Label-free quantitative phosphoproteomic profiling of cellular response induced by an insect cytokine paralytic peptide

Paralytic peptide (PP) participates in diverse physiological processes as an insect cytokine, such as immunity control, paralysis induction, regulation of cell morphology and proliferation. To investigate the molecular mechanism underlying those physiological activities, we systematically investigated the global phosphorylation events in fat body of silkworm larvae induced by PP through label-free quantitative phosphoproteomics. 2534 phosphosites were finally identified, of which the phosphorylation level of 620 phosphosites on 244 proteins was significantly up-regulated and 67 phosphosites on 43 proteins was down-regulated. Among those proteins, 13 were protein kinases (PKs), 13 were transcription factors (TFs) across 10 families and 17 were metabolism related enzymes. Meanwhile, Motif-X analysis of the phosphorylation sites showed that 16 motifs are significantly enriched, including 8 novel phosphorylation motifs. In addition, KEGG and functional interacting network analysis revealed that phosphorylation cascades play the crucial regulation roles in PP-dependent signaling pathways, and highlighted the potential central position of the mitogen-activated protein kinases (MAPKs) in them. These analyses provide direct insights into the molecule mechanisms of cellular response induced by PP.

Significance: PP as an insect cytokine participated in diverse functions including immunity control paralysis induction, regulation of cell morphology and proliferation. In this study, we performed firstly a label-free quantitative phosphoproteomics analysis. We found some new phosphorylation targets of PP-stimulation. Meanwhile, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and functional networks revealed that phosphorylation cascades play the crucial regulation roles in PP-dependent signaling pathways. In addition, the potential central position of the mitogen-activated protein kinases (MAPKs) was highlighted in PP-dependent signaling pathways. We think our findings may help us gain a systematic understanding of the cytokine-dependent response regulation in insects.