Characterization of a eukaryotic type serine/threonine kinase in Spodoptera litura nucleopolyhedrovirus-I (SpltNPV-I).
An open reading frame (ORF) of 819nt coding for a predicted protein of 272 amino acids was identified in the genome of Spodoptera litura nucleopolyhedrovirus (SpltNPV-I). Sequence derived amino acid sequence analysis of this ORF suggested it to be a eukaryotic type protein kinase having conserved I-XI subdomains of Hanks kinase. In addition to kinase catalytic domains, this hypothetical protein had two bromodomains which could play regulatory roles in transcription. The ORF was expressed as approximately 31 kDa apoprotein in E. coli and approximately 33 kDa glycoprotein in Sf9 cells, and was called SpltNPV-I pk1 or pk1. The protein was localized in the nuclei of infected cells of the SpltNPV-I permissive cell line, NIV-HA-197. The recombinant protein had autophosphorylation and substrate phosphorylation activities in presence of Mn(2+) or Mg(2+), and these activities were inhibited by staurosporine. Mutation of Lys-50 to Met but not Lys-44 to Gln abolished pk1 kinase activity. Kinetics of pk1 showed that the rate of phosphorylation of SpltNPV-I pk1>MBP>histone H1, and both MBP and histone H1 had the K(m)s of 3muM. Analysis of phosphorylated protein showed the phosphorylation of serine and threonine residues, but not tyrosine. All these results suggested that identified SpltNPV-I ORF codes for a serine/threonine kinase.
Mishra
Gourav
G
Comparative Genomics Unit, Institute of Genomics and Integrative Biology (CSIR), Delhi University Campus, Mall Road, Delhi 110 007, India.
Das
Rakha Hari
RH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005057:Eukaryotic Cells / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D017924:Nucleopolyhedroviruses / Q000201:enzymology* / Q000235:genetics; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D018411:Spodoptera / Q000821:virology*; D013347:Subcellular Fractions
D017346:Protein Serine-Threonine Kinases