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. 2010 Nov;10(21):3884-95.
doi: 10.1002/pmic.201000349.

Characterization of multiple alternative forms of heterogeneous nuclear ribonucleoprotein K by phosphate-affinity electrophoresis

Yayoi Kimura  1 , Kayoko Nagata, Nobutake Suzuki, Ryo Yokoyama, Yuko Yamanaka, Hiroshi Kitamura, Hisashi Hirano, Osamu Ohara
Affiliations

Characterization of multiple alternative forms of heterogeneous nuclear ribonucleoprotein K by phosphate-affinity electrophoresis

Yayoi Kimura et al. Proteomics. 2010 Nov.

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.

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