The 3-phosphoinositide–dependent protein kinase 1 is an essential upstream activator of protein kinase A in malaria parasites

Abstract

Cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) signalling is essential for the proliferation of Plasmodium falciparum malaria blood stage parasites. The mechanisms regulating the activity of the catalytic subunit PfPKAc, however, are only partially understood, and PfPKAc function has not been investigated in gametocytes, the sexual blood stage forms that are essential for malaria transmission. By studying a conditional PfPKAc knockdown (cKD) mutant, we confirm the essential role for PfPKAc in erythrocyte invasion by merozoites and show that PfPKAc is involved in regulating gametocyte deformability. We furthermore demonstrate that overexpression of PfPKAc is lethal and kills parasites at the early phase of schizogony. Strikingly, whole genome sequencing (WGS) of parasite mutants selected to tolerate increased PfPKAc expression levels identified missense mutations exclusively in the gene encoding the parasite orthologue of 3-phosphoinositide–dependent protein kinase-1 (PfPDK1). Using targeted mutagenesis, we demonstrate that PfPDK1 is required to activate PfPKAc and that T189 in the PfPKAc activation loop is the crucial target residue in this process. In summary, our results corroborate the importance of tight regulation of PfPKA signalling for parasite survival and imply that PfPDK1 acts as a crucial upstream regulator in this pathway and potential new drug target.

Screening the Toxoplasma kinome with high-throughput tagging identifies a regulator of invasion and egress

“…TGGT1_239420 was previously linked to the development of artemisinin resistance in vitro 78 . Intriguingly, the Plasmodium falciparum ortholog of TGGT1_268210 (PfPDK1; Pf3D7_1121900), was classified as fitness-conferring in previous screens and has been linked to the regulation of protein kinase A (PKA) 14 , 79 .…”

Activation loop phosphorylation and cGMP saturation of PKG regulate egress of malaria parasites

“…Experiments with phosphopeptide libraries could potentially provide more insights into the substrate preference of PfPKG and whether activation loop mutants are subject to product inhibition, as is the case for PKCδ, possibly enabling the identification of physiological substrates. Future studies should explore whether activation loop phosphorylation is an autophosphorylation event or whether it is mediated by PDK1, which has been shown to phosphorylate the PfPKAc activation loop (T189) ( 49 ) as is the case with many AGC kinases ( 50 ). A structure of PfPKG with cGMP bound will be invaluable for understanding in detail the interactions of the different domains in the activated enzyme and how phosphorylation regulates this essential enzyme.…”

SPARK regulates AGC kinases central to theToxoplasma gondiiasexual cycle

“…The PKA signaling circuit differs in Plasmodium spp., where a sole PKA C subunit is indispensable for invasion but not egress (Patel et al, 2019;Wilde et al, 2019). However, SPARK inhibition would lead to a similarly inhibitory effect on parasite proliferation, as has been suggested recently in studies employing altered P. falciparum PDK1 and PKA alleles (Hitz et al, 2021). T. gondii has two additional paralogs of the PKA catalytic subunit, relative to Plasmodium spp., PKA C2 and PKA C3.…”