ELSEVIER Molecular and Biochemical Parasitology 66 (1994) 111-118 MOLECULAR AND BIOCHEMICAL PARASITOLOGY Expression and characterization of a rac family protein kinase of Entamoeba histolytica Xuchu Oue, Sharon L. Reed * Departments of Pathology and Medicine, University of California, 200 W. Arbor Dr. San Diego, CA 92103-8416, USA Received 13 January 1994; accepted 29 March 1994 Abstract We previously reported the isolation from Entamoeba histolytica of a novel rac family protein kinase gene, termed Ehracl, for "related to cAMP-dependent protein kinases and protein kinase Cs". To study the function and properties of this kinase gene further, we fused the full-length coding region and the truncated catalytic domain of the Ehracl gene in frame with the gene encoding glutathione S-transferase in the pGEX-KG vector and expressed the fusion in Escherichia coli. The thrombin-cleaved and uncleaved fusion proteins, GST-Ehracl and GST-Ehracl-c (catalytic domain), were purified and found to exhibit similar protein kinase activities. The Ehracl fusion kinase was found to phosphorylate serine/threonine residues exclusively in vitro. The preferred substrate for the enzyme was histone H1 with a K m of approx. 14/zM. Histone H3 and kemptide were phosphorylated at about half the rate of histone H1. Protamine, enolase, bovine serum albumin, and poly (Glu:Tyr) were not substrates for the enzyme. The protein kinase activity was higher in the presence of Mn 2+ than Mg 2+. Neither cAMP, Ca 2+, nor CaZ+/calmodulin stimulated enzyme activity. The pH optimum of the enzyme was 7.5. The Ehracl kinase can utilize GTP as well as ATP as a phosphate donor with an apparent K m of 80 /.tM. Enzyme activity was inhibited 30-40% by a crude cAMP-dependent protein kinase inhibitor from rabbit and by thiol reagents. The expression and purification of enzymatically active Ehracl protein kinase should allow further analysis of the regulation and signal transduction pathways of E. histolytica. Key words: Entamoeba histolytica; Protein kinase expression; rac Family kinase; Fusion protein I. Introduction Abbreviations: Ehracl, E. histolytica kinase related to PKAs and PKCs; GST, glutathione S-transferase; G-beads, glutathione agarose beads; IPTG, isopropyl 1-thio-/3-D-galactoside; MBP, myelin basic protein; NEM, N-ethylmaleimide; PCR, polymerase chain reaction; PK, protein kinase; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; PKI, protein kinase inhibitor from rabbit muscle; rac, kinase related to PKA and PKC. * Corresponding author. Tel: 619-543-6146 Fax: 619-543-6614 The protozoan parasite, Entamoeba histolytica in- fects 500 million people worldwide and causes 40-50 million cases of amebic colitis or liver abscesses, which lead to 80000 deaths annually [1]. We have been interested in the biochemical signals which determine key events in the life cycle of E. histolyt- ica, particularly invasion or encystation, which are 0166-6851/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDIO166-6851(94)OOO75-X