The roles of macromolecular synthesis and phosphorylation in the regulation of a protein kinase activity transiently stimulated by nerve growth factor.
Past work identified and characterized an apparently novel protein kinase activity (designated HMK) that is highly and transiently stimulated in PC12 pheochromocytoma cells by nerve growth factor (NGF). In vitro, HMK phosphorylates both high molecular weight microtubule-associated proteins and myelin basic protein. This study investigates the potential mechanisms of HMK regulation in intact PC12 cells and reveals the following. 1) HMK activation is independent of macromolecular synthesis while the subsequent post-induction suppression requires both RNA and protein synthesis. 2) Neither cAMP-dependent nor Ca2+/phospholipid-dependent protein kinases appear to play a role in regulation of HMK activity by NGF. 3) In vitro, HMK activity is inactivated by protein phosphatase 2A. 4) In vivo, HMK activation by NGF is inhibited by the kinase inhibitor, K-252a. (5) Vanadate, a tyrosine phosphatase inhibitor, induces HMK activity in intact cells, while okadaic acid, a serine/threonine phosphatase inhibitor, is much less efficacious. 6) Application of okadaic acid to vanadate-pretreated cells synergistically stimulates HMK activity to a level comparable to that achieved with NGF. (7) Activation of HMK by NGF is not significantly affected when cells are pretreated with okadaic acid. However, the subsequent NGF-promoted deactivation of HMK is greatly accelerated by okadaic acid. (8) NGF down-regulated HMK activity can be heterologously restimulated by exposure to vanadate and okadaic acid. These data suggest that phosphorylation plays a critical role in both the up- and down-regulation of HMK activity in NGF-treated cells. Moreover, suppression of HMK activity requires ongoing macromolecular synthesis and appears to occur by inactivation rather than degradation.
Tsao
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Department of Pathology, Columbia University College of Physicians and Surgeons, New York, New York 10032.
Greene
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LA
D000818:Animals; D002227:Carbazoles / Q000494:pharmacology; D002460:Cell Line; D003609:Dactinomycin / Q000494:pharmacology; D004789:Enzyme Activation; D004988:Ethers, Cyclic / Q000494:pharmacology; D026121:Indole Alkaloids; D007700:Kinetics; D009414:Nerve Growth Factors / Q000494:pharmacology*; D019319:Okadaic Acid; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
D002227:Carbazoles; D004988:Ethers, Cyclic; D026121:Indole Alkaloids; D009414:Nerve Growth Factors; D047428:Protein Kinase Inhibitors; D003609:Dactinomycin; D019319:Okadaic Acid; D014638:Vanadates; C049985:staurosporine aglycone; D011494:Protein Kinases