The wheat germ cell-free based screening of protein substrates of calcium/calmodulin-dependent protein kinase II delta

Abstract

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 CaMKIIδ using a wheat cell-free system. The cell-free mixture expressing CaMKIIδ 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 CaMKIIδ phosphorylates eukaryotic translation initiation factor 4B and stress-induced phosphoprotein 1 (STIP1), the latter on Ser189. Furthermore, constitutively-active CaMKIIδ phosphorylated STIP1 in HeLa cells and dramatically promoted nuclear localization of STIP1, suggesting that calcium signals via CaMKIIδ may regulate subcellular localization of STIP1. This approach may be a useful tool for target screening of protein kinases. Structured summary MINT- 6538664: CAMK2D (uniprotkb: Q13557) phosphorylates (MI: 0217) STIP1 (uniprotkb: P31948) by protein kinase assay (MI: 0424) MINT- 6538652: CAMK2D (uniprotkb: Q13557) phosphorylates (MI: 0217) EIF4B (uniprotkb: P23588) by protein kinase assay (MI: 0424)