title: Concurrent binding and modifications of AUF1 and HuR mediate the pH-responsive stabilization of phosphoenolpyruvate carboxykinase mRNA in kidney cells.
abstract: Onset of metabolic acidosis leads to a pronounced increase in renal expression of phosphoenolpyruvate carboxykinase (PEPCK). This response, which is mediated in part by stabilization of PEPCK mRNA, is effectively modeled by treating LLC-PK(1)-F(+)-9C cells with an acidic medium. siRNA knockdown of HuR prevented the pH-responsive increase in PEPCK mRNA half-life suggesting that HuR is necessary for this response. A recruitment assay, using a reporter mRNA in which the pH response elements of the PEPCK 3'-UTR were replaced with six MS2 stem-loop sequences, was developed to test this hypothesis. The individual recruitment of a chimeric protein containing the MS2 coat protein and either HuR or p40AUF1 failed to produce a pH-responsive stabilization. However, the concurrent expression of both chimeric proteins was sufficient to produce a pH-responsive increase in the half-life of the reporter mRNA. siRNA knockdown of AUF1 produced slight increases in basal levels of PEPCK mRNA and protein, but partially inhibited the pH-responsive increases. Complete inhibition of the latter response was achieved by knockdown of both RNA-binding proteins. The results suggest that binding of HuR and AUF1 has opposite effects on basal expression, but may interact to mediate the pH-responsive increase in PEPCK mRNA. Two-dimensional gel electrophoresis indicated that treatment with acidic medium caused a decrease in phosphorylation of HuR, but may increase phosphorylation of the multiple AUF1 isoforms. Thus, the pH-responsive stabilization of PEPCK mRNA requires the concurrent binding of HuR and AUF1 and may be mediated by changes in their extent of covalent modification.
authors.affiliation: Dept. of Biochemistry and Molecular Biology, Colorado State Univ., Ft. Collins, CO 80523-1870, USA.
authors.forename: Lakshmi
authors.initials: L
authors.lastname: Gummadi
authors.forename: Lynn
authors.initials: L
authors.lastname: Taylor
authors.forename: Norman P
authors.initials: NP
authors.lastname: Curthoys
mesh_terms: D000138:Acidosis / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D051959:ELAV Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D023421:Models, Animal; D011485:Protein Binding; D020033:Protein Isoforms; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology; D013552:Swine
chemical_list: D051959:ELAV Proteins; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; D020033:Protein Isoforms; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C069767:phosphoenolpyruvate carboxylase kinase; D017346:Protein Serine-Threonine Kinases