Protein Kinase A Dependent Phosphorylation of Apical Membrane Antigen 1 Plays an Important Role in Erythrocyte Invasion by the Malaria Parasite
Figure 3
Two dimensional gel analyses confirm that P. falciparum AMA1 is phosphorylated at S610 in vivo.
P. falciparum proteins were metabolically labelled with 32[P], detergent extracted and resulting 2DE blots developed using Deep Purple stain to visualize total protein (A). Autoradiography detected in vivo phosphorylated proteins (B), and Western Blotting with an anti-AMA1 antibody detected the AMA1 tail (C). Isoelectric point and molecular sizes are indicated on the top and left. 2D spots corresponding to the 83 kDa precursor (AMA183) or post-translationally processed ∼66 kDa AMA1 fragment (AMA66) are highlighted by bounding boxes, respectively. Magnification of the AMA166 region reveals a series of five discrete 32[P]-labelled (a, c, d) or -unlabelled (b, e) protein spots recognized by anti-AMA1 antibody (arrows). (D-I) all show ∼66 kDa species of AMA1 separated by 2DE and analysed by Western Blot. 3D7 parasites either expressed a transgenic TY1-tagged wild type W2mef AMA1 (AMA1-WT-TY1; D, E), or mutant forms where the W2mef transgene carried the AMA1-S610A mutation (AMA1-S610A-TY1; F, G) or mutations at all putative phosphorylation sites in the cytoplasmic tail AMA1-PM-TY1; H, I). Blots were probed with a mouse monoclonal antibody against TY1 to detect transgenic epitope-tagged AMA1 followed and by an anti-AMA1 antibody against the AMA1 tail to detect both endogenously expressed 3D7 AMA1 as well as the W2mef transgenic species.