Journal
PROTEOMICS
Volume 10, Issue 21, Pages 3884-3895Publisher
WILEY-BLACKWELL
DOI: 10.1002/pmic.201000349
Keywords
2-D phosphate-affinity gel electrophoresis; Alternative splicing; Animal Proteomics; Heterogeneous nuclear ribonucleoprotein K; Mass spectrometry; Phosphorylation
Funding
- Special Coordination Funds for Promoting Science and Technology Creation of Innovation Centers for Advanced Interdisciplinary Research Areas
- Grants-in-Aid for Scientific Research [21790334] Funding Source: KAKEN
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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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