Plasmodium falciparum cyclin G associated kinase as a drug target

Abstract

Kinases have become attractive drug targets in disease and several selective inhibitors are currently in clinical use. A P. falciparum kinase gene (PFL2280W) was identified in silico and investigated as a potential novel drug target. PFL2280W shows 57% similarity to mammalian cyclin G associated kinase (cGAK) which is essential for the survival of developing and mature mice.The P. falciparum 3D7 strain was maintained in culture and DNA was isolated and used to amplify the catalytic domain and flanking region of the PfcGAK gene by PCR. The PCR product was cloned into a vector that yielded a fusion protein with a N‐terminal HA and a C‐terminal His (6X) tag respectively. Protein expression was induced in E. coli and isolated by affinity chromatography on Ni+ beads. The protein samples were analysed by SDS‐PAGE and Western blotting using antibodies against the 6xHis and HA tags. The function of the fusion proteins was assessed in a standard kinase assay using radiolabelled ATP and exogenous substrates.Recombinant PfcGAK showed kinase activity using casein as substrate. PfcGAK was shown to undergo autophosphorylation. PfcGAK has a typical serine/threonine catalytic domain except for the absence of the conserved glycine triad in subdomain 1. Bioinformatic analysis of PfcGAK revealed an unique repetitive nanomeric amino acid sequence of 81 amino acids (KLQND/ED/VSG) which occurs outside the catalytic domain. In one clone this repetitive sequence was deleted in frame, which enhanced the production of the recombinant protein. PfcGAK shows the presence of a conserved GOLD domain which indicates that it may function in the Golgi complex, protein sorting or protein‐protein interactions. The carboxyl terminus has a predicted prenylation site, implying that the protein may be anchored in a membrane. Further studies are required to elucidate the potential of PfcGAK as a drug target in P.falciparum. .