Figure 2
S610 is targeted by PfPKA.
(A) Autoradiograph and SDS-PAGE of recombinant AMA1-GST variants treated with bovine heart PKA in the presence of cAMP and 32P-γ-ATP. The AMA1 variants either had S610 mutated to alanine (AMA1S610A) or had all phospho-sites except for S610 (AMA1S610) mutated. Purified GST serves as negative and AMA1WT as positive control. (B) Signal intensities were quantified using Image Gauge software (Fujifilm, Image Gauge V4.0). Signal intensity of the GST sample was subtracted from each sample and they were then normalized against the signal intensity of the AMA1WT sample. Error bars correspond to standard deviation of two independent experiments done in triplicate. (C) SDS-PAGE and autoradiograph of in vitro phosphorylation of GST, AMA1WT, AMA1S610A and AMA1S610 after incubation with schizont material in the presence of 32P-γ-ATP and either with (+) or without (-) cAMP. (D) 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 phosphorylation at S610. Parasites were treated with H-89 for 2 hours during egress and invasion and a mouse anti-PfAMA1 antibody was used to capture PfAMA1 from culture lysates. Phosphorylation of S610 was detected in an ELISA format using the anti-PfAMA1S610p antibody. Histograms were generated after normalizing against uninfected (0%, background) and untreated (100%) culture signals. Lambda phosphatase-treatment was used to denote zero phosphorylation and chloroquine treatment was used to exclude parasite growth arrest as a cause for reduced phosphorylation.
