Phosphorylation of a 25 kDa protein is induced by thermostable direct hemolysin of Vibrio parahaemolyticus.
Thermostable direct hemolysin (TDH) is a possible virulence factor produced by Vibrio parahaemolyticus. Although TDH has a variety of biological activities, including hemolytic activity, the biochemical mechanism of action remains uncertain. Here we analysed biochemical events, especially phosphorylation, caused by TDH in erythrocytes, and found that TDH caused significant phosphorylations of proteins on erythrocyte membrane. Phosphorylation of proteins was studies using [gamma-32P] ATP and SDS-PAGE. A number of protein kinase inhibitors were tested, to determine which types of kinases were involved in the phosphorylation events. TDH induced the phosphorylation of two proteins on membranes of human erythrocyte that are sensitive to TDH. The estimated molecular weight of these proteins was 25 and 22.5 kDa. Interestingly, the 22.5 kDa, but not the 25 kDa protein, was phosphorylated on the membrane of TDH-insensitive (resistant) horse erythrocytes. Moreover, a mutant TDH (R7), which retained binding ability but lost hemolytic activity, also phosphorylated only the 22.5 kDa protein on human erythrocyte membranes. Among the protein kinase inhibitors used the protein kinase C inhibitors, (staurosporine and calphostin C) showed marked inhibition of phosphorylation of 25kDa protein. In addition to phosphorylation, these protein kinase C inhibitors suppressed hemolysis by TDH. These results indicate that the phosphorylation of the 25 kDa protein seems to be essential for the hemolysis by TDH after it binds to erythrocyte membranes.
Yoh
M
M
Department of Bacterial Infections, Research Institute for Microbial diseases, Osaka University, Japan.
Tang
G Q
GQ
Iida
T
T
Morinaga
N
N
Noda
M
M
Honda
T
T
D001427:Bacterial Toxins; D004910:Erythrocyte Membrane / Q000187:drug effects* / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D006461:Hemolysis / Q000187:drug effects / Q000502:physiology; D006801:Humans; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000097:blood* / Q000737:chemistry; D008970:Molecular Weight; D009154:Mutation; D010766:Phosphorylation; D014736:Vibrio parahaemolyticus / Q000235:genetics / Q000472:pathogenicity*; D014774:Virulence
D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D008565:Membrane Proteins; C068489:thermostable direct hemolysin