Abstract
Our understanding of the key phosphorylation-dependent signalling pathways in the human malaria parasite, Plasmodium falciparum, remains rudimentary. Here we address this issue for the essential cGMP-dependent protein kinase, PfPKG. By employing chemical and genetic tools in combination with quantitative global phosphoproteomics, we identify the phosphorylation sites on 69 proteins that are direct or indirect cellular targets for PfPKG. These PfPKG targets include proteins involved in cell signalling, proteolysis, gene regulation, protein export and ion and protein transport, indicating that cGMP/PfPKG acts as a signalling hub that plays a central role in a number of core parasite processes. We also show that PfPKG activity is required for parasite invasion. This correlates with the finding that the calcium-dependent protein kinase, PfCDPK1, is phosphorylated by PfPKG, as are components of the actomyosin complex, providing mechanistic insight into the essential role of PfPKG in parasite egress and invasion.
Highlights
Our understanding of the key phosphorylation-dependent signalling pathways in the human malaria parasite, Plasmodium falciparum, remains rudimentary
By employing a chemical genetic approach in conjunction with quantitative global phosphoproteomics, we describe here 107 phosphorylation events in the human malaria parasite that are dependent on PfPKG activity
We reveal the cellular targets of cGMP-signalling in malaria, but we provide a map of the physiological processes that are likely regulated by PfPKG
Summary
Our understanding of the key phosphorylation-dependent signalling pathways in the human malaria parasite, Plasmodium falciparum, remains rudimentary. One of the barriers to exploiting these opportunities is the paucity of information regarding the role of essential protein kinases and the nature of phospho-signalling cascades in Plasmodium parasites[11] We address these issues by combining chemical genetics and global phospho-proteomic approaches to reveal the phosphorylation events mediated by the P. falciparum guanosine 30,50-cyclic monophosphate (cGMP)dependent protein kinase, PfPKG (PF3D7_1436600). This protein kinase is known to play an essential role in asexual blood stages, where it is involved in both late stage schizogony[14] and egress of the parasite from red blood cells (RBCs)[15,16], and in the mosquito stages, with demonstrated roles in gametogenesis[17] and ookinete motility[18,19] as well as late liver stage development[20]. PfPKG is reported to be important in maintaining elevated calcium levels, possibly through the regulation of phosphoinositide biosynthesis, necessary for ookinete motility in P. berghei and blood stage schizogony in P. falciparum[18]
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