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

ABSTRACT: The CCL5/CCR5 axis plays a pivotal role in tumor progression and metastasis. We previously reported CCR5 promotes 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 melanoma cells following CCL5 stimulation at three time points (5, 10 and 30 min). Temporal analysis revealed that CCL5 treatment modulated 256, 134, and 83 phosphosites at these respective time points, with a predominant bias toward upregulation. In total, 393 phosphosites across 315 phosphoproteins exhibited significant regulation. To specifically dissect CCR5-mediated phosphorylation events (distinct from other CCL5 receptors), we generated CCR5 knockout B16/F10 cells and performed comparative phosphoproteomic analysis against wild type cells at the 5-min CCL5 stimulation timepoint. This approach identified 52 phosphoproteins regulated by the CCL5/CCR5 axis, whose temporal phosphorylation dynamics were illustrated through heatmap visualization. Gene Ontology (GO) enrichment analysis revealed that CCL5/CCR5 axis participates in various biological processes, including gene expression, cell cycle, DNA repair and cytoskeleton organization. Notably, the phosphorylation of three cell cycle- associated proteins: Cep131, Khdrbs1 and Mak6 was analyzed in detail. All three proteins exhibited transient phosphorylation induction within 5-15 min of CCL5 stimulation, followed by attenuation at 15-30 min, and the phosphorylation activation was dependent on the CCR5. Overall. these findings provide a comprehensive phosphorylation resource for dissecting the molecular mechanisms underlying CCL5/CCR5-facilitated tumor progression, and provide important cues for identifying potential therapeutic targets for melanoma or other related diseases.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

DISEASE(S): Skin Cancer

SUBMITTER: Jie Liu  

LAB HEAD: jie liu

PROVIDER: PXD066097 | Pride | 2026-07-14

REPOSITORIES: Pride