Identification of the minimum segment in which the threonine246 residue is a potential phosphorylated site by protein kinase A for the LukS‐specific function of staphylococcal leukocidin

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Abstract
Staphylococcal leukocidin and γ‐hemolysin consist of LukF and LukS for leukocidin and LukF and Hlg2 for γ‐hemolysin. In this report, we identify the minimum segment responsible for the LukS‐specific function of leukocidin. After chemical analysis and homology study of the amino acid sequence of the C‐terminal region between LukS and Hlg2, we found a unique 5‐residue sequence I242K243R244S245T246 in LukS in which the 4‐residue KRST is identical with that of the phosphorylated segment of a protein phosphorylated by protein kinase A. To elucidate whether the 5‐residue segment is essential for the LukS function, we created plasmids containing a series of mutant genes corresponding to the 5‐residue sequence and expressed them in Escherichia coli. The mutant proteins were purified and assayed for their leukocytolytic activity with LukF. The mutant MLS‐TS, in which the T246 in the 5‐residue sequence was replaced by S, showed leukocidin activity 10 times higher than that of the intact LukS. However, neither mutant MLS‐TY nor MLS‐TA, in which T246 was replaced by Y or A, respectively, showed leukocidin activity. The 5‐residue segment was found to be deleted in Hlg2. The mutant of Hlg2, in which the 5‐residue segment was inserted at the position that the segment is deleted, showed leukocidin activity. The boiled LukS, MLS‐TS, and MHS‐Z were strongly phosphorylated with [γ‐32P]ATP in the presence of protein kinase A in a cell‐free system. Thus, we conclude that the 5‐residue segment I242K243R244S245T246 is the pivotal segment of LukS responsible for the LukS function of staphylococcal leukocidin.

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