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.
Publication types
-
Research Support, Non-U.S. Gov't
-
Research Support, U.S. Gov't, P.H.S.
MeSH terms
-
Alkaloids / pharmacology
-
Amino Acid Sequence
-
Animals
-
Base Sequence
-
Blotting, Western
-
Casein Kinase II
-
Cell Line
-
Cloning, Molecular
-
Cricetinae
-
DNA Primers
-
Electrophoresis, Polyacrylamide Gel
-
Escherichia coli
-
Genes, Viral
-
Histidine
-
Kidney
-
Measles virus / genetics
-
Measles virus / metabolism*
-
Molecular Sequence Data
-
Phosphoproteins / metabolism*
-
Phosphorylation
-
Polymerase Chain Reaction
-
Protein Kinase C / antagonists & inhibitors
-
Protein Serine-Threonine Kinases / metabolism*
-
Recombinant Proteins / chemistry
-
Recombinant Proteins / isolation & purification
-
Recombinant Proteins / metabolism
-
Restriction Mapping
-
Sequence Tagged Sites
-
Serine
-
Staurosporine
-
Viral Proteins / chemistry
-
Viral Proteins / isolation & purification
-
Viral Proteins / metabolism*
Substances
-
Alkaloids
-
DNA Primers
-
P protein, Sendai virus
-
Phosphoproteins
-
Recombinant Proteins
-
Viral Proteins
-
Serine
-
Histidine
-
Casein Kinase II
-
Protein Serine-Threonine Kinases
-
Protein Kinase C
-
Staurosporine