Selective inhibition of nerve growth factor-stimulated protein kinases by K-252a and 5'-S-methyladenosine in PC12 cells.
K-252a, a protein kinase inhibitor isolated from the culture broth of Nocardiopsis sp., inhibits the nerve growth factor (NGF)-stimulated phosphorylation of microtubule-associated protein 2 (MAP2) and Kemptide (synthetic Leu-Arg-Arg-Ala-Ser-Leu-Gly) by blocking the activation of two independent kinases in PC12 cells: MAP2/pp250 kinase and Kemptide kinase. The NGF-stimulated activation of these kinases is inhibited in a dose-dependent manner following treatment of the cells with K-252a. Although these kinases also are activated by epidermal growth factor (EGF) and 12-O-tetradecanoyl-phorbol 13-acetate, K-252a has no inhibitory effect when these agents are used. Half-maximal inhibition of the activation of both kinases was observed at 10-30 nM K-252a. K-252a was shown to directly inhibit the activity of MAP2/pp250 kinase and Kemptide kinase when added to the phosphorylation reaction mixture in vitro; however, half-maximal inhibition under these conditions was observed at greater than or equal to 50 nM K-252a. These data suggest that K-252a exerts its effects at a step early in the cascade of events following NGF binding. The effects of K-252a are similar to those reported for 5'-S-methyladenosine (MTA) and other methyltransferase inhibitors. Treatment of PC12 cells with MTA inhibited NGF-, but not EGF-mediated activation of MAP2/pp250-kinase (Ki greater than 500 microM). MTA, when added to the phosphorylation reaction mixture in vitro, directly inhibited kinase activity (Ki = 50 microM), suggesting that the effects of MTA may be the result of its action on protein kinases rather than methyltransferases.
Smith
D S
DS
Department of Neurology, Medical University of South Carolina, Charleston 29425.
King
C S
CS
Pearson
E
E
Gittinger
C K
CK
Landreth
G E
GE
D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000310:Adrenal Gland Neoplasms / Q000201:enzymology*; D000818:Animals; D002227:Carbazoles / Q000494:pharmacology*; D000242:Cyclic AMP / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology; D026121:Indole Alkaloids; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009414:Nerve Growth Factors / Q000494:pharmacology*; D009842:Oligopeptides / Q000494:pharmacology; D010673:Pheochromocytoma / Q000201:enzymology*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D002227:Carbazoles; D026121:Indole Alkaloids; D008869:Microtubule-Associated Proteins; D009414:Nerve Growth Factors; D009842:Oligopeptides; D047428:Protein Kinase Inhibitors; D004815:Epidermal Growth Factor; C037642:kemptide; C049985:staurosporine aglycone; D000242:Cyclic AMP; D011494:Protein Kinases; D000241:Adenosine; D013755:Tetradecanoylphorbol Acetate