Quantitative phosphoproteomic profiling of CCL5/CCR5 signaling cascade in melanoma cells

The CCL5/CCR5 axis plays a pivotal role in tumor progression and metastasis. We previously reported CCR5 promoted melanoma EMT and metastasis by upregulating TGFβ1 expression via the PI3K/AKT/GSK3β pathway. However, the full spectrum of downstream events triggered by CCR5 activation remains poorly understood.

Here we employed quantitative phosphoproteomics to profile dynamic phosphorylation events in B16/F10 cells following CCL5 stimulation at three time points (5, 10 and 30 min). Moreover, to specifically dissect CCR5-mediated phosphorylation events distinct from other CCL5 receptors, we performed comparative phosphoproteomics between CCR5 knockout and wild type cells at the 5-min stimulation timepoint.

Temporal analysis revealed that CCL5 treatment modulated 256, 134, and 83 phosphosites at these respective time points. In total, 393 phosphosites across 315 phosphoproteins exhibited significant regulation. The comparative phosphoproteomics between CCR5 knockout and wild type cells identified 52 phosphoproteins and their temporal dynamics were illustrated. Gene Ontology enrichment analysis revealed that CCL5/CCR5 axis participated in various biological processes, particularly cell cycle regulation. Notably, upon CCL5 stimulation, three cell cycle-associated proteins-CEP131, KHDRBS1, and MAPK6-underwent phosphorylation activation, but this activation was remarkably prevented by CCR5 knockout. Overall. these findings provide a comprehensive phosphorylation resource for dissecting the molecular mechanisms underlying CCL5/CCR5-facilitated tumor progression and identifying potential therapeutic targets.