Modular Phosphoprotein Signatures Link Rac1 Inhibition to Neurite Morphogenesis in a Dose-Dependent Manner

INTRODUCTION: Precise regulation of neurite initiation, elongation, and branching is critical for neuronal network formation. Rac1, a key regulator of cytoskeletal remodeling, influences neurite morphogenesis through protein phosphorylation-mediated signaling, but the global phosphorylation landscape that governs Rac1-mediated morphogenesis remains unknown. METHOD: To address this knowledge gap, we performed phosphoproteomics profiling of primary rat cortical neurons treated with 3, 10, or 30 µm of a Rac1 inhibitor for 48 h to evaluate phosphoprotein dynamics. Phosphorylation levels of 167 signaling proteins were quantified using a targeted phospho-antibody array and correlated with neuron count, neurite count and length, and branch point count. RESULTS: Correlation analysis identified morphology-specific phosphoproteins, such as Tau (Thr181), GluR1 (Ser863), TrkB (Tyr515), and Merlin (Ser10), whose phosphorylation levels were significantly altered across three Rac1 inhibitor concentrations, and multiple phosphorylation sites showed dose-specific correlations with neurite morphology features. CONCLUSION: These results define a correlation-based framework linking phosphoprotein signaling to neurite morphology and offer novel insights into neurodevelopmental processes, neuronal disorders, and developmental neurotoxicity.