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 413 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 STIPI. This approach may be a useful tool for target screening of protein kinases.