Premium
Characterization of multiple alternative forms of heterogeneous nuclear ribonucleoprotein K by phosphate‐affinity electrophoresis
Author(s) -
Kimura Yayoi,
Nagata Kayoko,
Suzuki Nobutake,
Yokoyama Ryo,
Yamanaka Yuko,
Kitamura Hiroshi,
Hirano Hisashi,
Ohara Osamu
Publication year - 2010
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201000349
Subject(s) - heterogeneous nuclear ribonucleoprotein , phosphorylation , ribonucleoprotein , alternative splicing , rna splicing , heterogeneous ribonucleoprotein particle , biology , microbiology and biotechnology , endogeny , gel electrophoresis , small nuclear ribonucleoprotein , biochemistry , gene , rna , messenger rna
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.