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.