Analysis of the CRK3 Kinase of Leishmania mexicana

Control of cell cycle progression in eukaryotes is governed by members of a highly conserved family of serine threonine protein kinases, the cyclin-dependent kinases (CDKs), which associate with a regulatory cyclin partner protein to attain full activity. The CRK3 gene product of Leishmania mexicana is one of two putative CDKs so far identified in this organism. The possible function of CRK3 and the requirement of the CRK3 gene for Leishmania survival was investigated using molecular genetic techniques. Attempts to create null mutants lacking an intact CRK3 locus failed repeatedly and instead a series of transgenic cell lines were generated that had undergone changes in ploidy or genomic rearrangements. However, both alleles could be successfully disrupted when extra copies of CRK3 were introduced on an episome into a heterozygote mutant, prior to disruption of the second chromosomal CRK3 allele. Together these results provide evidence that CRK3 is an essential gene in the promastigote form of Leishmania mexicana. CDKs are regulated not only by cyclin binding, but also by inhibitory and activatory phosphorylation events. Inhibition of Leishmania mexicana phosphotyrosine phosphatase activity by bpV(phen) resulted in inhibition of cell cycle progression, and led to an accumulation of cells in the 01 and S-phases of the cell cycle. Cells treated with bpV(phen) had a low level of CRK3 activity and upon release from inhibition CRJC3 activity increased. Inhibition of a phosphotyrosine phosphatase activity could be involved in regulation of CRK3 activity, either directly or indirectly. The CDK inhibitor flavopiridol is a potent inhibitor of mammalian cdkl, cdk2 and cdk4, enzymes, which all have a role in cell cycle progression. Flavopiridol was found to be a potent inhibitor of L. mexicana CRX3 activity and inhibits purified CRK3 with an IC50 value of 100 nM. Flavopiridol inhibited growth of L. mexicana promastigotes in liquid culture, with 50% inhibition of growth achieved with a concentration of 250 nM. Inhibition of growth is due to inhibition of cell cycle progression, as cells were found to accumulate in the G2 phase of the cell cycle, probably due to inhibition of CRK3 activity. Flavopiridol-induced growth inhibition is reversible up to 24 hours after addition of the drug. Release from flavopiridol inhibition resulted in a partially synchronous re-entry into the cell cycle. This method may be used to obtain cell samples enriched for cells in particular phases of the cell cycle. In contrast to several plant and animal CDKs, CRK3 failed to complement for loss of function of CDC28 activity in the Saccharomyces cerevisiae cdc28-1Nts cdc28-4ts and cdc28-13ts mutants, suggesting that mechanisms of cell cycle control in Leishmania may be less conserved than those of other eukaryotes. The findings that CRK3 is an essential gene in Leishmania, that CRK3 is inhibited by specific inhibitors and that CRK3 has features that distinguish it from mammalian homologues, make CRK3 a novel drug target of some promise