Rapid phosphorylation of heterogeneous nuclear ribonucleoprotein C1/C2 in response to physiologic levels of hydrogen peroxide in human endothelial cells.
Hydrogen peroxide (H(2)O(2)) has been implicated as a signaling agent in numerous signal transduction pathways in mammalian cells. However, to date, no sensor for low concentrations (<10 microm) of H(2)O(2) 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 H(2)O(2). A protein doublet with a molecular mass of 39-41 kDa and a pI of approximately 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 H(2)O(2), 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 microm H(2)O(2) and were maximal with 5-8 microm H(2)O(2). The H(2)O(2)-stimulated phosphorylation was inhibited by catalase, but not by the transcriptional inhibitor actinomycin D.
Stone
James R
JR
Department of Pathology, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Collins
Tucker
T
D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D004730:Endothelium, Vascular / Q000378:metabolism*; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D015151:Immunoblotting; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D012261:Ribonucleoproteins / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin; D014471:Umbilical Veins
C439788:C1 HNRNP; C503635:HNRNPC protein, human; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D010446:Peptide Fragments; D012261:Ribonucleoproteins; D006861:Hydrogen Peroxide; D014357:Trypsin