Exploring the Plasmodium falciparum cyclic-adenosine monophosphate (cAMP)-dependent protein kinase (PfPKA) as a therapeutic target

Abstract

One of the prototype mammalian kinases is PKA and various roles have been defined for PKA in malaria pathogenesis. The recently described phospho-proteomes of Plasmodium falciparum introduced a great volume of phospho-peptide data for both basic research and identification of new anti-malaria therapeutic targets. We discuss the importance of phosphorylations detected in vivo at different sites in the parasite R and C subunits of PKA and highlight the inhibitor sites in the parasite R subunit. The N-terminus of the parasite R subunit is predicted to be very flexible and we propose that phosphorylation at multiple sites in this region likely represent docking sites for interactions with other proteins, such as 14-3-3. The most significant observation when the P. falciparum C subunit is compared to mammalian C isoforms is lack of phosphorylation at a key site tail implying that parasite kinase activity is not regulated so tightly as mammalian PKA. Phosphorylation at sites in the activation loop could be mediating a number of processes from regulating parasite kinase activity, to mediating docking of other proteins. The important differences between Plasmodium and mammalian PKA isoforms that indicate the parasite kinase is a valid anti-malaria therapeutic target.

Fig. 1

Sequence alignment of C and C mammalian cAMP-dependent protein kinase C-subunits (PKA) with Plasmodium and Toxoplasma gondii isoforms. Sequences are labeled according to species and isoform: (h), Homo sapiens, PKA (P17612 KAPCA_HUMAN), PKA (P22694 KAPCB_HUMAN); (b) = bovine, (Bos taurus) PKA (P00517 KAPCA_BOVIN); (p), pig (Sus scrofa) PKA (P36887 KAPCA_PIG), PKA (P05383 KAPCB_PIG); (P.f.), Plasmodium falciparum isolate 3D7 (gene PfPKAc, uniprot Q7K6A0_PLAF7); (P.v.), Plasmodium vivax (gene PVX_086975, uniprot A5KE97_PLAVI); (P.k.), Plasmodium knowlesi strain H (gene PKH_073290, uniprot B3L322_PLAKH); (P.y.), Plasmodium yoelii yoelli (gene PY052325, uniprot Q7RE33_PLAYO); (T.g.) Toxoplasma gondii VEG (gene TGVEG_066990, uniprot B9Q7X8_TOXGO). The structure of PKA (1ATP) is shown colored by subdomains as annotated on the sequence.

Fig. 2

Unique N and C terminal tails of AGC type kinases. (A) Cartoon illustrating the distinctive N-terminal tail of PKA kinases highlighting the myristylation site (yellow spheres), the N-terminal Ser10 phosphorylation site and the Trp30 residue on the A-helix that are conserved in mammalian isoforms. (B) Illustrates the protein:protein interaction sites of the conserved AGC-type C-terminal tail. (C) AGC kinases share a distinct C-terminal tail consisting of putative protein-binding sites and the conserved PxxP and HF-motif. PDB IDs and colors of each C-tail are indicated in the alignment in panel D. (D) Alignment of representative AGC C-terminal tails indicate conservation between two representative plasmodia forms (PfPKA and PyyAKT2) with a variety of mammalian AGC isoforms. Sequences are labeled according to species and isoform: PfPKA (O15906, Pf), PyyAKT/PKB (Q7RSF6, Pyy), PKA (P05132, mouse), PKC-iota (P41743, human), PKC-theta (Q04759, human), AKT2 (P31751, human), ROCK1 (Q13464, human), ROCK2 (Q28021, bovine), PDK1 (O15530, human), GRK2 (P21146, bovine), GRK6 (P43250, human). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

The P. falciparum PKA regulatory subunit N-terminus is highly divergent from mammalian isoforms. (A) Sequence alignment of various isoforms. (B) Disorder prediction by disprot-Database of protein disorder (http://www.disprot.org/) for PfPKA-R and mammalian PKA-R shows significant differences in the N-terminal region. (C) Structural schematics of mammalian and Plasmodia R-subunits.

Fig. 4

Core domain alignment of cAMP-dependent protein kinase (PKA) R-subunits of mammalian, yeast, Plasmodia and Toxoplasma isoforms. Structure shown is of RI (1RGS) colored according to the representative sequence. Sequences are labeled according to species and isoform: RI (P10644, human), RIβ (P31321, human), RII (P13861, human), RIIβ (P31323, human), bcy1 (P07278, yeast), PfR (Q8T323, P. falciparum), PkR (B3LCL9, P. knowlesi), PvR (gi 156103253, P. vivax), PbR (gi68068779, P. berghei), PyyR (gi 83318121, P. yoelii yoelli), PccR (gi 70947112, P. chabaudi chabaudi), TgR (gi 12698442, T. gondii).

Fig. 5

Predicted myristylation and palmitylation sites in the P. falciparum R-subunit and P. falciparum encoded acyltransferases.

Fig. 6

Putative phosphorylation sites of the catalytic and regulatory subunits of PfPKA and TgPKA, as detected by Treeck et al, 2011. (A) Sequences of PfPKA-C and TgPKA-C and (B) PfPKA-R and TgPKA-R with key conserved residues highlighted by a white circle and phosphorylation sites indicated by red dots. S149 in PfPKA-R is a bona fide PKA substrate. Spectra of PfPKA-R peptides, PepR149 (QKKRLSVS) harboring S149, PepR68 (SRGFSLS) harboring S68 and Kemptide (LRRASLG) following in vitro phosphorylation by bovine PKA. (C) MS trace of Kemptide used as positive control. The presented spectrum is a composite of two distinct spectra, since the phosho Kemptide (m/z = 852.5) was eluted around 1 min before its non-phosphorylated counterpart (m/z = 772.5). (D) MS trace of pepR68 used as negative control. PepR68 was visible in MS on a few consecutive fractions at around 28min elution. The presented spectrum shows the non-phosphorylated pepR68 (m/z = 753.4), while the phosphorylated form was not detected in any of the fractions. (E) MS trace of pepR149. Non-phosphorylated PepR149 was visible in MS on a few consecutive fractions at 20min elution. The presented spectrum is an intermediary fraction between the two elution peaks. Phosphorylated PepR149 (m/z = 1025.55) was eluted 30 s before its non-phosphorylated counterpart (m/z = 945.59). (D) MS/MS trace of CID-fragmented phosphoPepR149 (m/z = 1025.55); (F1) full spectrum showing an intense signal corresponding to H2PO4 (98amu) loss; (F2) zoomed spectrum below neutral loss m/z showing fragmented ions’ assignation of b and y ions according to Roepstorff and Fohlman [62], nomenclature correlating to PepR14. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

The inhibitor-site and phosphate (PBC) cassettes of the P. falciparum R-subunit compared to mammalian isoforms (A) Comparison of mammalian and PfPKA-R inhibitor site. (B) Alignment of isoform-specific phosphate-binding cassettes. (C) Cyclic-nucleotide bindign domain A (CNB-A) of RIα and critical residues involved in protein:protein interactions and the capping of cAMP.