-130,000. Finally, evidence is presented to indicate that the kinase, not the substrate, is the component responsible for the decrease in phosphorylation seen after treatment of the cells with nerve growth factor.
D000310:Adrenal Gland Neoplasms / Q000201:enzymology*; D000818:Animals; D047388:Casein Kinases; D002460:Cell Line; D003035:Cobalt / Q000494:pharmacology; D008970:Molecular Weight; D009414:Nerve Growth Factors / Q000378:metabolism*; D010673:Pheochromocytoma / Q000201:enzymology*; D010766:Phosphorylation; D011188:Potassium / Q000494:pharmacology; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D012964:Sodium / Q000494:pharmacology; D013997:Time Factors; D015032:Zinc / Q000494:pharmacology
D009414:Nerve Growth Factors; D003035:Cobalt; D012964:Sodium; D011494:Protein Kinases; D047388:Casein Kinases; D015032:Zinc; D011188:Potassium