Protein kinase C alpha-dependent phosphorylation of the mRNA-stabilizing factor HuR: implications for posttranscriptional regulation of cyclooxygenase-2

Doller, Anke; Huwiler, Andrea; Müller, Roswitha; Radeke, Heinfried H; Pfeilschifter, Josef; Eberhardt, Wolfgang (2007). Protein kinase C alpha-dependent phosphorylation of the mRNA-stabilizing factor HuR: implications for posttranscriptional regulation of cyclooxygenase-2. Molecular biology of the cell, 18(6), pp. 2137-48. Bethesda, Md.: American Society for Cell Biology 10.1091/mbc.E06-09-0850

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In this study, we investigated the molecular mechanisms underlying the ATP analogue adenosine-5'-O-(3-thio)triphosphate-induced nucleocytoplasmic shuttling of the mRNA stabilizing factor HuR in human (h) mesangial cells (MC). Using synthetic protein kinase C (PKC) inhibitors and small interfering RNA approaches, we demonstrated that knockdown of PKC alpha efficiently blocked the ATP-dependent nuclear HuR export to the cytoplasm. The functional importance of PKC alpha in HuR shuttling is highlighted by the high cytosolic HuR content detected in hMC stably overexpressing PKC alpha compared with mock-transfected cells. The ATP-induced recruitment of HuR to the cytoplasm is preceded by a direct interaction of PKC alpha with nuclear HuR and accompanied by increased Ser phosphorylation as demonstrated by coimmunoprecipitation experiments. Mapping of putative PKC target sites identified serines 158 and 221 as being indispensable for HuR phosphorylation by PKC alpha. RNA pull-down assay and RNA electrophoretic mobility shift assay demonstrated that the HuR shuttling by ATP is accompanied by an increased HuR binding to cyclooxygenase (COX)-2 mRNA. Physiologically, the ATP-dependent increase in RNA binding is linked with an augmentation in COX-2 mRNA stability and subsequent increase in prostaglandin E(2) synthesis. Regulation of HuR via PKC alpha-dependent phosphorylation emphasizes the importance of posttranslational modification for stimulus-dependent HuR shuttling.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Pharmacology

UniBE Contributor:

Huwiler, Andrea

ISSN:

1059-1524

ISBN:

17392515

Publisher:

American Society for Cell Biology

Language:

English

Submitter:

Factscience Import

Date Deposited:

04 Oct 2013 14:54

Last Modified:

04 May 2014 23:15

Publisher DOI:

10.1091/mbc.E06-09-0850

PubMed ID:

17392515

Web of Science ID:

000246977600016

URI:

https://boris.unibe.ch/id/eprint/23006 (FactScience: 38537)

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