Characterization of Plasmodium falciparum Atypical Kinase PfPK7^– Dependent Phosphoproteome

Abstract

PfPK7 is an "orphan" kinase displaying regions of homology to multiple protein kinase families. PfPK7 functions in regulating parasite proliferation/development as evident from the phenotype analysis of knockout parasites. Despite this regulatory role, the functions of PfPK7 in signaling pathways are not known. To better understand PfPK7-regulated phosphorylation events, we performed isobaric tag-based quantitative comparative phosphoproteomics of the schizont and segmenter stages from wild-type and pfpk7 parasite lines. This analysis identified 3,875 phosphorylation sites on 1,047 proteins. Among these phosphorylation events, 146 proteins with 239 phosphorylation sites displayed reduction in phosphorylation in the absence of PfPK7. Further analysis of the phosphopeptides revealed three motifs whose phosphorylation was down regulated in the pfpk7 cell line in both schizonts and segmenters. Decreased phosphorylation following loss of PfPK7 indicates that these proteins may function as direct substrates of PfPK7. We demonstrated that PfPK7 is active toward three of these potential novel substrates; however, PfPK7 did not phosphorylate many of the other proteins, suggesting that decreased phosphorylation in these proteins is an indirect effect. Our phosphoproteomics analysis is the first study to identify direct substrates of PfPK7 and reveals potential downstream or compensatory signaling pathways.

MoMo: Discovery of statistically significant post-translational modification motifs

“…To test the accuracy of the p-values reported by motif-x * , we examined whether they follow the expected uniform distribution when the input peptides are random. We sorted together p-values of all the motifs reported by motif-x * when run with 10,000 shuffled versions of the Pease et al [15] Supplemental Data 2 dataset, and we plotted the reported p-value as a function of its rank in the sorted list ( Fig. 1).…”

“…First, we reproduced the motif-x results from Fig. 3C in Pease et al [15]. Running motif-x * with the same input files (the 196 sequences from Pease et al…”

“…However, we found that many motifs reported by motif-x * (and, hence, by motif-x) are not statistically significant, and that the motif scores it reports are not highly correlated with motif significance. For example, of the 54 motifs found by motif-x * in the 3875 peptides in Supplemental Data 2 from Pease et al [15] using the (default) shuffled foreground peptides as background peptides (Fig. 4A), only 32 (59.2%) are statistically significant (p=0.05), as estimated using the Monte Carlo simulation.…”

“…Conversely, when shuffled versions of the foreground peptides are used as the background peptides, some highly significant motifs reported by motif-x * have motif scores close to zero, and less than those of some non-significant motifs. As a second example, we ran motif-x * on the much smaller dataset (196 peptides) used by Pease et al [15] (their Supplemental Data 3 file), and used peptides from the Plasmodium falciparum proteome as the background, to reproduce their Fig. 3C, as discussed at the start of the Results section.…”

“…Supplemental Data 3 that are centered on 'S' and do not contain an 'x'; the Plasmodium falciparum proteome as background) and motif-x * parameters equivalent to what Pease et al used with motif-x (--width 13 --score-threshold 0.00001 --min-occurrences 10 --db-background), motif-x * outputs exactly the same three reported motifs.Next, as a further check, we ran both versions of motif-x on the 3875 peptides in the Supplemental Data 2 file from Pease et al[15]. The results produced by motif-x * are slightly different from the reported results, unless we supply the --harvard switch to motif-x * .…”