The wheat germ cell-free based screening of protein substrates of calcium/calmodulin-dependent protein kinase II delta.
Calcium/calmodulin-dependent protein kinase II (CaMKII) plays a crucial role in mediating calcium signaling. Here, we demonstrate a method for screening substrates phosphorylated by human CaMKII delta using a wheat cell-free system. The cell-free mixture expressing CaMKII delta was incubated with HeLa extracts and radiolabeled ATP. From analysis of two-dimensional electrophoresis gels and mass spectrometry, two proteins were found. The cell-free based in vitro kinase assay revealed that CaMKII delta phosphorylates eukaryotic translation initiation factor 4B and stress-induced phosphoprotein 1 (STIP1), the latter on Ser189. Furthermore, constitutively-active CaMKII delta phosphorylated STIP1 in HeLa cells and dramatically promoted nuclear localization of STIP1, suggesting that calcium signals via CaMKII delta may regulate subcellular localization of STIP1. This approach may be a useful tool for target screening of protein kinases.
Masaoka
Takashi
T
Cell-free Science and Technology Research Center, Ehime University, Matsuyama 790-8577, Japan.
Nishi
Mayuko
M
Ryo
Akihide
A
Endo
Yaeta
Y
Sawasaki
Tatsuya
T
D021581:Active Transport, Cell Nucleus; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism*; D002474:Cell-Free System; D004252:DNA Mutational Analysis; D039642:Eukaryotic Initiation Factors / Q000378:metabolism; D005854:Germ Cells / Q000737:chemistry / Q000166:cytology; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008722:Methods; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D013379:Substrate Specificity; D014908:Triticum / Q000737:chemistry / Q000166:cytology
D039642:Eukaryotic Initiation Factors; D006360:Heat-Shock Proteins; C079373:STIP1 protein, human; C031799:eIF-4B; C517660:CAMK2D protein, human; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2