Protein Kinase A Dependent Phosphorylation of Apical Membrane Antigen 1 Plays an Important Role in Erythrocyte Invasion by the Malaria Parasite
Figure 1
Recombinant AMA1 C-terminal tail is phosphorylated in vitro by P. falciparum (3D7 line) parasite lysates in a calcium and cAMP dependent manner.
(A) Schematic representation of AMA1 and the GST-fusion protein used. Signal peptide (blue), prosequence (PS), ectodomains I, II & III, transmembrane domain (grey), cytoplasmic tail (C) and thrombin cleavage site are indicated. (B, C) Auto-radiographs showing phosphorylation of recombinant AMA1 tail by parasite lysates in the presence of 1.5 mM EGTA/1 mM EDTA or 2 mM CaCl2 or 1 µM cAMP. The AMA1 tail was incubated with schizont (S), merozoite (M) or red blood cell (RBC) lysates and 32[P]-γ -ATP. After washing the GST part was cleaved off with thrombin. As a loading control the membrane was probed with an anti-AMA1 antibody detecting the AMA1 tail. Molecular sizes are indicated on the left. (D) Quantitation of signal intensities in panel C with Image Gauge software. In the absence of additional EGTA/EDTA or cAMP the strength of the phosphorylation signal in untreated schizont lysate was set to 100% and all other signals are relative to that. The numbers of experimental replicates in in vitro phosphorylation assays are found in the Supplementary data (Text S1). Error bars correspond to standard deviation.