Molecular Glue and Phosphorylation-Dependent 14-3-3 Recruitment to p53 with an Engineered Interaction Cassette.

The tumor suppressor p53 is regulated by phosphorylation-dependent protein-protein interactions, including via binding to 14-3-3 adaptor proteins, which can tune p53 activity. Molecular glue (MG)-induced stabilization of 14-3-3/client interactions offers an attractive strategy to probe such networks, but cellular engagement is often constrained by context-dependent phosphorylation and interaction occupancy. Here, we engineered phosphorylation- and MG-dependent 14-3-3 interaction cassettes (IC1 and IC2) and fused them to p53 to promote recruitment of endogenous 14-3-3 proteins in human cells. Biochemical characterization establishes high-affinity binding of the phosphorylated cassettes to 14-3-3 and enhanced in vitro stabilization by the 14-3-3 molecular glue 3'-deacetylated fusicoccin-A (FC-A). In HEK293T cells, Flag-p53-IC1 and Flag-p53-IC2 co-immunoprecipitated native 14-3-3 proteins. Mutation of the cassette's phospho-accepting serine to alanine abolished binding, confirming phosphorylation dependent recruitment. Transcriptomic profiling of transiently transfected cells reveals cassette-dependent remodeling of a p53-associated gene expression landscape. Together, these results establish a modular, MG- and phosphorylation-dependent platform for engaging 14-3-3 in a p53 context and for evaluating how chemical stabilization translates from biochemical interaction control to cellular pathway-level readouts.