Regulation of cellular states via targeted phosphorylation of p53 using a nanobody-coupled kinase system

Phosphorylation participates in numerous signal transduction processes, including proliferation, differentiation, apoptosis, and cellular response to stimuli. Understanding its regulatory mechanisms is essential for advancing therapeutic interventions. In this study, we developed a target protein phosphorylation (TPP) system, consisting of a nanobody fused to a kinase domain, to investigate its ability to phosphorylate target proteins and regulate their cellular characteristics. We first verified that the nanobody-coupled kinase effectively phosphorylates GFP. Subsequently, we focused on p53 phosphorylation, identifying specific phosphorylation sites targeted by the system. This phosphorylation resulted in stabilization of p53 protein levels, inducing p21 expression, delaying cell cycle progression and suppressing cell growth. Furthermore, combining the TPP system with chemotherapeutic drugs (5-Fluorouracil and Oxaliplatin) enhanced cytotoxicity in colorectal cancer cells. The TPP system achieved p53 phosphorylation without external stimuli, inducing a DNA-damaged state in cells. In vivo, doxycycline-induced expression of the TPP system in a xenograft mouse model significantly inhibited tumor growth. This work demonstrates the ability to phosphorylate key regulatory proteins and alter cellular states, suggesting applications in studying phosphorylation-related pathways and developing therapies for diseases associated with dysregulated phosphorylation.