Rapid phosphorylation of heterogeneous nuclear ribonucleoprotein C1/C2 in response to Physiologic levels of hydrogen peroxide in human endothelial cells

Hydrogen peroxide (H2O2) has been implicated as a signaling agent in numerous signal transduction pathways in mammalian cells. However, to date, no sensor for low concentrations (<10 mu M) of H2O2 has been identified. Using a functional proteomic approach, nuclear extracts from human umbilical vein endothelial cells were analyzed by two-dimensional PAGE with or without prior treatment with a low concentration of H2O2. A protein doublet with a molecular mass of 39-41 kDa and a pI of similar to 5.0 was observed to be consistently altered by the treatment. Using proteolytic peptide mass fingerprinting, the protein was identified as heterogeneous nuclear ribonucleoprotein C1/C2, a nuclear restricted, pre-mRNA-binding protein. Upon two-dimensional PAGE, each heterogeneous nuclear ribonucleoprotein-C splice form was present as multiple spots because of differing levels of phosphorylation. Upon treatment with H2O2, there was an increase in phosphorylation at 10-20 min, which partially reversed by 30 min. Subsequently, at 60 min after treatment, a population of unphosphorylated protein was transiently present. The effects were observed with as little as 1 mu M H2O2 and were maximal with 5-8 mu M H2O2. The H2O2-stimulated phosphorylation was inhibited by catalase, but not by the transcriptional inhibitor actinomycin D.