Pfnek‐1, a NIMA‐related kinase from the human malaria parasite Plasmodium falciparum

Abstract

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.

The kinomes of apicomplexan parasites

“…MAP kinases are activated by phosphorylation of their activation loops by MAP kinase kinases (MAPKK), which belong to the STE group and are entirely lacking in apicomplexans as previously suggested [10,93]. In fact, STE kinases are sparsely represented in apicomplexans (and entirely absent from Theileria), and do not appear to have homologues in other alveolates or metazoan species.…”

“…Interestingly, the NEKs of Apicomplexa show no clear relationship to higher eukaryotic NEKs. Four NEK orthologous groups exist in apicomplexans: Pfnek-1 (PFL1370w) is an essential enzyme that phosphorylates one of the two parasite MAPKs (Pfmap-2) and may play a role in its regulation [85]. Pfnek-1 homologues are found in all apicomplexans and other alveolates (but not metazoans).…”

PfPKB, a Novel Protein Kinase B-like Enzyme from Plasmodium falciparum

“…Smears were blocked in phosphate-buffered saline containing 0.05% saponin and 5% bovine serum albumin. Subsequently, incubations with antiPfPKB and/or anti-MSP1 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] anti-sera (1:100 -1:200 dilution) were performed for 2 h at room temperature or at 4°C overnight. Anti-mouse IgG labeled with fluorescein isothiocyanate and anti-rabbit IgG labeled with Texas Red were used to localize merozoite surface protein 1 (MSP1) and PfPKB, respectively.…”

“…Systematic analysis of molecules involved in cell signaling events that occur during the course of P. falciparum development need to be pursued. A recently published genome sequence (1) and earlier studies suggest that several homologues of eukaryotic signaling proteins, such as protein kinases and phosphatases, are conserved in P. falciparum (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). The major challenge is to understand how these enzymes integrate in cellular machinery of Plasmodium and what roles they play in parasite development.…”

An evolutionary perspective on the kinome of malaria parasites

“…Some Plasmodium kinases in a given family appear to contain domains/motifs that are characteristic of other kinase families; a striking example is that of the NIMA-related kinase Pfnek-1: despite overall homology and clear phylogenetic linkage to the NIMA family, Pfnek-1 possesses a MAPKK-like activation motif SMAHS, which is clearly reminiscent of the SMANS motif of mammalian MEK1 and distinct from the FXXT motif usually found at the corresponding position in other members of the NIMA family [42]. Interestingly, Pfnek-1 (and another NIMA-related kinase, Pfnek-3 [43]) has been tentatively implicated in the regulation of an atypical mitogen-activated protein kinase (MAPK), Pfmap-2 [42], which itself has an atypical activation motif …”