PfPK6, a novel cyclin-dependent kinase/mitogen-activated protein kinase-related protein kinase from Plasmodium falciparum

We have isolated a novel protein kinase cDNA, PfPK6, by differential displa y RT-PCR (DDRT-PCR) of mRNA obtained from different asexual erythrocytic st ages of Plasmodium falciparum, which shows sequence similarity to both cycl in-dependent kinase (CDK) and mitogen-activated protein kinase (MAPK) famil y members. The 915 bp open reading frame (ORF) is interrupted by seven intr ons and encodes a 305-residue polypeptide with a predicted molecular mass o f 35848 Da. Several cDNA clones with some of the intron sequences were isol ated, indicating alternate or defective splicing of PfPK6 transcripts becau se the gene seems to be a single copy located on chromosome 13. The similar ity of the catalytic domain of PfPK6 to those of CDK2 and MAPK is 57.3 % an d 49.6 %, respectively. The signature PSTAIRE (single-letter amino acid cod es) CDK( motif is changed to SKCILRE in PfPK6). The TXY residues that are p hosphorylated in MAPKs for their activation are (TPT)-P-173 in PfPK6. Three size classes of PfPK6 transcripts of 6.5, 2.0 and 1.1 kb are up-regulated during the transition of P. falciparum from ring to trophozoite. Western bl ot analysis suggested the expression of a 35 kDa polypeptide in trophozoite s and schizonts. Immunofluorescence studies indicated both nuclear and cyto plasmic localization of PfPK6 in trophozoite, schizont and segmenter stages . In vitro, recombinant PfPK6 phosphorylated itself and also exogenous subs trates, histone and the small subunit of the malarial ribonucleotide reduct ase (R2). The kinase activity of PfPK6 is sensitive to CDK inhibitors web a s olomoucine and roscovitine. PfPK6 showed a preference for Mn2+ over Mg2ions as a cofactor. The Lys(38) --> Arg mutant is severely defective in its interaction with ATP and bivalent cations and somewhat defective in cataly tic rate for R2 phosphorylation.