Phosphorylation of simian cytomegalovirus assembly protein precursor (pAPNG.5) and proteinase precursor (pAPNG1): multiple attachment sites identified, including two adjacent serines in a casein kinase II consensus sequence.
The assembly protein precursor (pAP) of cytomegalovirus (CMV), and its homologs in other herpesviruses, functions at several key steps during the process of capsid formation. This protein, and the genetically related maturational proteinase, is distinguished from the other capsid proteins by posttranslational modifications, including phosphorylation. The objective of this study was to identify sites at which pAP is phosphorylated so that the functional significance of this modification and the enzyme(s) responsible for it can be determined. In the work reported here, we used peptide mapping, mass spectrometry, and site-directed mutagenesis to identify two sets of pAP phosphorylation sites. One is a casein kinase II (CKII) consensus sequence that contains two adjacent serines, both of which are phosphorylated. The other site(s) is in a different domain of the protein, is phosphorylated less frequently than the CKII site, does not require preceding CKII-site phosphorylation, and causes an electrophoretic mobility shift when phosphorylated. Transfection/expression assays for proteolytic activity showed no gross effect of CKII-site phosphorylation on the enzymatic activity of the proteinase or on the substrate behavior of pAP. Evidence is presented that both the CKII sites and the secondary sites are phosphorylated in virus-infected cells and plasmid-transfected cells, indicating that these modifications can be made by a cellular enzyme(s). Apparent compartmental differences in phosphorylation of the CKII-site (cytoplasmic) and secondary-site (nuclear) serines suggest the involvement of more that one enzyme in these modifications.
Plafker
S M
SM
Virology Laboratories, Department of Pharmacology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Woods
A S
AS
Gibson
W
W
D000595:Amino Acid Sequence; D000818:Animals; D002213:Capsid / Q000737:chemistry; D047390:Casein Kinase II; D002460:Cell Line; D016384:Consensus Sequence; D003587:Cytomegalovirus / Q000502:physiology*; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D010766:Phosphorylation; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*; D019065:Virus Assembly
D004792:Enzyme Precursors; D011498:Protein Precursors; D014764:Viral Proteins; C058313:assembly protein, Cytomegalovirus; D012694:Serine; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D012697:Serine Endopeptidases; C071910:assemblin