ABSTRACT
Previous evidence showed that b- and a-type flagellins of Pseudomonas aeruginosa are modified in vivo by phosphorylation at tyrosine. This research was designed to demonstrate phosphorylation of flagellin at tyrosine in vitro. Evidence presented showed that flagellin is labelled by [gamma-32P]-ATP, but not by [alpha-32P]-ATP, when incubated with cell envelope fractions. Results suggested that autophosphorylation of a 42 kDa membrane protein occurred. No activity was detected in cytoplasmic fractions. Flagellin protein was identified by flagella-specific monoclonal antibody (mAb) and was labelled with anti-phosphotyrosine mAb. Confirmation of tyrosine kinase activity was shown by labelling of synthetic poly(Glu:Tyr) as a substrate with [gamma-32P]-ATP. Labelling of poly(Glu:Tyr) was heat sensitive and time dependent. Labelled phosphotyrosine was observed in partial acid hydrolysates of substrates. Using poly(Glu:Tyr) as substrate, tyrosine kinase activity was shown to be inhibited by sulphydryl reagents. It appears that tyrosine kinase and flagellin phosphorylation occur in several Pseudomonas spp. Location of phosphotyrosine in a conserved region of flagellin may serve as a cell signal so that intact flagellin is appropriately exported.
Subject(s)
Flagellin/metabolism , Protein-Tyrosine Kinases/metabolism , Pseudomonas aeruginosa/enzymology , Adenosine Triphosphate , Ethylmaleimide/pharmacology , Mercuric Chloride/pharmacology , Phosphorylation/drug effects , Phosphotyrosine , Protein-Tyrosine Kinases/antagonists & inhibitors , Substrate Specificity , Tyrosine/analogs & derivatives , Tyrosine/analysis , Tyrosine/metabolismABSTRACT
Both a- and b-type purified flagellins from a number of Pseudomonas aeruginosa strains grown in radiolabeled phosphate were shown to be phosphorylated. Analysis of partial acid-hydrolyzed flagellar filaments revealed that 32Pi was in phosphotyrosine. Three 32P-phosphopeptides apparently are common to a- and b-type flagellins, but a fourth peptide was found only in b-type hydrolysates. P. aeruginosa PAK flagellin, containing only two tyrosines, both in the variable region, was readily labeled and gave the same peptide pattern as flagellins containing additional tyrosines. Data showing that a- and b-type flagellins gave positive immunoblots with antiphosphotyrosine monoclonal antibody and that release of P(i) by alkaline phosphatase occurred indicated that unmodified tyrosine phosphate exists in flagellin.
