The Calcium-Dependent Protein Kinase 3 of Toxoplasma Influences Basal Calcium Levels and Functions beyond Egress as Revealed by Quantitative Phosphoproteome Analysis
Figure 5
Overexpression of TgCDPK1 partially rescues the egress defect.
A) TgCDPK1 expression in engineered parasites. Lysates from MBE1.1 parasites with and without TgCDPK1-HA expressed off the GRA2 promoter were separated by gel electrophoresis and blotted. Antibodies against TgCDPK1 were used to detect both the endogenous TgCDPK1 and TgCDPK1-HA expression (the endogenous protein migrates slightly faster than the HA-tagged version). The mass (in kDa) and migration of size markers are shown to the left RON4 expression was detected by blotting with rabbit anti-RON4 and served as a loading control. B) Overexpression of TgCDPK1 partially rescues egress. Parasites were treated with 1 µM A23187 and egress monitored over 10 minutes. Wild type (RH), TgCDPK3 mutant (MBE1.1) and MBE1.1 over-expressing TgCDPK1 (MBE1.1::CDPK1) were compared. C) TgCDPK1 and TgCDPK3 have overlapping substrate preferences. Peptide microarrays spotted with random 13-mer peptides with a central serine residue have been incubated with recombinant TgCDPK1 or TgCDPK1 and 32P-ATP. All peptides where ranked according to the ability of the respective kinase to phosphorylate any given peptide, with the highest phosphorylation ranked 1st. The scale is log10.