The heat-induced hsp 70 promoter activity is negatively regulated by serine phosphatases: evidence from the effects of okadaic acid.
We examined the effects of okadaic acid (OA), a potent and specific inhibitor of serine phosphatases 2A and 1, on the transient expression of an hsp 70 promoter-reporter gene construct in IMR-90 human diploid lung fibroblasts. We showed that OA markedly potentiated the heat-induced but not the basal expression of pHBCAT, a full-length human hsp-70-promoter-driven CAT gene construct. This effect of OA was dose and time dependent and promoter specific. Importantly, the potentiating effects of OA appeared to be independent of the binding of the activated heat shock transcription factor (HSTF) to its consensus DNA sequence, the heat shock element (HSE). Thus, OA had no effect on the HSTF DNA-binding activity as measured by mobility shift assay, and mutation of the HSE sequence did not obliterate the stimulatory effects of OA on reporter gene expression under a heat shock condition, although heat shock by itself was without effect. Analysis of the status of phosphorylation of the largest subunit of RNA polymerase II provided evidence that this effect of OA is attributable, at least in part, to the increased phosphorylation of RNA polymerase II. These results provided evidence that the heat-induced hsp 70 promoter activity is negatively regulated by serine phosphatases. We propose that the heat-induced transcriptional activation of hsps is associated with phosphorylation of component(s) of the transcription complex; one of the likely candidates being the transcriptionally engaged RNA polymerase II. OA, by inhibiting phosphatase 2A and 1 activity, enhanced this phosphorylation and potentiated the transcriptional activation of hsps.
Chang
N T
NT
Department of Biological Sciences, Rutgers, State University of New Jersey, Piscataway 08855-1059.
Liu
A Y
AY
D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000235:genetics; D004988:Ethers, Cyclic / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D006360:Heat-Shock Proteins / Q000235:genetics*; D006358:Hot Temperature; D006801:Humans; D008969:Molecular Sequence Data; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D054648:Protein Phosphatase 2; D014162:Transfection
D004988:Ethers, Cyclic; D006360:Heat-Shock Proteins; D019319:Okadaic Acid; D004247:DNA; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2