RRC ID |
38221
|
著者 |
Kimura Y, Nagata K, Suzuki N, Yokoyama R, Yamanaka Y, Kitamura H, Hirano H, Ohara O.
|
タイトル |
Characterization of multiple alternative forms of heterogeneous nuclear ribonucleoprotein K by phosphate-affinity electrophoresis.
|
ジャーナル |
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.
|
巻・号 |
10(21)
|
ページ |
3884-95
|
公開日 |
2010-11-1
|
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 |
3.254
|
引用数 |
20
|
WOS 分野
|
BIOCHEMICAL RESEARCH METHODS
BIOCHEMISTRY & MOLECULAR BIOLOGY
|
リソース情報 |
ヒト・動物細胞 |
|