The crk3 Gene of Leishmania mexicanaEncodes a Stage-regulated cdc2-related Histone H1 Kinase That Associates with p12cks1

Karen M Grant,Paul Hassan,J Simon Anderson,Jeremy C Mottram

doi:10.1074/jbc.273.17.10153

Abstract

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.

Highlights

Cdc2, one of the cyclin-dependent kinase (CDK)1 family of serine-threonine protein kinases, is an essential mitotic acti
The predicted protein encoded by the leishmanial crk3 gene shows the greatest degree of homology to the cdc2 family of serine/threonine protein kinases when compared with protein sequence data bases
This includes equivalent residues to human cdc2 at Thr-14 and Tyr-15, in the ATP-binding domain, and Thr-161 (Fig. 2). These three residues are highly conserved both within the CDK family and between species. This implies that CRK3 activity may be controlled by similar post-translational mechanisms as exist in other eukaryotes, no homologues of these regulatory enzymes have been identified in Leishmania to date

Summary

Introduction

Cdc2, one of the cyclin-dependent kinase (CDK)1 family of serine-threonine protein kinases, is an essential mitotic acti-. CRK3 has stage-regulated histone H1 kinase activity that binds p13suc1, features of the leishmanial SBCRK and mammalian cdc2. Both the original lysate and the eluate from the column (CRK3-depleted) were incubated with p13suc1 or control beads, as described above, and the bound protein was assayed for histone H1 kinase activity together with the washed NiNTA agarose beads.

Results

Conclusion