cAMP-dependent stabilization of phosphoenolpyruvate carboxykinase mRNA in LLC-PK1-F+ kidney cells.
Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes a rate-limiting step in hepatic and renal gluconeogenesis. In the kidney, PEPCK expression is enhanced during metabolic acidosis and in response to ANG II and parathyroid hormone. The effect of the latter hormone is mediated, in part, by cAMP. Treatment of subconfluent cultures of LLC-PK1-F+ cells, a gluconeogenic line of porcine proximal tubule-like cells, with cAMP produces a pronounced increase in the level of PEPCK mRNA. The luciferase activity of pLuc/3'-PCK-1, a reporter construct that contains the 3'-UTR of the PEPCK mRNA, was increased three- to fourfold by coexpression of the catalytic subunit of protein kinase A (PKA). This result indicates that cAMP-dependent stabilization may contribute to the increased expression of PEPCK mRNA in LLC-PK1-F+ cells. Various pLuc/3' constructs containing different segments of the 3'-UTR of PEPCK mRNA were used to map the cAMP response to two segments that were previously shown to bind AUF1 and to function as instability elements. A tetracycline-responsive promoter system was used to quantify the effect of forskolin on the half-lives of chimeric beta-globin-PEPCK (TbetaG-PCK) mRNAs. The half-life of the labile betaG-PCK-1 mRNA was increased eightfold by addition of forskolin. In contrast, the half-lives of the constructs containing the individual instability elements were increased only twofold. Therefore, the multiple instability elements present within the 3'-UTR may function synergistically to mediate both the rapid degradation and the cAMP-induced stabilization of PEPCK mRNA. The latter process may result from a PKA-dependent phosphorylation of AUF1.
Dhakras
Purabi S
PS
Department of Biochemistry and Molecular Biology, Colorado State University, Campus Delivery 1870, Fort Collins, CO 80523-1870, USA.
Hajarnis
Sachin
S
Taylor
Lynn
L
Curthoys
Norman P
NP
D020413:3' Untranslated Regions; D000818:Animals; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000737:chemistry / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases; D004795:Enzyme Stability / Q000187:drug effects*; D005914:Globins / Q000378:metabolism; D006207:Half-Life; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D / Q000378:metabolism; D018374:LLC-PK1 Cells; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000235:genetics* / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020218:Response Elements; D015183:Restriction Mapping; D013552:Swine; D014162:Transfection
D020413:3' Untranslated Regions; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D005576:Colforsin; D005914:Globins; D000242:Cyclic AMP; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D019764:Phosphoenolpyruvate Carboxykinase (ATP)