Characterization of multiple alternative forms of heterogeneous nuclear ribonucleoprotein K by phosphate-affinity electrophoresis.
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.
Laboratory for Immunogenomics, RIKEN Research Center for Allergy and Immunology, Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, Japan.
Yayoi
Y
Kimura
Kayoko
K
Nagata
Nobutake
N
Suzuki
Ryo
R
Yokoyama
Yuko
Y
Yamanaka
Hiroshi
H
Kitamura
Hisashi
H
Hirano
Osamu
O
Ohara
D017398:Alternative Splicing; D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D002474:Cell-Free System; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D034641:Heterogeneous-Nuclear Ribonucleoprotein K / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008070:Lipopolysaccharides / Q000494:pharmacology; D013058:Mass Spectrometry; D051379:Mice; D010766:Phosphorylation; D014176:Protein Biosynthesis / Q000187:drug effects; D020033:Protein Isoforms; D040901:Proteomics / Q000379:methods*
D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D008070:Lipopolysaccharides; D020033:Protein Isoforms