Pfnek3 functions as an atypical MAPKK in Plasmodium falciparum.
Eukaryotes generally rely on signal transduction by mitogen-activated protein kinases (MAPKs) for activating their regulatory pathways. However, the presence of a complete MAPK cascade in Plasmodium falciparum is debatable because a search of the entire genome did not portray known MAPK kinase (MAPKK) sequences. Via homology PCR experiments, only two copies of plasmodial MAPK homologues (Pfmap1 and Pfmap2) have been identified but their upstream activators remain unknown. In an earlier experiment, Pfnek3 was found to be an unusual activator of Pfmap2 in in vitro experiments, despite its molecular identity as a malarial protein kinase from the NIMA (Never in Mitosis, Aspergillus) family. In this study, the role of Pfnek3 as a likely upstream MAPKK is defined through molecular and biochemical characterization. Since a previous report proposes a TSH motif as an activation site of Pfmap2, its site-directed mutants, T290A, S291A, and H292K were constructed to elucidate the involvement of Pfnek3 in phosphorylating and activating Pfmap2 in a battery of kinase assays. The results suggested that residue T290 is the site of phosphorylation by Pfnek3. This supposition was further supported by liquid chromatography mass spectrometry. Although P. falciparum does not appear to possess a conventional MAPK cascade, they may rely on other kinases such as Pfnek3 to carry out similar phosphorylation to activate its signaling pathways.
Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, MD4A, 5 Science Drive 2, Singapore 117597, Singapore.
Huiyu
H
Low
Yu Min
YM
Lye
Tiow-Suan
TS
Sim
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D004789:Enzyme Activation; D013058:Mass Spectrometry; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D010963:Plasmodium falciparum / Q000201:enzymology*; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D013912:Threonine / Q000378:metabolism
D010455:Peptides; D015800:Protozoan Proteins; D013912:Threonine; D020929:Mitogen-Activated Protein Kinase Kinases