Doctoral Dissertations

Date of Award

12-1993

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

Stephen J. Kennel

Committee Members

K. Bruce Jacobson, Kai-Lin Lee, Larry C. waters

Abstract

Insulin exerts pleiotropic effects in cells by regulating phosphorylation and hence function of specific proteins through 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 in response to insulin was examined with particular emphasis on the role of serine/threonine specific protein phosphatases in a hepatoma cell line. This stable cell (Dll) line contains a chimeric transcription reporter gene construct, pG33/0.5-CAT, contains DNA sequences from -480 to +21 base pairs relative to the transcription start site of Gene 33 that is known to enhance expression of bacterial chloramphenicol acetyltransferase (CAT) in response to insulin. Using okadaic acid, an inhibitor of serine/threonine phosphatases 1 and 2A, it was shown that expression of PEPCK mRNA is inhibited in a dose and time dependent fashion. Conversely, expression of Gene 33 mRNA was transiently enhanced by okadaic acid. At the gene level, okadaic acid, like insulin, prevents PEPCK mRNA accumulation by inhibiting transcription of the gene. The rates of decline of PEPCK mRNA levels following simultaneous treatment with insulin and okadaic acid are indistinguishable from those mediated by either effector alone. These data suggest that the inhibition of PEPCK gene transcription by insulin is mediated by the inhibition of serine/threonine phosphatases. In contrast, following a one hour lag, okadaic acid enhances the rate of Gene 33 transcription and augments the post transcriptional stability of the mRNA resulting in elevated Gene 33 mRNA levels two hours post-treatment. These data suggest that the phosphoproteins which control Gene 33 expression are distinctly different from those influencing PEPCK gene expression. Time course studies examining the simultaneous effects of okadaic acid and insulin on Gene 33 expression indicated okadaic acid acts synergistically with insulin to enhance the accumulation of Gene 33 mRNA. The combination of insulin-induced early Gene 33 transcription, delayed transcriptional activation by okadaic acid, and the okadaic acid-enhanced post-transcriptional stability of Gene 33 mRNA are probably responsible for this synergism. Expression of the reporter gene construct, pG33/0.5-CAT, did not effectively respond to okadaic acid and/or insulin treatment and consequently 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 certain protein phosphatases act as negative and positive mediators of gene expression. This further strengthens the view that phosphorylation cascades are major mechanisms by which insulin regulates gene expression.

Download
Files over 3MB may be slow to open. For best results, right-click and select "save as..."

Share

COinS