Reduced Activity of Mutant Calcium-Dependent Protein Kinase 1 Is Compensated in Plasmodium falciparum through the Action of Protein Kinase G

Abstract

We used a sensitization approach that involves replacement of the gatekeeper residue in a protein kinase with one with a different side chain. The activity of the enzyme with a bulky gatekeeper residue, such as methionine, cannot be inhibited using bumped kinase inhibitors (BKIs). Here, we have used this approach to study Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1). The methionine gatekeeper substitution, T145M, although it led to a 47% reduction in transphosphorylation, was successfully introduced into the CDPK1 locus using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9. As methionine is a bulky residue, BKI 1294 had a 10-fold-greater effect in vitro on the wild-type enzyme than on the methionine mutant. However, in contrast to in vitro data with recombinant enzymes, BKI 1294 had a slightly greater inhibition of the growth of CDPK1 T145M parasites than the wild type. Moreover, the CDPK1 T145M parasites were more sensitive to the action of compound 2 (C2), a specific inhibitor of protein kinase G (PKG). These results suggest that a reduction in the activity of CDPK1 due to methionine substitution at the gatekeeper position is compensated through the direct action of PKG or of another kinase under the regulation of PKG. The transcript levels of CDPK5 and CDPK6 were significantly upregulated in the CDPK1 T145M parasites. The increase in CDPK6 or some other kinase may compensate for decrease in CDPK1 activity during invasion. This study suggests that targeting two kinases may be more effective in chemotherapy to treat malaria so as not to select for mutations in one of the enzymes.

Plasmodial Kinase Inhibitors: License to Cure?

“…Upregulation of kinases such as Pf CDPK5 and Pf CDPK6 that are downstream from Pf PKG has been shown previously for the CDPK1 mutant T145M. 41 …”

Epistasis studies reveal redundancy among calcium-dependent protein kinases in motility and invasion of malaria parasites

“…The presence of CDPK1 at the pellicle of P. berghei is consistent with its proposed function in phosphorylating a number of proteins possibly involved in motility and invasion 35 , 36 . Furthermore, there is evidence that CDPK1 functionally interacts with PKG in P. falciparum merozoites 37 and is important for erythrocyte invasion 38 . On the other hand, P. falciparum asexual blood stages can adapt to the loss of CDPK1 21 , and in P. berghei , neither CDPK1-KO nor the double CDPK1-KO/CDPK4-KO nor PKG T619Q -3xHA/CDPK1-KO parasites show a significant growth defect (Supplementary Data 1 ).…”

“…These features may allow for a balance between evolvability to adapt to species- or stage-specific requirements and robustness of calcium-dependent signalling. For example, the requirement for CDPKs is slightly different between P. berghei and P. falciparum for gametogenesis 20 , 21 or merozoite invasion 21 , 37 , 45 , suggesting that if the same network is involved in both processes, the wiring might be slightly different due to species-specific factors. On the other hand, requirement for the same kinases at unrelated developmental stages may also constrain the evolvability of CDPK networks.…”

Plasmodium falciparum Calcium-Dependent Protein Kinase 2 Is Critical for Male Gametocyte Exflagellation but Not Essential for Asexual Proliferation

“…For this, we tested the transcript levels of 11 different genes (10 kinases and Hsp90) in the KO C1 and the WT parasites at the late schizont stage (42 to 48 hpi). The 10 kinase genes were selected based on an earlier study ( 17 ). The HSP90 gene was selected to evaluate the stress response in the KO parasites.…”

“…The reactions were run on the Bio-Rad CFX Connect instrument with the following program: initial denaturation at 95°C for 3 min, followed by 40 cycles of 95°C for 10 s, 52°C for 20 s, and 62°C for 30 s). The transcript expression of each target gene was normalized to expression levels of two housekeeping genes: threonine-tRNA ligase and glyceraldehyde-3-phosphate dehydrogenase, as reported earlier ( 17 ). The expression of each target gene in the Pf CDPK2 KO C1 parasite, relative to that in the WT, was calculated using Bio-Rad CFX Manager, and the data were analyzed using the R statistical analysis package (version 3.3.2) ( 47 ).…”