PLASMODIUM-FALCIPARUM CALCIUM-DEPENDENT PROTEIN-KINASE PHOSPHORYLATES PROTEINS OF THE HOST ERYTHROCYTIC MEMBRANE

The unusual Ca2+-dependent protein kinase from Plasmodium falciparum (PfCPK) [1], whose gene structure and expression in bacteria have been reported [1], was purified to homogeneity. The purified recombinant kinase has a native molecular mass of 62 000, is activated by Ca2+ (K-0.5, = 15 mu M) in the presence of Mg2+ or Mn2+, and can associate with Ca-45(2+). The activation by Ca2+ could be partially replaced by Mn2+, but not by Zn2+ or Mg2+. PfCPK preferentially phosphorylated casein and histone H1. The K-m and V-max for Mg(2+)ATP were 26 mu M and 70 nmol min(-1) mg(-1) respectively, with casein as substrate; and 34 mu M and 143 nmol min(-1) mg(-1), respectively, with histone H1 as substrate. The kinase undergoes autophosphorylation on both serine and threonine residues. Calmodulin antagonists (calmidazolium, trifluoperazine, N-[6-aminohexyl]-5-chloro-1-napthalene-sulfonamide, and ophiobolin A) could inhibit the kinase activation, but much higher concentrations of the antagonists are needed than was required to inhibit calmodulin-mediated effects. PfCPK preferentially phosphorylates proteins of the host erythrocytic membrane in vitro but phosphorylates parasitic proteins only to a minor extent. The selectivity of the phosphorylation may be partially controlled by phosphatidylserine which is bound to some of the erythrocytic membrane proteins. Using a rabbit polyclonal antiserum against the recombinant protein, the kinase was found to be mainly expressed in the ring and schizont stages, and mainly localized in the parasitic membrane-organelle fraction and partially localized on the erythrocytic membrane.