CDPK3

TgCDPK3 is a Ca2+-dependent protein kinase that serves as a central transducer of calcium signals governing Toxoplasma gondii egress from host cells (PMID:23149386, PMID:23209419). It is an active autophosphorylating kinase whose catalytic lysine is essential, and it phosphorylates substrates in a Ca2+-dependent manner (PMID:19699312). The enzyme is anchored to the inner face of the plasma membrane through dual acylation (N-myristoylation and palmitoylation), and both this membrane targeting and its catalytic activity are required for its role in Ca2+-dependent egress (PMID:23226109, PMID:24550330). During egress, TgCDPK3 directly phosphorylates Myosin A (TgMyoA) at serines 21 and 743 to initiate rapid gliding motility, with phosphomimetic substitutions rescuing the motility and egress defects of cdpk3 mutants (PMID:26544049), and it drives egress in part by phosphorylating and thereby relieving the suppressive activity of the negative regulator SCE1 (PMID:28258212). It further shapes the egress response by controlling microneme secretion in an intracellular, high-K+-dependent manner, negatively regulating dense granule exocytosis, quenching the Ca2+ flux, and participating in a positive cAMP–Ca2+ feedback loop that accelerates the response (PMID:23226109, PMID:30673152, PMID:27781359, PMID:36265000). Beyond egress, under extracellular stress TgCDPK3 loses N-myristoylation, relocalizes to the cytosol, and acts as an eEF2 kinase that phosphorylates elongation factor 2 to inhibit translational elongation (PMID:37272703). In the type II ME49 strain, TgCDPK3 also engages host factors — interacting with host Arginase-1 to bias macrophages toward M1 polarization and with autophagy proteins Atg3/Atg5 to promote IRG/GBP recruitment to the parasitophorous vacuole — both of which restrict intracellular proliferation (PMID:40394721, PMID:35757711).

1. 2009 Medium

   Establishing that TgCDPK3 is a genuine Ca2+-dependent kinase was the prerequisite for any signalling role, showing it autophosphorylates and phosphorylates substrates only when Ca2+ is present and only with an intact catalytic lysine.

   Evidence Recombinant kinase assays with [γ-32P]ATP, Ca2+ titration, catalytic lysine mutagenesis, and co-IP of the aldolase TgALD1

   PMID:19699312

   Open questions at the time

   • TgALD1 interaction confirmed only in a mammalian overexpression system, not endogenously in Toxoplasma
   • physiological substrates in the parasite not identified at this stage
2. 2012 High

   Multiple 2012 studies converged to define TgCDPK3's cellular role, showing it is specifically required for ionophore-induced egress but not invasion, that four independent loss-of-function alleles reproduce the egress defect, and that its egress function depends on plasma-membrane targeting via dual acylation.

   Evidence Chemical-genetic analog-sensitive kinase inhibition, forward genetic screening with whole-genome sequencing, knockout/complementation with acylation-site mutants, and egress/invasion assays

   PMID:23149386 PMID:23226109 PMID:23209419

   Open questions at the time

   • direct kinase substrates driving egress not yet identified
   • mechanism linking membrane localization to activity unresolved
3. 2012 Medium

   The requirement for TgCDPK3 in microneme secretion was shown to be context-dependent, tied to the high-K+ intracellular environment rather than a universal secretory step.

   Evidence Knockout with microneme secretion assays comparing intracellular vs. extracellular parasites and ionic-environment manipulation

   PMID:23226109

   Open questions at the time

   • ionic-environment mechanism inferred rather than directly demonstrated
   • molecular sensor of K+ not identified
4. 2014 Medium

   Heterologous complementation demonstrated that the egress function is a conserved property of this kinase family, requiring both membrane localization and catalytic activity.

   Evidence Complementation of a TgCDPK3 mutant with Plasmodium falciparum PfCDPK1 using localization and kinase-dead mutants plus egress assays

   PMID:24550330

   Open questions at the time

   • substrates of the complementing kinase not defined
   • single-lab result
5. 2015 High

   Identification of TgMyoA as a direct substrate provided the mechanistic link between CDPK3 activity and the motility that drives egress, resolving how the kinase triggers movement.

   Evidence BioID proximity labeling, peptide-array and in vitro kinase assays mapping S21/S743, and genetic rescue with phosphomimetic TgMyoA mutants

   PMID:26544049

   Open questions at the time

   • whether MyoA phosphorylation is the sole egress-relevant substrate not established
   • in vivo phosphorylation kinetics not resolved
6. 2016 Medium

   Live Ca2+ imaging placed CDPK3 within the temporal dynamics of the egress signalling cascade, implicating it in quenching the Ca2+ flux prior to egress.

