Mutant/mutation
In this mutant the wild type pkg gene is replaced with a modified allele pkg (T619Q-HA), in which the threonine gatekeeper residue was mutated to a larger glutamine residue together with a C-terminal triple HA epitope tag. The mutant does not contain a selectable marker. The positive/negative selectable marker cassette has been removed by negative selection with 5-FC.

Protein (function)
Cyclic GMP-dependent protein kinases (PKGs) are the major mediators of the cGMP signal transduction pathway and regulate a variety of physiological effects. PKG is is a serinethreonine kinase that transfers the γ-phosphate of ATP, in a cGMP-dependent manner, to a variety of substrate proteins.

Work in Toxoplasma gondii and Eimeria tenella, coccidian parasites that are related to Plasmodium, identified PKG as the primary target for two structurally distinct anticoccidal compounds, the trisubstituted pyrrole compound 1 (C1) and the imidazopyridine-based inhibitor compound 2 (C2). Both compounds achieve high selectivity over PKG of humans by exploiting an unusually small gatekeeper residue within the active site of all apicomplexan PKG enzymes. Mutating the threonine gatekeeper residue of apicomplexan PKG to a larger residue renders parasites resistant to both inhibitors.

This leads to the hypothesis that a major function for PKG is to control intracellular Ca2+ levels in malaria parasites, through the regulation of phosphoinositide metabolism by lipid kinases. This study presents evidence in support of this idea by showing in three life cycle stages and two Plasmodium species that activation of PKG is critically required to regulate cytosolic Ca2+ levels. PKG emerges as a universal regulator that controls ookinete gliding, gametocyte activation, and schizont rupture.

Phenotype
Mutant parasites show normal development throughout the complete life cycle. Expression of PKG(T619Q-HA) in ookinetes

Additional information
See also mutant RMgm-999: A mutant expressing a C-terminal HA-tagged version of PKG

Gliding motility of ookinetes expressing PKG-HA was strongly inhibited by the inhibitor C2 (imidazopyridine-based inhibitor compound 2) with a half-maximal effect of ~100 nM. Expression of PKG(T619Q-HA), in contrast, conferred complete resistance to C2 up to at least 5 mM, demonstrating that PKG is the critical target for C2 and essential for ookinete gliding.

Inhibition of gliding motility by C2 was as potent as disrupting cGMP production genetically by deleting guanylyl cyclase beta (GCβ; PBANKA_113670). In contrast,  interfering with degradation of cyclic nucleotides through complete deletion of phosphodiesterase delta, putative (PDEδ; PBANKA_133370)  had the opposite effect, resulting in a marked increase in average gliding speed

Other mutants
RMgm-999: A mutant expressing a C-terminal HA-tagged version of PKG