Phosphorylation and binding of AUF1 to the 3'-untranslated region of cardiomyocyte SERCA2a mRNA.
Experimental animals and patients with cardiac hypertrophy and heart failure display abnormally slowed myocardial relaxation, which is associated with downregulation of sarco(endo)plasmic reticulum calcium ATPase 2a (SERCA2a), the cardiomyocyte sarcoplasmic reticulum Ca2+ pump. We previously showed that SERCA2a downregulation can be simulated in cultured neonatal rat ventricular myocytes (NRVM) by treatment with the hypertrophic agonist phorbol myristate acetate (PMA) or by overexpression of the novel protein kinase C (PKC) isoenzymes PKCdelta and PKCepsilon. PKC activation, in turn, decreased SERCA2a promoter activity and destabilized the SERCA2a mRNA. Here we demonstrate by using an RSV beta-galactosidase reporter system that a 609-nt fragment of the SERCA2a mRNA 3'-untranslated region (UTR), containing five adenylate-uridylate (AU)-rich regions, may be responsible for destabilizing the message following PMA treatment. UV cross-linking analysis demonstrated that several proteins found in the NRVM cell extracts bind to the 609-nt fragment. In addition, protein binding was transiently increased in response to PMA stimulation. 3'-UTR mRNA pull-down assays and Western blot analysis indicated that the AU binding protein AUF1 interacted with the SERCA2a 3'-UTR. AUF1 binding activity was predominantly found in the nuclear fraction, and PMA-induced AUF1 binding was associated with increased threonine phosphorylation of AUF1. These data suggest that the phosphorylation, binding, and location of AUF1 affect the posttranscriptional regulation of the SERCA2a message in NRVM.
Blum
Juliana L
JL
The Cardiovascular Institute, Loyola Univ. Medical Center, Bldg 110, Rm. 5222, 2160 South First Ave., Maywood, IL 60153, USA.
Samarel
Allen M
AM
Mestril
Ruben
R
D020413:3' Untranslated Regions / Q000378:metabolism*; D000818:Animals; D000831:Animals, Newborn; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000235:genetics* / Q000378:metabolism*; D002478:Cells, Cultured; D006352:Heart Ventricles / Q000166:cytology / Q000378:metabolism; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism*; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases
D020413:3' Untranslated Regions; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; C000630988:Hnrnpd protein, rat; D012333:RNA, Messenger; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases