EXPRESSION AND CHARACTERIZATION OF A RAC FAMILY PROTEIN-KINASE OF ENTAMOEBA-HISTOLYTICA

We previously reported the isolation from Entamoeba histolytica of a n ovel rac family protein kinase gene, termed Ehrac1, for ''related to c AMP-dependent protein kinases and protein kinase Cs''. To study the fu nction and properties of this kinase gene further, we fused the full-l ength coding region and the truncated catalytic domain of the Ehrac1 g ene in frame with the gene encoding glutathione S-transferase in the p GEX-KG vector and expressed the fusion in Escherichia coli. The thromb in-cleaved and uncleaved fusion proteins, GST-Ehrac1 and GST-Ehrac1-c (catalytic domain), were purified and found to exhibit similar protein kinase activities. The Ehrac1 fusion kinase was found to phosphorylat e serine/threonine residues exclusively in vitro. The preferred substr ate for the enzyme was histone H1 with a K-m of approx. 14 mu M. Histo ne H3 and kemptide were phosphorylated at about half the rate of histo ne H1. Protamine, enolase, bovine serum albumin, and poly (Glu:Tyr) we re not substrates for the enzyme. The protein kinase activity was high er in the presence of Mn2+ than Mg2+. Neither cAMP, Ca2+, nor Ca2+/cal modulin stimulated enzyme activity. The pH optimum of the enzyme was 7 .5. The Ehra1 kinase can utilize GTP as well as ATP as a phosphate don or with an apparent K-m of 80 mu M. Enzyme activity was inhibited 30-4 0% by a crude cAMP-dependent protein kinase inhibitor from rabbit and by thiol reagents. The expression and purification of enzymatically ac tive Ehrac1 protein kinase should allow further analysis of the regula tion and signal transduction pathways of E. histolytica.