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 PC 12 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 factoi (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 tc the phosphorylation reaction mixture in vitro; however, half‐maximal inhibition under these conditions was observed at ±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± 500 μM). MTA, when added to the phosphorylation reaction mixture in vitro, directly inhibited kinase activity (Ki= 50 μM), suggesting that the effects of MTA may be the result of its action on protein kinases rather than methyltransferases.