   Evidence GCaMP6 Ca2+ biosensor live imaging with genetic and pharmacological perturbation of CDPK1 and CDPK3

   PMID:27781359

   Open questions at the time

   • molecular basis of signal quenching not defined
   • CDPK1 vs CDPK3 contributions not fully separated
7. 2017 High

   A suppressor screen revealed that CDPK3 promotes egress in part by inactivating a brake, identifying SCE1 as a negative egress regulator whose CDPK3-dependent phosphorylation relieves suppression.

   Evidence Forward genetic suppressor screen in a cdpk3 KO background, whole-genome sequencing, global phosphoproteomics, and SCE1 knockout

   PMID:28258212

   Open questions at the time

   • direct CDPK3-SCE1 phosphorylation not shown in vitro
   • downstream effectors of SCE1 unidentified
8. 2018 Medium

   Phosphoproteomics extended the CDPK3 substrate repertoire beyond the motor to metabolic control, identifying the tyrosine transporter ApiAT5-3 S56 as a Ca2+-induced CDPK3-dependent phosphosite.

   Evidence WT vs Δcdpk3 phosphoproteomics, apiAT5-3 conditional knockout, S56A phosphosite mutagenesis, metabolomics, and heterologous transporter characterization

   PMID:30402958

   Open questions at the time

   • direct kinase-substrate relationship rests on phosphoproteomic inference
   • S56 phosphorylation is beneficial but not essential, leaving its precise role open
9. 2019 Medium

   CDPK3 was shown to mediate the reciprocal Ca2+-dependent control of secretory organelles, being required for Ca2+-mediated suppression of dense granule exocytosis.

   Evidence CDPK3 knockout with dense granule secretion assays under Ca2+-elevated conditions, compared to CDPK1 knockout

   PMID:30673152

   Open questions at the time

   • substrates mediating dense granule suppression not identified
   • single method for CDPK3-specific contribution
10. 2022 Medium

    Sub-minute phosphoproteomics revealed CDPK3 as a partial driver of a positive cAMP-Ca2+ feedback loop that accelerates the egress response, linking calcium and cyclic-nucleotide signalling.

    Evidence Conditional knockout, sub-minute phosphoproteomics timecourse after Ca2+ ionophore, lipidomics, and PDE inhibition

    PMID:36265000

    Open questions at the time

    • feedback loop inferred from phosphoproteomic correlations
    • direct enzymatic targets in the cAMP pathway not identified
11. 2022 Medium

    CDPK3 was shown to have a host-directed function during latency, with the ME49 kinase engaging host autophagy proteins to recruit anti-parasitic effectors to the vacuole.

    Evidence Co-IP of TgCDPK3 with host Atg3/Atg5, ME49 CDPK3 knockout, IRG/GBP localization imaging, and proliferation/plaque assays

    PMID:35757711

    Open questions at the time

    • mechanism by which the CDPK3-Atg interaction promotes IRG/GBP targeting not resolved
    • single lab
12. 2023 Medium

    A distinct stress-conditional role was uncovered: loss of N-myristoylation relocalizes CDPK3 to the cytosol where it acts as the first protozoan eEF2 kinase to inhibit translational elongation.

    Evidence Homology analysis to yeast RCK2, stress-conditional co-IP of CDPK3 and eEF2, eEF2 phosphorylation and elongation-rate assays in cdpk3 KO, and localization/myristoylation analysis

    PMID:37272703

    Open questions at the time

    • signal driving loss of N-myristoylation under stress not defined
    • single lab
13. 2025 Medium

    The host-directed repertoire was extended to immune reprogramming, with CDPK3 interacting with host Arginase-1 to reduce its activity and bias macrophages toward M1 polarization.

    Evidence Mass spectrometry, GST pull-down, co-IP, co-localization, arginase activity assay, FACS/ELISA for macrophage markers, and proliferation/plaque assays

    PMID:40394721

    Open questions at the time

    • structural basis of the CDPK3-Arg1 interaction not defined
    • whether CDPK3 phosphorylates Arg1 not established

• How CDPK3 is spatially and temporally switched between its membrane-anchored egress function and its cytosolic eEF2-kinase and host-directed functions, and the full physiological substrate set, remain open.
• the molecular signal controlling stress-induced demyristoylation and relocalization is unknown
• no comprehensive validated in vivo substrate catalog
• relationship between egress substrates and host-directed functions unresolved