Human parainfluenza virus type 3 phosphoprotein: identification of serine 333 as the major site for PKC zeta phosphorylation.
The human parainfluenza virus type 3 P protein is an RNA polymerase subunit involved in both transcription and replication during the life cycle of the virus. Our laboratory has recently shown that the P protein is phosphorylated both in vitro and in vivo by the cellular protein kinase C (PKC) isoform zeta and that this phosphorylation is essential for viral replication. To identify the site(s) of phosphorylation, we have used CNBr cleavage, phosphoamino acid analysis, and two-dimensional tryptic peptide mapping of the in vitro and in vivo phosphorylated P protein. We demonstrate that when bacterially expressed unphosphorylated P is labeled in vitro with either commercial PKC or purified recombinant PKC zeta P protein has one major phosphorylation site. By site-directed mutagenesis of PKC consensus sites in the P protein, the primary phosphorylation site is found to be Ser 333. The same site appeared to be modified when viral P protein was phosphorylated in vitro by the PKC packaged within the virion and in the P protein of progeny virion labeled in vivo.
Huntley
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Department of Molecular Biology, Research Institute, Cleveland Clinic Foundation, Ohio 44195, USA.
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Murray
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Banerjee
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D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D006801:Humans; D008969:Molecular Sequence Data; D010224:Parainfluenza Virus 3, Human / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D013379:Substrate Specificity; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D000099221:Protein Kinase C zeta
D017931:DNA Primers; D010750:Phosphoproteins; D011493:Protein Kinase C; D012694:Serine; D014764:Viral Proteins; D000099221:Protein Kinase C zeta; C095185:P protein, Human parainfluenza virus 3