Purification and mechanism of activation of a nerve growth factor-sensitive S6 kinase from PC12 cells.
A nerve growth factor (NGF)-sensitive S6 kinase was purified by alkaline lysis of PC12 cells. The activity in lysates from NGF-treated cells was 10-20-fold higher than that from controls. Half-maximal stimulation of the S6 kinase by NGF treatment occurred in approximately 5 min, and the activity returned almost to basal levels by 2 h. A rapid purification method was devised in which crude extract was applied directly to a PBE 94 column after buffer exchange on a PD-10 column (Sephadex G-25 M). The activated S6 kinase was purified at least 673-fold with a recovery of approximately 70%. The S6 kinase has an apparent molecular weight of 45,000 and is highly specific for S6. It is not inhibited by the specific inhibitor of cAMP-dependent protein kinases, or by chlorpromazine or sodium vanadate, nor is it activated by Ca2+/calmodulin. It was inhibited by EGTA, beta-glycerophosphate, or NaF. Phosphorylation occurred solely on serine residues. The S6 kinase activity from control cells and from NGF-treated cells eluted at pH 5.69 and 5.58, respectively, during PBE 94 column chromatography. Pretreatment of crude extract from NGF-stimulated cells with alkaline phosphatase resulted in an elution of the enzyme at the position of S6 kinase from control cells and a concomitant decrease in activity. These results indicate that phosphorylation is involved in the mechanism of S6 kinase activation.
D000310:Adrenal Gland Neoplasms / Q000201:enzymology*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002460:Cell Line; D002746:Chlorpromazine / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D009414:Nerve Growth Factors / Q000302:isolation & purification / Q000378:metabolism*; D010673:Pheochromocytoma / Q000201:enzymology*; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D019893:Ribosomal Protein S6 Kinases; D012969:Sodium Fluoride / Q000494:pharmacology; D014638:Vanadates; D014639:Vanadium / Q000494:pharmacology
D002147:Calmodulin; D009414:Nerve Growth Factors; D014639:Vanadium; D014638:Vanadates; D004533:Egtazic Acid; D012969:Sodium Fluoride; D011494:Protein Kinases; D019893:Ribosomal Protein S6 Kinases; D002118:Calcium; D002746:Chlorpromazine