Characterization of a polypeptide-dependent membrane protein kinase that specifically phosphorylates NS protein of vesicular stomatitis virus in vitro.
Phosphorylation of membrane-associated proteins by protein kinases in the membrane fraction from HeLa S3 cells was rapidly increased when the cells were infected with vesicular stomatitis virus (VSV). SDS-PAGE followed by autoradiography revealed polypeptides with molecular sizes of Mr. 53,000, 44,000, 42,000, 35,000, 30,000 and 27,000 in the kinase fraction from uninfected cells to be highly phosphorylated. Virus-coding NS protein (Mr. 40,000) was phosphorylated when the membrane fraction from virus-infected cells was incubated with [gamma-32P]ATP in the presence of histone H1 and Mg2+. Under these conditions, histone H1 functioned as a stimulator for NS protein phosphorylation by the kinases. One (kinase III) of the membrane-associated kinases was partially purified from HeLa S3 cells using FPLC (type Mono Q) after DEAE-cellulose column chromatography. The enzymatic properties of kinase III were similar to those reported for a polypeptide-dependent protein kinase (protein kinase P), because (a) both kinases highly phosphorylated beta-casein, although no phosphorylation was observed with histones; (b) several endogenous substrates from HeLa S3 cell membrane were phosphorylated by the kinases in the presence of basic proteins, such as histones, protamine and poly-Lys; (c) their activity was insensitive to a low concentration (19 micrograms/ml) of heparin, which highly inhibited casein kinase II activity; and (d) the kinases were extractable from the plasma membrane using Triton X-100. In addition, provided evidence suggests that kinase III may play an important role in an early stage of VSV replication through its specific phosphorylation of NS protein and membrane proteins in virus infected cells.
Ikeuchi
T
T
Virus Section, Research Institute of Daiichi Seiyaku Co., Ltd., Tokyo, Japan.
Yokoyama
M
M
Ohtsuki
K
K
D000818:Animals; D001345:Autoradiography; D002213:Capsid / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000032:analysis*; D014721:Vesicular stomatitis Indiana virus / Q000378:metabolism*; D014758:Viral Core Proteins / Q000378:metabolism*; D017361:Viral Nonstructural Proteins
D008565:Membrane Proteins; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; D011494:Protein Kinases