Tandem phosphorylation of serines 221 and 318 by protein kinase Cdelta coordinates mRNA binding and nucleocytoplasmic shuttling of HuR.
Stabilization of mRNA by the ubiquitous RNA binding protein human antigen R (HuR), a member of the embryonic lethal abnormal vision (ELAV) protein family, requires canonical binding to AU-rich element (ARE)-bearing target mRNA and export of nuclear HuR-mRNA complexes to the cytoplasm. In human mesangial cells (HMC) both processes are induced by angiotensin II (AngII) via protein kinase Cdelta (PKCdelta)-triggered serine phosphorylation of HuR. By testing different point-mutated Flag-tagged HuR proteins, we found that Ser 318 within RNA recognition motif 3 (RRM3) is essential for AngII-induced binding to ARE-bearing mRNA but irrelevant for nucleocytoplasmic HuR shuttling. Conversely, mutation at Ser 221 within the HuR hinge region prevents AngII-triggered HuR export without affecting mRNA binding of HuR. Using phosphorylation state-specific antibodies, we found a transient increase in HuR phosphorylation at both serines by AngII. Functionally, PKCdelta mediates the AngII-induced stabilization of prominent HuR target mRNAs, including those of cyclin A, cyclin D(1), and cyclooxygenase-2 (COX-2), and is indispensable for AngII-triggered migration and wound healing of HMC. Our data suggest a regulatory paradigm wherein a simultaneous phosphorylation at different domains by PKCdelta coordinates mRNA binding and nucleocytoplasmic shuttling of HuR, both of which events are essentially involved in the stabilization of HuR target mRNAs and relevant cell functions.
Doller
Anke
A
Pharmazentrum Frankfurt/ZAFES, Klinikum der Johann Wolfgang Goethe-Universität Frankfurt am Main, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany.
Schlepckow
Kai
K
Schwalbe
Harald
H
Pfeilschifter
Josef
J
Eberhardt
Wolfgang
W
D000804:Angiotensin II / Q000494:pharmacology; D000954:Antigens, Surface / Q000378:metabolism*; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D019925:Cyclin A / Q000235:genetics / Q000378:metabolism; D019938:Cyclin D1 / Q000235:genetics / Q000378:metabolism; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism*; D017354:Point Mutation / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D051745:Protein Kinase C-delta / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D038621:Regulatory Sequences, Ribonucleic Acid / Q000235:genetics; D013997:Time Factors
D000954:Antigens, Surface; D019925:Cyclin A; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; C098770:ELAVL1 protein, human; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D038621:Regulatory Sequences, Ribonucleic Acid; D000804:Angiotensin II; D019938:Cyclin D1; D010768:Phosphoserine; D051745:Protein Kinase C-delta