Identification and characterization of serine/threonine protein kinase activity intrinsic to the L protein of vesicular stomatitis virus New Jersey.
A photoaffinity analogue of ATP, 8-azido-adenosine 5'-triphosphate (8-N3ATP), was used to probe ATP-binding sites in native transcription complexes of vesicular stomatitis virus (VSV) (New Jersey serotype). The analogue was found to be a substrate for a serine/threonine protein kinase that phosphorylated both the NS and L proteins of native complexes. The analogue failed to interact with the RNA polymerase, another ATP-utilizing activity associated with the transcription complex. Kinetic analyses of both ATP and 8-N3ATP utilization by the protein kinase yielded biphasic saturation curves. Photolysis of 8-N3ATP in the presence of VSV transcription complexes resulted in selective labelling of the L protein. The photolabelling of L was saturable and apparently biphasic. Photolabelling of the L protein was significantly reduced by competition with ATP whereas other nucleoside triphosphates (GTP, UTP and CTP) were ineffective competitors. The stoichiometry of photolabelling was 0.2 at 10 microM-8N3ATP and 1.3 at 100 microM-ATP. These data provide chemical evidence for a virus-encoded serine/threonine protein kinase which resides on the L protein.
Hammond
D C
DC
School of Biological Sciences, Lucille P. Markey Cancer Center, University of Kentucky, Lexington 40506-0225.
Haley
B E
BE
Lesnaw
J A
JA
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000345:Affinity Labels; D001386:Azides / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D007700:Kinetics; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D012324:RNA-Dependent RNA Polymerase; D018116:Vesiculovirus / Q000201:enzymology*; D014764:Viral Proteins / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; D014764:Viral Proteins; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; C040530:L protein, vesicular stomatitis virus; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases