RRC ID 38221
Author Kimura Y, Nagata K, Suzuki N, Yokoyama R, Yamanaka Y, Kitamura H, Hirano H, Ohara O.
Title Characterization of multiple alternative forms of heterogeneous nuclear ribonucleoprotein K by phosphate-affinity electrophoresis.
Journal Proteomics
Abstract The phosphorylation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) is thought to play an important role in cell regulation and signal transduction. However, the relationship between hnRNP K phosphorylation and cellular events has only been indirectly examined, and the phosphorylated forms of endogenous hnRNP K have not been biochemically characterized in detail. In this study, we extensively examined the phosphorylated forms of endogenous hnRNP K by direct protein-chemical characterization using phosphate-affinity electrophoresis followed by immunoblotting and MS. Phosphate-affinity electrophoresis enabled us to sensitively detect and separate the phosphorylated forms of hnRNP K. When we used 2-DE with phosphate-affinity SDS-PAGE in the second dimension, the nuclear fraction contained more than 20 spots of endogenous hnRNP K on the 2-D map. We determined that the multiple forms of hnRNP K were produced mainly by alternative splicing of the single hnRNP K gene and phosphorylation of Ser116 and/or Ser284. Furthermore, the subcellular localization of these proteins revealed by the 2-D gel correlated with their phosphorylation states and alternative splicing patterns. The results also indicated that the multiple forms of hnRNP K were differentially modulated in response to external stimulation with bacterial lipopolysaccharide or serum.
Volume 10(21)
Pages 3884-95
Published 2010-11
DOI 10.1002/pmic.201000349
PMID 20960454
MeSH Alternative Splicing Animals Cell Fractionation Cell Line Cell Nucleus / chemistry Cell Nucleus / metabolism Cell-Free System Cytoplasm / chemistry Cytoplasm / metabolism Electrophoresis, Gel, Two-Dimensional / methods* Electrophoresis, Polyacrylamide Gel HeLa Cells Heterogeneous-Nuclear Ribonucleoprotein K / chemistry* Heterogeneous-Nuclear Ribonucleoprotein K / metabolism Humans Immunoblotting Lipopolysaccharides / pharmacology Mass Spectrometry Mice Phosphorylation Protein Biosynthesis / drug effects Protein Isoforms Proteomics / methods*
IF 3.106
Times Cited 13
WOS Category BIOCHEMICAL RESEARCH METHODS BIOCHEMISTRY & MOLECULAR BIOLOGY
Resource
Human and Animal Cells