AMA1 appears to be phosphorylated at multiple sites soon after synthesis in late schizonts, as the 83?kDa proprotein form is phosphorylated alongside the processed 66?kDa form, but the purpose of this is currently unknown16. the cytoplasmic domain of various AMA1 from Apicomplexa. The conservation is scored and colour coded by PRALINE (www.ibi.vu.nl). Amino acids predicted to be phosphorylated in by NetPhos (www.cbs.dtu.dk/services/NetPhos) and confirmed by mass spectrometry17,18,21 are highlighted. S610 was previously shown to be essential for efficient erythrocyte invasion. (B) The invasion ability of the different MKT 077 AMA1-TY1 parasite strains expressing AMA1 with single mutations in each phosphorylation site was investigated by an invasion assay. Assays were performed in the presence of 100?g/mL R1 peptide. Re-invasion was normalised to 3D7 and AMA1WT-TY1, which were used as controls. Error bars correspond to standard errors. Assays were performed in triplicates in three independent experiments. S610 is targeted by PKA While extensive data by Leykauf phosphorylation assays. Wild-type AMA1 (GST-AMA1WT) and phosphorylation-defective S610A (AMA1S610A) were compared with AMA1 with a single acceptor site at S610 left intact and all other phosphorylation sites mutated to non-phosphorylatable alanines (AMA1S610). Phosphorylation of AMA1S610A by purified bovine PKA (Fig. 2A,B) or by parasite extracts stimulated with cyclic AMP (Fig. 2C,D) was drastically reduced compared to AMA1WT, while AMA1S610 was phosphorylated to comparable levels as AMA1WT. This indicates that PKA is responsible for S610 phosphorylation parasites. Open in a separate window Figure 2 S610 is targeted by phosphorylation of GST, AMA1WT, AMA1S610A and AMA1S610 after incubation with schizont material in the presence of 32P–ATP and either with (+) or without (-) cAMP. (D) MKT 077 Densitometric quantification with error bars corresponds to standard deviation of two independent experiments done in triplicates. (E) Sandwich ELISA demonstrating H-89-induced inhibition of native AMA1 MKT 077 phosphorylation at S610. Parasites MKT 077 were treated with H-89 for 2?hours during egress and invasion and a mouse anti-genome and that are expressed in blood stages. The latter kinase is essential and might be required during schizogony as well as for other life cycle stages23. also has a cdk5 homolog MKT 077 called protein kinase 5 which appears to have nuclear functions24. A GSK3 homologue (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-gsk3-3repl-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 phosphorylation samples (upper panel) as well as coomassie stained loading Mouse monoclonal to RAG2 (lower panel) of AMA1WT and AMA1PM incubated with human GSK3 (hGSK3). (F) Differential phosphorylation of AMA1 variants with single phosphorylation sites (AMA1S588, AMA1S601, AMA1S610, AMA1T612, AMA1T613) by hGSK3. SDS-PAGE and autoradiograph of the phosphorylation samples (upper panel) as well as coomassie stained loading (lower panel) are shown. Thus, we examined phosphorylation of the AMA1 CPD by human GSK3 and found that, indeed, phosphorylation assays with parasite extracts, but, while AMA1WT and AMA1S610 were phosphorylated, presumably by residual PKA, AMA1T613 displayed no signal (Fig. 4A). We therefore hypothesized that S610, and likely its phosphorylation, must be present to prime the phosphorylation of T613. To test this hypothesis we generated an AMA1.