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 hum an malaria parasite Plasmodium falciparum. This enzyme displays maximal hom ology to the never-in-mitosis/Aspergillus (NIMA)/NIMA-like kinase (Nek) fam ily of protein kinases, whose members are involved in eukaryotic cell divis ion processes. Similar to other P. falciparum protein kinases and many enzy mes of the NIMA/Nek family, Pfnek-1 possesses a large C-terminal extension in addition to the catalytic domain. Bacterially expressed recombinant Pfne k-1 protein is able to autophosphorylate and phosphorylate a panel of prote in 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 N IMA/Nek protein kinases is substituted in Pfnek-1 by a SMAHS motif, which i s reminiscent of a MAP/ERK kinase (MEK) activation site. Mutational analysi s 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 M APK homologue, in vitro, and (b) that coincubation of Pfnek-1 and Pfmap-2 r esults in a synergistic increase in exogenous substrate labelling. This sug gests 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, w hich opens the way to the screening of chemical libraries aimed at identify ing potential new antimalarials.