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Insulin-Induced c-Jun N-Terminal Kinase Activation Is Negatively Regulated by Protein Kinase C ¹

Third Department of Medicine, Shiga University of Medical Science, Seta, Otsu, Shiga 520-2192, Japan

Address all correspondence and requests for reprints to: Hiroshi Maegawa, Third Department of Medicine, Shiga University of Medical Science, Seta, Otsu, Shiga, 520-2192, Japan. E-mail: maegawa{at}belle.shiga-med.ac jp.

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 insulin receptors. Insulin treatment led to increased SAPK/ERK kinase 1 (SEK1) phosphorylation, and then stimulated JNK activity in a dose- and time-dependent manner, as measured either by a solid-phase kinase assay using glutathione S-transferase (GST)-c-Jun fusion protein as a substrate, or by quantitation of the levels of phosphorylated JNK by Western blotting using anti-phospho-JNK antibody. Insulin-induced JNK activation was potentiated by either preincubating cells with 2 nM GF109203X (PKC inhibitor) or down-regulation of PKC by overnight treatment with 100 nM tetradecanoyl phorbol acetate. In contrast, brief preincubation with 100 nM tetradecanoyl phorbol acetate inhibited the insulin- induced JNK activation. Furthermore, we found that 5 µM rottlerin, a PKC inhibitor, enhanced insulin-induced JNK activation, but a PKCß inhibitor, LY333531, had no effect. Consistent with these findings, overexpression of PKC led to decreased insulin-induced JNK activation, whereas overexpression of PKCß had no effect. Although overexpression of wild-type PKC attenuated insulin-induced JNK activation, a kinase-dead PKC mutant did not cause such attenuation. Finally, we found that the magnitude of insulin-induced JNK activation was inversely correlated with the expression level of PKC among different cell lines. In conclusion, the expression of PKC may negatively regulate insulin-induced JNK activation.