Expression and characterization of a rac family protein kinase of Entamoeba histolytica.
We previously reported the isolation from Entamoeba histolytica of a novel rac family protein kinase gene, termed Ehrac1, 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 Ehrac1 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-Ehrac1 and GST-Ehrac1-c (catalytic domain), were purified and found to exhibit similar protein kinase activities. The Ehrac1 fusion kinase was found to phosphorylate serine/threonine residues exclusively in vitro. The preferred substrate for the enzyme was histone H1 with a Km of approx. 14 microM. 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 Mn2+ than Mg2+. Neither cAMP, Ca2+, nor Ca2+/calmodulin stimulated enzyme activity. The pH optimum of the enzyme was 7.5. The Ehrac1 kinase can utilize GTP as well as ATP as a phosphate donor with an apparent Km of 80 microM. 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 Ehrac1 protein kinase should allow further analysis of the regulation and signal transduction pathways of E. histolytica.
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Department of Pathology, University of California, San Diego 92103-8416.
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D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D004748:Entamoeba histolytica / Q000201:enzymology*; D004791:Enzyme Inhibitors; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D017346:Protein Serine-Threonine Kinases / Q000032:analysis* / Q000235:genetics*; D051057:Proto-Oncogene Proteins c-akt; D011993:Recombinant Fusion Proteins / Q000032:analysis; D011994:Recombinant Proteins / Q000032:analysis; D013379:Substrate Specificity; D014162:Transfection
D017931:DNA Primers; D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt