The crk3 gene of Leishmania mexicana encodes a stage-regulated cdc2-related histone H1 kinase that associates with p12.
A cdc2-related protein kinase gene, crk3, has been isolated from the parasitic protozoan Leishmania mexicana. Data presented here suggests that crk3 is a good candidate to be the leishmanial cdc2 homologue but that the parasite protein has some characteristics which distinguish it from mammalian cdc2. crk3 is predicted to encode a 35.6-kDa protein with 54% sequence identity with the human cyclin-dependent kinase cdc2 and 78% identity with the Trypanosoma brucei CRK3. The trypanosomatid CRK3 proteins have an unusual, poorly conserved 19-amino acid N-terminal extension not present in human cdc2. crk3 is single copy, and there is 5-fold higher mRNA in the replicative promastigote life-cycle stage than in the non-dividing metacyclic form or mammalian amastigote form. A leishmanial suc-binding cdc2-related kinase (SBCRK) histone H1 kinase, has previously been described which binds the yeast protein, p13(suc1), and that has stage-regulated activity (Mottram J. C., Kinnaird, J., Shiels, B. R., Tait, A., and Barry, J. D. (1993) J. Biol. Chem. 268, 21044-21051). CRK3 from cell extracts of the three life-cycle stages was found to bind p13(suc1) and the leishmanial homologue p12(cks1). CRK3 fused with six histidines at the C terminus was expressed in L. mexicana and shown to have SBCRK histone H1 kinase activity. Depletion of histidine-tagged CRK3 from L. mexicana cell extracts, by Ni-nitrilotriacetic acid agarose selection, reduced histone H1 kinase activity binding to p13(suc1). These data imply that crk3 encodes the kinase subunit of SBCRK. SBCRK and histidine-tagged CRK3 activities were inhibited by the purine analogue olomoucine with an IC50 of 28 and 42 microM, respectively, 5-6-fold higher than human p34(cdc2)/cyclinB.
Grant
K M
KM
Wellcome Unit of Molecular Parasitology, University of Glasgow, The Anderson College, Glasgow G11 6NU, Scotland, United Kingdom.
Hassan
P
P
Anderson
J S
JS
Mottram
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005816:Genetic Complementation Test; D006801:Humans; D007701:Kinetin; D007894:Leishmania mexicana / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D015800:Protozoan Proteins / Q000378:metabolism*; D011687:Purines / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature
C099912:CKS1 protein, Leishmania mexicana; D004791:Enzyme Inhibitors; D015800:Protozoan Proteins; D011687:Purines; D011994:Recombinant Proteins; C090046:olomoucine; D016203:CDC2 Protein Kinase; D007701:Kinetin