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.