The Plasmodium falciparum Schizont Phosphoproteome Reveals Extensive Phosphatidylinositol and cAMP-Protein Kinase A Signaling

Abstract

The asexual blood stages of Plasmodium falciparum cause the most lethal form of human malaria. During growth within an infected red blood cell, parasite multiplication and formation of invasive merozoites is called schizogony. Here, we present a detailed analysis of the phosphoproteome of P. falciparum schizonts revealing 2541 unique phosphorylation sites, including 871 novel sites. Prominent roles for cAMP-dependent protein kinase A- and phosphatidylinositol-signaling were identified following analysis by functional enrichment, phosphoprotein interaction network clustering and phospho-motif identification tools. We observed that most key enzymes in the inositol pathway are phosphorylated, which strongly suggests additional levels of regulation and crosstalk with other protein kinases that coregulate different biological processes. A distinct pattern of phosphorylation of proteins involved in merozoite egress and red blood cell invasion was noted. The analyses also revealed that cAMP-PKA signaling is implicated in a wide variety of processes including motility. We verified this finding experimentally using an in vitro kinase assay and identified three novel PKA substrates associated with the glideosome motor complex: myosin A, GAP45 and CDPK1. Therefore, in addition to an established role for CDPK1 in the motor complex, this study reveals the coinvolvement of PKA, further implicating cAMP as an important regulator of host cell invasion.

The Global Phosphoproteome of Chlamydomonas reinhardtii Reveals Complex Organellar Phosphorylation in the Flagella and Thylakoid Membrane

“…For any site within six residues of the N-or C terminus of its largest contiguous sequence, the VBA program used the inferred protein sequence to extend the site neighborhood to give a phosphosite-centered 13-mer sequence. Sequences were categorized as basic, acidic, proline-directed, tyrosine, or "other" according to the rules of Huttlin and colleagues (23), well-articulated by Lasonder and colleagues (25). Motifs were determined for each category using Motif-X (http://motif-x.med.harvard.…”

Phosphoproteomic analysis of the resistant and susceptible genotypes of maize infected with sugarcane mosaic virus

“…It has been reported that physical interactions between proteins can be regulated by phosphorylation (Lasonder et al 2012). To better elucidate virus infection processes regulated by phosphorylation, a protein-protein interaction network (Fig.…”

Applying chemical genetic tools to the study of phospho-signalling pathways in malaria parasites

“…To date there have been five mass spectrometry malaria parasite phosphoproteomes published that have established that protein phosphorylation plays a wide regulatory role in malaria, controlling key processes such as metabolism and cell growth, as well as malaria-specific processes such as egress, invasion and cytoadherence [16][17][18][19][20]. The basic work flow for a global phosphoproteome study is as follows:-1) extraction of most of the proteins in the parasite infected cell; 2) digestion of the proteins to small peptides with, for example, trypsin (an enzyme which cuts at lysine or arginine residues); 3) chromatographic fractionation of the peptides to simplify the complexity of the sample; 4) enrichment of phosphopeptides using an enrichment technique such as IMAC (immobilized metal affinity chromatography) or TiO 2 and 5) identification of the peptides and the phosphorylation sites on a mass spectrometer .…”

“…Lasonder et al, [17] also identified the phosphoproteome of schizont stage of the parasite where they reported phosphopeptides with a mascot score of at least 15, 1% false discovery rate and 75%…”

“…Applying these criteria identified 2541 phosphorylation sites on 919 parasite proteins [17]. Comparing the phosphoproteomes of the schizont stage of P. falciparum from Lasonder, Treeck and Solyakov remarkably only identifies 450 phosphorylation sites that were common to all three phosphoproteomes.…”