Figure 3. T₆₁₃ is phosphorylated by GSK3.

(A–D) Expression and localization of PfGSK3 and its PKA dependent phosphorylation of AMA1. (A) Schematic drawing of the GSK3-GFP 3′replacement approach in 3D7 parasites and diagnostic PCR revealing plasmid integration. The gsk3 gene has a six exon structure and an open reading frame of 2472 base pairs. Approximately 1 kb of the 3′ end was fused with the coding sequence of GFP (black) and cloned into a pARL derivate (pARL-gsk3-3’repl-gfp). The human dihydrofolate reductase (hDHFR, grey box) of the plasmid allowed selection of transgenic parasites. Position of oligonucleotides used for diagnostic PCR are shown with blue and red arrows. Sizes are indicated in kilo bases (kb). (B) Expression of PfGSK3-GFP in late stage parasites was analyzed by Western blot analysis using anti-GFP specific antibodies. Anti-Aldolase specific antibodies were used as a loading control. (C,D) Epifluorescence (C) and confocal (D) localization of PfGSK3-GFP in late trophozoites (LT) schizonts (S) and merozoites (M) revealed perinuclear and cytosolic distribution. Nuclei are stained with DAPI (blue). Scale bars, 2 μm. E. SDS-PAGE and autoradiograph of in vitro phosphorylation samples (upper panel) as well as coomassie stained loading (lower panel) of AMA1_WT and AMA1_PM incubated with human GSK3β (hGSK3β). (F) Differential in vitro phosphorylation of AMA1 variants with single phosphorylation sites (AMA1_S588, AMA1_S601, AMA1_S610, AMA1_T612, AMA1_T613) by hGSK3β. SDS-PAGE and autoradiograph of the in vitro phosphorylation samples (upper panel) as well as coomassie stained loading (lower panel) are shown.