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Protein Kinase A Dependent Phosphorylation of Apical Membrane Antigen 1 Plays an Important Role in Erythrocyte Invasion by the Malaria Parasite

Figure 2

PfPKA phosphorylates the AMA1 tail exclusively on residue S610.

(A) Multiple alignment of AMA1 C-terminal cytoplasmic domains including protein sequences of nine different Plasmodium species, T. gondii, B. bovis and two Theileria species. The conservation is scored and colour coded by PRALINE (www.ibi.vu.nl). Amino acids predicted to be phosphorylated in P. falciparum AMA1 by NetPhos (www.cbs.dtu.dk/services/NetPhos) are scaled up in font size according to their relative predicted probabilities above the alignment and numbered regarding the protein sequence. As shown below the alignment the GST-fusion protein of the AMA1 tail was mutated at residues S601, S610, T612, T613, Y621 and Y622, respectively. (B, C) SDS-PAGE and radiograph of phosphorylation samples. GST-fusion proteins of the AMA1 tail and mutants were incubated with schizont lysate and 32[P]- γ -ATP. As a loading control the membrane was probed with an anti-AMA1-C antibody. (C) H89 (50 µM) and KT5720 (10 µM), two inhibitors to PKA reverse the effect of cAMP (1 µM) on AMA1 tail phosphorylation. (D, E) Quantitation of signal intensities in (C) with Image Gauge software. The phosphorylation signal strength in untreated schizont lysate was set to 100% and all other signals were 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. (F) PfPKA-HA was immuno-precipitated from lysate of 3D7PfPKA-HA transgenic parasites using protein-G-sepharose. PfPKA-HA was detected by Western Blot analysis using anti-HA antibodies and parasite lysate, washed fraction or sepharose beads. (G) SDS-PAGE and radiograph of in vitro phosphorylation of AMA1-GST using either protein-G-sepharose incubated with 3D7PfPKA-HA or with wild type (WT) parasites in the presence of 32[P]- γ -ATP.

Figure 2

doi: https://doi.org/10.1371/journal.ppat.1000941.g002