Protein kinase CK1α regulates mRNA binding by heterogeneous nuclear ribonucleoprotein c in response to physiologic levels of hydrogen peroxide

At low concentrations, hydrogen peroxide (H2O2) is a positive endogenous regulator of mammalian cell proliferation and survival; however, the signal transduction pathways involved in these processes are poorly understood. In primary human endothelial cells, low concentrations of H2O2 stimulated the rapid phosphorylation of the acidic C-terminal domain (ACD) of heterogeneous nuclear ribonucleoprotein C (hnRNP-C), a nuclear restricted pre-mRNA-binding protein, at Ser(240) and at Ser(225)-Ser(228). A kinase activity was identified in mouse liver that phosphorylates the ACD of hnRNP-C at Ser(240) and at two sites at Ser(225)-Ser(228). The kinase was purified and identified by tandem mass spectrometry as protein kinase CK1 alpha (formerly casein kinase 1 alpha). Protein kinase CK1 alpha immunoprecipitated from primary human endothelial cell nuclei also phosphorylated the ACD of hnRNP-C at these positions. Pretreatment of endothelial cells with the protein kinase CK1-specific inhibitor IC261 prevented the H2O2-stimulated phosphorylation of hnRNP-C. Utilizing phosphoserine-mimicking Ser-to-Glu point mutations, the effects of phosphorylation on hnRNP-C function were investigated by quantitative equilibrium fluorescence RNA binding analyses. Wild-type hnRNP-C1 and hnRNP-C1 modified at the basal sites of phosphorylation (S247E and S286E) both avidly bound RNA with similar binding constants. In contrast, hnRNP-C1 that was also modified at the CK1 alpha phosphorylation sites exhibited a 14-500-fold decrease in binding affinity, demonstrating that CK1 alpha-mediated phosphorylation modulates the mRNA binding ability of hnRNP-C.