Role of Protein Kinases in Transport Regulation

DESCRIPTION (Verbatim from the application): Both acute and chronic regulation of the Na,K-ATPase by prostaglandins will be examined using the Madin Darby Canine Kidney (MDCK) cell line in hormonally defined serum free medium. Two classes of variant MDCK cells will be utilized, includingProstaglandin El (PGE1) independent MDCK cells (with elevated cyclic AMP (cAMP)), which no longer require PGE1 for growth, and dibutyryl cAMP resistant MDCK cells (with a defect in CAMP dependent protein kinase (PKA), which no longer respond to PGE1 by an increase Na,K-ATPase activity). The hypothesis will be evaluated that following long term PGE1 treatment, the Na,K-ATPase is regulated at the transcriptional level via the protein kinase C (PKC) as well as the PKA pathways, unlike the case with a short term PGE1 treatment, in which the Na,K-ATPase is regulated post-translationally. In order to evaluate this hypothesis: (1) Transcriptional regulation of the Na,K-ATPase beta subunit gene will be examined by means of transient transfection studies with normal and variant MDCK cells, utilizing the pH beta1-1141 Luc plasmid. The role of the PKA, PKC and calcium pathways will be examined. Regulatory regions in the Na,K-ATPase beta subunit promoter are to be defined, and trans-acting factors identified. (2) To determine the effect of chronic PGE1 treatment on transcriptional regulation of endogenous genes, nuclear-runoff transcription studies will be conducted with normal and variant MDCK cells. Control studies will also be conducted with primary rabbit kidney proximal tubule cells. Post-transcriptional effects of PGE1 will be evaluated by means of Northern analysis, an assessment of effects on mRNA stability. Na,K-ATPase biosynthesis and degradation rates will be determined. (3) To determine the mechanism underlying acute effects of PGE1, PGE2 and 8Br-cAMP, the possibility will be evaluated that the Na,K-ATPase is phosphorylated by these agents. The possible involvement of an inhibitory phosphatase, in regulating the phosphorylation of the Na,K-ATPase will also be examined.