PK9

PK9 (PF3D7_1315100) is a catalytically active atypical serine/threonine protein kinase of Plasmodium that couples kinase signaling to the ubiquitin-conjugation machinery during parasite development (PMID:17452636). Recombinant PK9 autophosphorylates at T82, T265, and T269 and phosphorylates an exogenous substrate in vitro, establishing intrinsic kinase activity (PMID:17452636). Its identified physiological substrate is the E2 ubiquitin-conjugating enzyme UBC13, which PK9 phosphorylates at the conserved regulatory residue S106 to suppress UBC13's ubiquitin-conjugating activity (PMID:17452636); in P. falciparum cells, pharmacological inhibition of PK9 lowers K63-linked polyubiquitination and enlarges liver-stage parasites, linking PK9 activity to regulation of K63-linkage ubiquitination and liver-stage development (PMID:30595530). In P. falciparum, PK9 is expressed from roughly 18 hours post-infection and localizes to the parasitophorous vacuolar membrane and cytosol (PMID:17452636), while in P. berghei PK9 and UBC13 co-localize in the cytosol of erythrocytic and liver stages (PMID:35490588). Species divergence is notable: genetic knockout of PK9 in P. berghei leaves UBC13-S106 phosphorylation intact and does not affect viability at any life cycle stage, indicating PK9 is dispensable there and that a redundant kinase maintains UBC13-S106 phosphorylation in that species (PMID:35490588).

1. 2007 High

   Whether PK9 is an active enzyme was unknown; demonstrating autophosphorylation and substrate phosphorylation established it as a bona fide catalytically active kinase and mapped its autoregulatory sites.

   Evidence In vitro kinase assay with recombinant PfPK9 and histone H1, with phosphopeptide mapping to T82/T265/T269

   PMID:17452636

   Open questions at the time

   • Functional consequence of the three autophosphorylation sites for activity or regulation not defined
   • No structural model of the kinase domain
   • Endogenous activation trigger unknown
2. 2007 High

   The physiological target of PK9 was unknown; identifying UBC13 as a substrate phosphorylated at S106 and showing this suppresses its conjugating activity connected PK9 to control of the ubiquitin system.

   Evidence In vitro kinase assay with recombinant PfPK9 and PfUBC13, phosphosite mapping to S106, and ubiquitin-conjugating activity assay

   PMID:17452636

   Open questions at the time

   • In vitro suppression not yet tied to a cellular phenotype at this stage
   • Whether UBC13 is the sole substrate not established
   • Stoichiometry and kinetics of the reaction not defined
3. 2007 Medium

   The timing and site of PK9 action were unknown; a temporal expression and localization profile placed PK9 at the parasitophorous vacuolar membrane and cytosol during blood-stage infection.

   Evidence Time-course western blot and subcellular fractionation/immunolocalization in P. falciparum

   PMID:17452636

   Open questions at the time

   • Localization not functionally linked to UBC13 phosphorylation
   • Mechanism of PVM targeting unknown
   • Single-lab observation
4. 2018 Medium

   Whether PK9's in vitro effect on UBC13 translates to cellular ubiquitination and parasite biology was unknown; selective inhibitors showed PK9 activity supports K63-linked ubiquitination and liver-stage development in P. falciparum.

   Evidence Takinib/HS220 inhibitor treatment with K63-linked ubiquitination assay and fluorescence imaging of liver-stage parasites in hepatocytes

   PMID:30595530

   Open questions at the time

   • Pharmacological rather than genetic perturbation, leaving off-target effects possible
   • In vitro data showed PK9 suppresses UBC13 activity while cellular inhibition decreases K63 ubiquitination, so the directionality of regulation is unresolved
   • Molecular link between PK9 activity and the liver-stage size/number phenotype not defined
5. 2022 Medium

   Whether PK9 is essential and the exclusive kinase for UBC13-S106 was unknown; genetic knockout in P. berghei showed PK9 is dispensable across the life cycle and that UBC13-S106 phosphorylation persists via a redundant kinase.

   Evidence Genetic knockout of Pk9 in P. berghei with western blot for UBC13-S106 phosphorylation, life-cycle phenotyping, and cytosolic co-localization imaging of UBC13 and PK9

   PMID:35490588

   Open questions at the time

   • Identity of the redundant UBC13-S106 kinase not determined
   • Species divergence from P. falciparum pharmacological data unexplained
   • Whether P. falciparum PK9 is genetically dispensable not directly tested

• The physiological role and essentiality of PK9 across Plasmodium species remain unresolved, particularly reconciling the P. falciparum inhibitor phenotypes with dispensability in P. berghei.
• No genetic loss-of-function study of PK9 in P. falciparum
• Identity of the redundant UBC13-S106 kinase unknown
• Upstream signals activating PK9 and the biological output of UBC13 regulation undefined