Tyrosine kinase activity in Pseudomonas aeruginosa.
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.
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Department of Microbiology, University of Tennessee, Knoxville 37996-0845.
Nichols
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Montie
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D000255:Adenosine Triphosphate; D005033:Ethylmaleimide / Q000494:pharmacology; D005408:Flagellin / Q000378:metabolism*; D008627:Mercuric Chloride / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013379:Substrate Specificity; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism
D005408:Flagellin; D019000:Phosphotyrosine; D014443:Tyrosine; D008627:Mercuric Chloride; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D005033:Ethylmaleimide