Post-translational phosphorylation of Pseudomonas aeruginosa flagellin by an envelope protein kinase

Previous results indicated that Pseudomonas aeruginosa flagellar filament protein was phosphorylated in vivo at tyrosine. However, use of more rigorous, 2-dimensional electrophoresis techniques could not confirm this finding, and also as previously shown as neither P-serine nor P-threonine could be identified. We have reported identification of a putative kinase in vitro in envelope preparations which phosphorylated both flagellin protein and the synthetic random co-polymer (Glu-tyr) (South et al., 1994). More recent in vitro investigations indicated that flagellin was phosphorylated at threonine. Phosphoserine was identified in flagellin after partial enzymatic degradation and dephosphorylation by bacterial alkaline phosphatase (BAP) prior to labeling. The putative membrane protein kinase (Mr 42 KDa) also was phosphorylated at threonine and serine. Evidence of protein kinase activity included rapid labeling at 10 °C and sequential transfer of phosphate to flagellin at 37 °C. This system could utilize Pi from [γ-³²]ATP or [γ-³²]GTP. Inhibition of kinase activity by a ser/thr kinase inhibitor, H-7 and the -SH inhibitor, N-ethylmaleimide (NEM) was shown. Using phosphoamino acid specific phosphatases it was shown that a ser/thr phosphatase (Lambda), but not tyrosine specific phosphatase (Yop) removed prelabeled phosphate from flagellin or the kinase. In vitro experiments using selected serine, threonine or tyrosine containing synthetic peptides showed extent of labeling ser >>thr> tyr (Nichols and Montie unpublished data), or thr >>ser >tyr in the peptide. These in vitro results indicate that the 42 KDa membrane protein kinase is primarily a ser/thr kinase that phosphorylates flagellin at threonine and serine. However, since several synthetic polypeptides can be phosphorylated at tyrosine in vitro, the kinase may phosphorylate in vivo some substrates including flagellin, secondarily at tyrosine. Phosphorylation of membrane-associated flagellin, readily occurs which suggests that the membrane protein kinase may be important for export of flagellin precursors. Attempts are being made to analyze the endogenous flagellin for phosphoamino acids and N-terminal of the membrane protein kinase.