CDPK2

TgCDPK2 is a Ca2+-dependent protein kinase that serves as a critical regulator of amylopectin metabolism in Toxoplasma gondii and is essential for viable tissue cyst development (PMID:26651943). It localizes to amylopectin stores through a carbohydrate-binding module 20 (CBM20), and its catalytic activity is switched on by Ca2+ acting through its EF-hands, coupling calcium signaling to storage-carbohydrate turnover (PMID:26651943). Once activated, CDPK2 phosphorylates enzymes of amylopectin catabolism, including glycogen phosphorylase (TgGP) at Ser25; the phosphomimetic TgGP S25E bypasses CDPK2 and rescues amylopectin accumulation in Δcdpk2 parasites, placing CDPK2 directly upstream of TgGP-driven degradation (PMID:28851850). Loss of CDPK2 causes massive amylopectin hyperaccumulation from both increased synthesis and lost degradation, producing gross morphological defects and complete ablation of cyst formation in mice; this lethality is entirely attributable to amylopectin overproduction, as deleting starch synthase in a Δcdpk2 background abolishes the accumulation and restores cyst formation and virulence (PMID:26651943, PMID:34129780). Although its mRNA is expressed in tachyzoites, TgCDPK2 protein is not detectably present there and does not account for the KT5926-sensitive kinase controlling tachyzoite motility (PMID:11154702).

1. 2001 Medium

   Established that TgCDPK2, despite tachyzoite mRNA expression, is not the kinase governing tachyzoite motility, distinguishing it from TgCDPK1 and redirecting attention away from a motility role.

   Evidence RT-PCR for mRNA plus immunodetection/protein fractionation in Toxoplasma tachyzoites

   PMID:11154702

   Open questions at the time

   • Did not identify the stage or condition where TgCDPK2 protein is expressed
   • Left the actual cellular function of CDPK2 unknown
2. 2015 High

   Defined CDPK2 as a Ca2+-regulated kinase targeted to amylopectin via a CBM20 domain and required to restrain amylopectin levels, revealing its true role in storage-carbohydrate metabolism.

   Evidence Genetic knockout, CBM20/EF-hand domain dissection, phosphoproteomics, and carbohydrate analysis in Toxoplasma

   PMID:26651943

   Open questions at the time

   • Direct catabolic substrates were not individually validated at this stage
   • How EF-hand Ca2+ binding mechanistically activates the kinase not resolved
3. 2015 High

   Demonstrated that CDPK2 is essential specifically for bradyzoite viability, since its loss drives amylopectin granule accumulation that abolishes cyst formation in vivo.

   Evidence Genetic knockout with mouse infection model and electron microscopy/morphological analysis

   PMID:26651943

   Open questions at the time

   • Did not establish which specific catabolic enzyme(s) mediate the phenotype
4. 2017 High

   Placed CDPK2 mechanistically upstream of glycogen phosphorylase by showing that Ser25 phosphorylation of TgGP activates degradation, with the phosphomimetic S25E rescuing amylopectin accumulation in Δcdpk2.

   Evidence Genetic epistasis with S25A/S25E/S25stop TgGP mutants, amylopectin assays, and mouse brain cyst counts in the Pru strain

   PMID:28851850

   Open questions at the time

   • Whether TgGP is the sole relevant substrate not established
   • Direct in vitro demonstration of CDPK2 phosphorylating TgGP Ser25 not detailed
5. 2021 High

   Proved that the lethality of CDPK2 loss stems entirely from amylopectin overproduction, since ablating starch synthase in Δcdpk2 removes the accumulation and restores cyst formation and virulence.

   Evidence Double genetic knockout (ΔSS/Δcdpk2) with mouse infection, cyst formation, and virulence assays

   PMID:34129780

   Open questions at the time

   • Does not resolve upstream signals that trigger Ca2+-dependent CDPK2 activation in bradyzoites
   • Full set of catabolic enzymes controlled by CDPK2 remains incompletely defined

• The upstream Ca2+ signal that activates CDPK2 during bradyzoite differentiation and the complete complement of its amylopectin-catabolic substrates remain to be defined.
• Trigger for Ca2+-dependent activation in cysts unknown
• Substrates beyond TgGP not fully enumerated