Insulin-induced c-Jun N-terminal kinase activation is negatively regulatedby protein kinase C delta

We investigated the role of protein kinase C (PKC) in insulin-induced c-Jun N-terminal kinase (JNK) activation in rat 1 fibroblasts expressing human i nsulin receptors. Insulin treatment led to increased SAP/ERK kinase 1 (SEK1 ) phosphorylation, and then stimulated JNK activity in a dose- and time-dep endent manner, as measured either by a solid-phase kinase assay using gluta thione S-transferase (GST)-c-Jun fusion protein as a substrate, or by quant itation of the levels of phosphorylated JNK by Western blotting using anti- phospho-JNK antibody. Insulin-induced JNK activation was potentiated by eit her preincubating cells with 2 nM GF109203X (PKC inhibitor) or down-regulat ion of PKC by overnight treatment with 100 nM tetradecanoyl phorbol acetate . In contrast, brief preincubation with 100 nM tetradecanoyl phorbol acetat e inhibited the insulin-induced JNK activation. Furthermore, we found that 5 muM rottlerin, a PKC delta inhibitor, enhanced insulin-induced JNK activa tion, but a PKC beta inhibitor, LY333531, had no effect. Consistent with th ese findings, overexpression of PKC delta led to decreased insulin-induced JNK activation, whereas overexpression of PKC beta had no effect. Although overexpression of wild-type PKC delta attenuated insulin-induced JNK activa tion, a kinase-dead PKC delta mutant did not cause such attenuation. Finall y, we found that the magnitude of insulin-induced JNK activation was invers ely correlated with the expression level of PKC delta among different cell lines. In conclusion, the expression of PKC delta may negatively regulate i nsulin-induced JNK activation.