The Calcium-Dependent Protein Kinase 3 of Toxoplasma Influences Basal Calcium Levels and Functions beyond Egress as Revealed by Quantitative Phosphoproteome Analysis

Abstract

Calcium-dependent protein kinases (CDPKs) are conserved in plants and apicomplexan parasites. In Toxoplasma gondii, TgCDPK3 regulates parasite egress from the host cell in the presence of a calcium-ionophore. The targets and the pathways that the kinase controls, however, are not known. To identify pathways regulated by TgCDPK3, we measured relative phosphorylation site usage in wild type and TgCDPK3 mutant and knock-out parasites by quantitative mass-spectrometry using stable isotope-labeling with amino acids in cell culture (SILAC). This revealed known and novel phosphorylation events on proteins predicted to play a role in host-cell egress, but also a novel function of TgCDPK3 as an upstream regulator of other calcium-dependent signaling pathways, as we also identified proteins that are differentially phosphorylated prior to egress, including proteins important for ion-homeostasis and metabolism. This observation is supported by the observation that basal calcium levels are increased in parasites where TgCDPK3 has been inactivated. Most of the differential phosphorylation observed in CDPK3 mutants is rescued by complementation of the mutants with a wild type copy of TgCDPK3. Lastly, the TgCDPK3 mutants showed hyperphosphorylation of two targets of a related calcium-dependent kinase (TgCDPK1), as well as TgCDPK1 itself, indicating that this latter kinase appears to play a role downstream of TgCDPK3 function. Overexpression of TgCDPK1 partially rescues the egress phenotype of the TgCDPK3 mutants, reinforcing this conclusion. These results show that TgCDPK3 plays a pivotal role in regulating tachyzoite functions including, but not limited to, egress.

Analysis of Noncanonical Calcium-Dependent Protein Kinases in Toxoplasma gondii by Targeted Gene Deletion Using CRISPR/Cas9

“…For example, loss of TgCDPK3 also affects parasite egress, and under some conditions microneme secretion and motility (23). Recent studies indicate that TgCDPK3 acts upstream of TgCDPK1 by controlling calcium homeostasis, and consistent with this, TgCDPK1 overexpression can partially compensate for loss of CDPK3 (24). Although both TgCDPK1 and TgCDPK3 regulate egress in T. gondii, this function is provided by CDPK5 in P. falciparum (25).…”

“…Previous studies with other CDPKs have shown that in some cases they can compensate for each other or that they work in common pathways (23,24). Hence, we considered that the lack of phenotypes for noncanonical CDPKs might reflect redundant functions such that single-gene knockouts remain normal.…”

Designing selective inhibitors for calcium-dependent protein kinases in apicomplexans

“…For example, the functions of TgCDPK3 partially mimic those of TgCDPK1 and they can also be compensated for by activation of protein kinase G (PKG), a distinct kinase that is also required for both egress and entry of T. gondii 29, 42 . Recent studies implicate TgCDPK3 in control of calcium homeostasis, suggesting that it acts upstream of TgCDPK1 43 . Consistent with this finding, over-expression of TgCDPK1 can partially rescue the egress phenotype of Δ cdpk3 mutants 43 .…”

Targeted mass spectrometry: An emerging powerful approach to unblock the bottleneck in phosphoproteomics

“…This study revealed 700 phosphopeptides, including unreported phosphosites corresponding to proteins involved in cellular polarization and mating tip formation [33]. More recently, SCX/IMAC-based phosphoproteomic studies resulted in the identification of more than 13,000 phosphorylation sites in fly embryos [34], over 13,000 in the parasite Toxoplasma gondii [35] and nearly 36,000 unique phosphosites in 9 mouse tissues [36].…”