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

Abbreviations: CaMKIId, calcium/calmodulin-dependent protein kinase II delta, DHFR, dihydrofolate reductase, 2-DE, two-dimensional gel electrophoresis, MALDI-TOF-MS, matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry, bls, biotin ligation site, STIP1, stress-induced phosphoprotein 1, eIF4B, eukaryotic translation initiation factor 4B, mSTI1, murine stress-inducible protein 1, CA, constitutively active, KD, kinase dead

Keywords: Cell-free protein synthesis, Protein kinase, Phosphorylation, Substrate screening, Calcium/calmodulin-dependent protein kinase II, Stress-induced phosphoprotein 1