PDK1

PfPDK1 (PF3D7_1121900) is an upstream protein kinase that activates the cAMP/PKA signalling axis in Plasmodium falciparum blood-stage parasites by phosphorylating the activation-loop threonine (T189) of the PKA catalytic subunit (PfPKAc), a role established through whole-genome sequencing of suppressor mutants selected to tolerate lethal PfPKAc overexpression together with targeted mutagenesis and conditional knockdown (PMID:34879056). Its placement upstream of PKA is independently corroborated by the recovery of an activation-loop-proximal missense mutation (N324I) in parasites selected for resistance to a PDEβ inhibitor (PMID:39642214). Beyond asexual signalling, conditional knockdown links PfPDK1 to regulation of gametocyte deformability in the sexual blood stages (PMID:34879056). In vitro, PfPDK1 binds the Chk1 inhibitor CHIR-124 in a competitive Kinobead assay (Kd app ~0.1 µM), but knockdown does not alter CHIR-124 susceptibility, indicating it is not the cellular target of that compound (PMID:42287204). No structural model of PfPDK1 or direct biochemical characterization of its catalytic mechanism beyond the T189 phosphorylation assignment has been reported in the available corpus.

1. 2021 High

   It was unknown what activates the essential PKA catalytic subunit in P. falciparum; identifying PfPDK1 as the activating kinase placed it at the head of the cAMP/PKA axis.

   Evidence WGS of suppressor mutants tolerating lethal PfPKAc overexpression, targeted mutagenesis of the PfPKAc T189 activation-loop residue, and conditional knockdown

   PMID:34879056

   Open questions at the time

   • No in vitro reconstitution of PfPDK1 kinase activity on PfPKAc T189 with purified components
   • Upstream regulators and activation mechanism of PfPDK1 itself not defined
   • No structural basis for substrate recognition
2. 2021 Medium

   Whether PfPDK1 functions beyond asexual signalling was untested; knockdown revealed a role in gametocyte deformability, extending its relevance to sexual blood stages.

   Evidence Conditional knockdown with phenotypic readout of gametocyte deformability

   PMID:34879056

   Open questions at the time

   • No downstream molecular mechanism identified for the deformability phenotype
   • Unclear whether this phenotype acts through the PKA axis or an independent pathway
3. 2024 Medium

   Independent confirmation of PfPDK1's pathway placement came from a different selection pressure, linking activation-loop-region mutations to altered cAMP/PKA signalling under drug challenge.

   Evidence In vitro selection for PDEβ inhibitor resistance plus WGS identifying the N324I substitution downstream of the activation loop

   PMID:39642214

   Open questions at the time

   • Functional consequence of the specific N324I mutation not biochemically validated
   • Mechanism connecting PfPDK1 mutation to inhibitor resistance not resolved
4. 2026 Medium

   A candidate small-molecule interaction was tested; PfPDK1 binds CHIR-124 in vitro but this binding does not drive the compound's parasiticidal effect, refining understanding of its druggability.

   Evidence Competitive Kinobead pulldown (Kd app ~0.1 µM) plus GFP-DD degron conditional knockdown susceptibility assay

   PMID:42287204

   Open questions at the time

   • The true cellular target of CHIR-124 remains unidentified
   • Negative cellular result limits conclusion to in vitro binding only

• The catalytic mechanism, structural basis, and full substrate repertoire of PfPDK1 beyond PfPKAc T189 remain undefined.
• No purified-enzyme kinase assay or structure
• No additional substrates characterized
• Upstream activators of PfPDK1 unknown