Phosphoproteomic Profiling Identifies PAK4 S474 Phosphorylation Affects Docetaxel Chemosensitivity via Modulation of Microtubule Stabilization in Breast Cancer.

Background/Objectives: Docetaxel is a frontline chemotherapeutic agent for breast cancer; however, therapeutic resistance remains a major clinical challenge. Emerging evidence suggests that chemotherapy-induced adaptive phosphorylation events can promote survival signaling and contribute to drug resistance. However, the phosphoproteomic mechanisms underlying docetaxel-induced adaptive responses remain poorly characterized. Methods: We performed integrated quantitative proteomic and phosphoproteomic profiling in breast cancer cells following docetaxel exposure. Candidate kinases associated with phosphorylation remodeling were identified and validated using TCGA-BRCA and CPTAC clinical datasets. The functional significance of PAK4 phosphorylation was validated using phosphomimetic and phospho-deficient mutants, pharmacological inhibition, and assessment of microtubule stabilization. Results: Integrated phosphoproteomic analysis revealed extensive phosphorylation remodeling following docetaxel treatment and identified PAK4 as a candidate kinase associated with the adaptive response. Analysis of the CPTAC phosphoproteomic dataset showed that phosphorylation of PAK4 at S474 was elevated in breast cancer tissues, increased with tumor stage, and was associated with poorer overall survival. In breast cancer cells, docetaxel induced phosphorylation of PAK4 at S474 without altering total PAK4 expression. Functionally, phosphomimetic PAK4 (S474D) reduced docetaxel sensitivity, whereas phospho-deficient PAK4 (S474A) enhanced drug sensitivity. Pharmacological inhibition of PAK4 using LCH-7749944 significantly enhanced the inhibitory effect of docetaxel on cell viability and increased apoptosis in breast cancer cells. Mechanistically, PAK4 inhibition enhanced docetaxel-induced microtubule stabilization, as evidenced by increased α-tubulin acetylation and accumulation of stabilized microtubule structures. Conclusions: Our study demonstrates that docetaxel induces global phosphorylation network reprogramming in breast cancer cells and identifies PAK4 S474 phosphorylation as a key determinant of docetaxel sensitivity. Inhibition of PAK4 enhances microtubule stabilization and improves the efficacy of docetaxel, providing a potential combinatorial strategy to overcome taxane resistance.