Involvement of cellular casein kinase II in the phosphorylation of measles virus P protein: identification of phosphorylation sites

Abstract

The phosphoprotein P gene of measles virus (Edmonston strain) has been cloned in the Escherichia coli expression vector pET-3a with a histidine tag at the C-terminal end. The expressed protein was soluble, unphosphorylated, and constituted 10 to 20% of total cellular protein. Recombinant P protein purified by Ni-affinity chromatography was found to be efficiently phosphorylated in vitro by recombinant casein kinase II (CKII) or by the CKII activity present in the uninfected cell extract. A comparison of phosphopeptide analyses between the in vivo- and the in vitro-32P-labeled P proteins revealed that both proteins share common phosphorylation sites. In an attempt to identify the exact site of the CKII-mediated phosphorylation, we altered specific serine residues located within the CKII consensus motif to alanine by site-directed mutagenesis. The results indicate that Ser 86, Ser 151, and Ser 180 located within the N-terminal half of the P protein are involved in the CKII-mediated phosphorylation of the P protein.