Identification of a physiological phosphorylation site of the herpes simplex virus 1-encoded protein kinase Us3 which regulates its optimal catalytic activity in vitro and influences its function in infected cells.
Us3 is a serine/threonine protein kinase encoded by herpes simplex virus 1 (HSV-1). Here, we report the identification of a physiological Us3 phosphorylation site on serine at position 147 (Ser-147) which regulates its protein kinase activity in vitro. Moreover, mutation of this site influences Us3 function, including correct localization of the enzyme and induction of the usual morphological changes in HSV-1-infected cells. These conclusions are based on the following observations: (i) in in vitro kinase assays, a domain of Us3 containing Ser-147 was specifically phosphorylated by Us3 and protein kinase A, while a mutant domain in which Ser-147 was replaced with alanine was not; (ii) in vitro, alanine replacement of Ser-147 (S147A) in Us3 resulted in significant impairment of the kinase activity of the purified molecule expressed in a baculovirus system; (iii) phosphorylation of Ser-147 in Us3 tagged with the monomeric fluorescent protein (FP) VenusA206K (VenusA206K-Us3) from Vero cells infected with a recombinant HSV-1 encoding VenusA206K-Us3 was specifically detected using an antibody that recognizes phosphorylated serine or threonine residues with arginine at the -3 and -2 positions; and (iv) the S147A mutation influenced some but not all Us3 functions, including the ability of the protein to localize itself properly and to induce wild-type cytopathic effects in infected cells. Our results suggest that some of the regulatory activities of Us3 in infected cells are controlled by phosphorylation at Ser-147.
Kato
Akihisa
A
Department of Infectious Disease Control, International Research Center for Infectious Diseases, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
Tanaka
Michiko
M
Yamamoto
Mayuko
M
Asai
Risa
R
Sata
Tetsutaro
T
Nishiyama
Yukihiro
Y
Kawaguchi
Yasushi
Y
D000818:Animals; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics*; D002522:Chlorocebus aethiops; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli; D005455:Fluorescent Antibody Technique; D016679:Genome, Viral / Q000235:genetics*; D015151:Immunoblotting; D047468:Immunoprecipitation; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism*
D017931:DNA Primers; D014764:Viral Proteins; D012694:Serine; D017346:Protein Serine-Threonine Kinases; C095207:US3 protein, Human herpesvirus 1; D017868:Cyclic AMP-Dependent Protein Kinases