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 display RT-PCR (DDRT-PCR) of mRNA obtained from different asexual erythrocytic stages of Plasmodium falciparum, which shows sequence similarity to both cyclin-dependent kinase (CDK) and mitogen-activated protein kinase (MAPK) family members. The 915 bp open reading frame (ORF) is interrupted by seven introns and encodes a 305-residue polypeptide with a predicted molecular mass of 35848 Da. Several cDNA clones with some of the intron sequences were isolated, indicating alternate or defective splicing of PfPK6 transcripts because the gene seems to be a single copy located on chromosome 13. The similarity of the catalytic domain of PfPK6 to those of CDK2 and MAPK is 57.3% and 49.6%, respectively. The signature PSTAIRE (single-letter amino acid codes) CDK motif is changed to SKCILRE in PfPK6. The TXY residues that are phosphorylated in MAPKs for their activation are T(173)PT 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 blot analysis suggested the expression of a 35 kDa polypeptide in trophozoites and schizonts. Immunofluorescence studies indicated both nuclear and cytoplasmic localization of PfPK6 in trophozoite, schizont and segmenter stages. In vitro, recombinant PfPK6 phosphorylated itself and also exogenous substrates, histone and the small subunit of the malarial ribonucleotide reductase (R2). The kinase activity of PfPK6 is sensitive to CDK inhibitors such as olomoucine and roscovitine. PfPK6 showed a preference for Mn(2+) over Mg(2+) ions as a cofactor. The Lys(38)-->Arg mutant is severely defective in its interaction with ATP and bivalent cations and somewhat defective in catalytic rate for R2 phosphorylation.
Bracchi-Ricard
V
V
Department of Molecular Biology and Microbiology, University of Central Florida, Orlando, FL 32816, USA.
Barik
S
S
Delvecchio
C
C
Doerig
C
C
Chakrabarti
R
R
Chakrabarti
D
D
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D002874:Chromosome Mapping; D018844:Cyclin-Dependent Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004912:Erythrocytes / Q000469:parasitology; D015723:Gene Library; D007438:Introns; D007700:Kinetics; D020928:Mitogen-Activated Protein Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010802:Phylogeny; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015800:Protozoan Proteins; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D015800:Protozoan Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D011494:Protein Kinases; C408698:PK6 protein, Plasmodium falciparum; D018844:Cyclin-Dependent Kinases; D020928:Mitogen-Activated Protein Kinases