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

Mol Biol Cell. 2007 Jun;18(6):2137-48. doi: 10.1091/mbc.e06-09-0850. Epub 2007 Mar 28.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Active Transport, Cell Nucleus / physiology
  • Adenosine Triphosphate / metabolism
  • Antigens, Surface / genetics
  • Antigens, Surface / metabolism*
  • Cells, Cultured
  • Cyclooxygenase 2 / genetics
  • Cyclooxygenase 2 / metabolism*
  • ELAV Proteins
  • ELAV-Like Protein 1
  • Enzyme Inhibitors / metabolism
  • Humans
  • Interleukin-1beta / metabolism
  • Mesangial Cells / cytology
  • Mesangial Cells / metabolism
  • Mutagenesis, Site-Directed
  • Phosphorylation
  • Protein Kinase C-alpha / metabolism*
  • RNA Processing, Post-Transcriptional*
  • RNA Stability
  • RNA, Messenger / metabolism
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism*
  • Response Elements
  • Serine / metabolism

Substances

  • Antigens, Surface
  • ELAV Proteins
  • ELAV-Like Protein 1
  • ELAVL1 protein, human
  • Enzyme Inhibitors
  • Interleukin-1beta
  • RNA, Messenger
  • RNA, Small Interfering
  • RNA-Binding Proteins
  • Serine
  • Adenosine Triphosphate
  • Cyclooxygenase 2
  • Protein Kinase C-alpha