title: Bacteriophage T7 protein kinase: Site of inhibitory autophosphorylation, and use of dephosphorylated enzyme for efficient modification of protein in vitro.
abstract: Bacteriophage T7 encodes a serine/threonine-specific protein kinase that phosphorylates multiple cellular proteins during infection of Escherichia coli. Recombinant T7 protein kinase (T7PK), normally purified in phosphorylated form, exhibits a modest level of phosphotransferase activity. A procedure is described that provides dephosphorylated T7PK with an enhanced ability to phosphorylate protein substrates, including translation initiation factor IF1 and the nuclease domain of ribonuclease III. Mass spectrometric analysis identified Thr12 as the site of IF1 phosphorylation in vitro. T7PK undergoes Mg(2+)-dependent autophosphorylation on Ser216 in vitro, which also is modified in vivo. The inability to isolate the presumptive autophosphorylation-resistant T7PK Ser216Ala mutant indicates a toxicity of the phosphotransferase activity and suggests a role for Ser216 modification in limiting T7PK activity during infection.
authors.affiliation: Department of Chemistry, Temple University, 1901 North 13th St., Philadelphia, PA 19122, USA.
authors.forename: Swapna
authors.initials: S
authors.lastname: Gone
authors.forename: Allen W
authors.initials: AW
authors.lastname: Nicholson
mesh_terms: D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D020134:Catalytic Domain; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D039662:Prokaryotic Initiation Factors / Q000737:chemistry / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D043244:Ribonuclease III / Q000737:chemistry / Q000378:metabolism; D014764:Viral Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D039662:Prokaryotic Initiation Factors; D011994:Recombinant Proteins; D014764:Viral Proteins; C072445:0.7 protein, Enterobacteria phage T7; D017346:Protein Serine-Threonine Kinases; D043244:Ribonuclease III; D008274:Magnesium