Pfnek-1, a NIMA-related kinase from the human malaria parasite Plasmodium falciparum Biochemical properties and possible involvement in MAPK regulation.
We have cloned Pfnek-1, a gene encoding a novel protein kinase from the human malaria parasite Plasmodium falciparum. This enzyme displays maximal homology to the never-in-mitosis/Aspergillus (NIMA)/NIMA-like kinase (Nek) family of protein kinases, whose members are involved in eukaryotic cell division processes. Similar to other P. falciparum protein kinases and many enzymes of the NIMA/Nek family, Pfnek-1 possesses a large C-terminal extension in addition to the catalytic domain. Bacterially expressed recombinant Pfnek-1 protein is able to autophosphorylate and phosphorylate a panel of protein substrates with a specificity that is similar to that displayed by other members of the NIMA/Nek family. However, the FXXT motif usually found in NIMA/Nek protein kinases is substituted in Pfnek-1 by a SMAHS motif, which is reminiscent of a MAP/ERK kinase (MEK) activation site. Mutational analysis indicates that only one of the serine residues in this motif is essential for Pfnek-1 kinase activity in vitro. We show (a) that recombinant Pfnek-1 is able to specifically phosphorylate Pfmap-2, an atypical P. falciparum MAPK homologue, in vitro, and (b) that coincubation of Pfnek-1 and Pfmap-2 results in a synergistic increase in exogenous substrate labelling. This suggests that Pfnek-1 may be involved in the modulation of MAPK pathway output in malaria parasites. Finally, we demonstrate that recombinant Pfnek-1 can be used in inhibition assays to monitor the effect of kinase inhibitors, which opens the way to the screening of chemical libraries aimed at identifying potential new antimalarials.
Dorin
D
D
Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France.
Le Roch
K
K
Sallicandro
P
P
Alano
P
P
Parzy
D
D
Poullet
P
P
Meijer
L
L
Doerig
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D018797:Cell Cycle Proteins; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004912:Erythrocytes / Q000469:parasitology; D017125:Genes, Protozoan; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000072380:NIMA-Related Kinase 1; D000072379:NIMA-Related Kinases; D010766:Phosphorylation; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016053:RNA, Protozoan / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D018797:Cell Cycle Proteins; D017931:DNA Primers; D012333:RNA, Messenger; D016053:RNA, Protozoan; D011994:Recombinant Proteins; D011505:Protein-Tyrosine Kinases; C000606914:NEK1 protein, human; D000072380:NIMA-Related Kinase 1; D000072379:NIMA-Related Kinases; C442331:NIMA-related kinase 6; D017346:Protein Serine-Threonine Kinases; D020928:Mitogen-Activated Protein Kinases