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FEBS Lett. 1997 Sep 22;415(1):96-100.

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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Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, Sendai, Japan.


Staphylococcal leukocidin and gamma-hemolysin consist of LukF and LukS for leukocidin and LukF and Hlg2 for gamma-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 [gamma-32P]ATP in the presence of protein kinase A in a cell-free system. Thus, we conclude that the 5-residue segment 1242K243R244S245T246 is the pivotal segment of LukS responsible for the LukS function of staphylococcal leukocidin.

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