Involvement of cellular casein kinase II in the phosphorylation of measles virus P protein: identification of phosphorylation sites.
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.
Das
T
T
Department of Molecular Biology, Cleveland Clinic Foundation, Ohio 44195, USA.
Schuster
A
A
Schneider-Schaulies
S
S
Banerjee
A K
AK
D000470:Alkaloids / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D047390:Casein Kinase II; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005814:Genes, Viral; D006639:Histidine; D007668:Kidney; D008459:Measles virus / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D016324:Sequence Tagged Sites; D012694:Serine; D019311:Staurosporine; D014764:Viral Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D000470:Alkaloids; D017931:DNA Primers; C055859:P protein, Sendai virus; D010750:Phosphoproteins; D011994:Recombinant Proteins; D014764:Viral Proteins; D012694:Serine; D006639:Histidine; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D019311:Staurosporine