Effects of okadaic acid on phosphoenolpyruvate carboxykinase gene and Gene 33 expression: Implications of serine/threonine phosphorylation cascades in insulin mediated regulation of gene expression

Insulin exerts pleiotropic effects in cells by regulating activities of serine/threonine kinases and phosphatases. These enzymes, via phosphorylation cascades, regulate the functions of specific proteins in response to insulin. Expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene and Gene 33 by insulin action was examined with emphasis on the role of serine/threonine specific protein phosphatases in a hepatoma cell line. This stable cell (D11) line contains a chimeric transcription reporter gene construct, pG33/0.5-CAT, with DNA sequences from ;Okadaic acid, an inhibitor of serine/threonine phosphatases 1 and 2A, inhibited expression of PEPCK mRNA in a dose and time dependent fashion. Conversely, expression of Gene 33 mRNA was enhanced by okadaic acid. Okadaic acid prevented PEPCK mRNA accumulation by inhibiting transcription. The rates of decline of PEPCK mRNA levels following treatment with insulin and okadaic acid are indistinguishable from those mediated by either effector alone. These data suggest that inhibition of PEPCK gene transcription by insulin is mediated by inhibition of serine/threonine phosphatases. Okadaic acid enhances the rate of Gene 33 transcription and augments the stability of the mRNA two hours post-treatment.;These data suggest that phosphoproteins which control Gene 33 expression are different from those influencing PEPCK gene expression. Time course studies examining effects of okadaic acid and insulin on Gene 33 expression indicated that okadaic acid acts synergistically with insulin to enhance accumulation of Gene 33 mRNA. The combination of insulin-induced early Gene 33 transcription, delayed transcriptional activation by okadaic acid, and okadaic acid-enhanced stability of Gene 33 mRNA are responsible for this synergism.;Expression of the pG33/0.5-CAT did not effectively respond to okadaic acid and/or insulin treatment and was not used to study the effects of okadaic acid and insulin on Gene 33 expression. Since okadaic acid mediated inhibition of specific serine/threonine phosphatases mimicked the actions of insulin on the expression of PEPCK gene and Gene 33, it was hypothesized that protein phosphatases act as negative and positive mediators of gene expression. This strengthens the view that phosphorylation cascades are major mechanisms by which insulin regulates gene expression.