Direct phospho-proteoform delivery reveals a degradation-prone monomeric state of 14-3-3

Site-specific phosphorylation controls protein function, interactions, and cellular fate, but the effects of individual phospho-proteoforms remain difficult to define in cells. Existing methods rely on kinase-mediated phosphorylation, which often generates heterogeneous mixtures that are phosphatase-sensitive, or on phosphomimetic substitutions that frequently fail to reproduce authentic phospho-states. Here we establish direct phospho-proteoform delivery, which combines genetic code expansion-enabled production of proteins containing the nonhydrolyzable phosphoserine analog nhpS in E. coli with electroporation-based delivery of purified proteins into human cells. This strategy creates an experimental framework for testing defined phospho-proteoforms in cells with control over identity, dose, and timing, independent of intracellular kinase, phosphatase, and expression systems. Using this capability, we tested whether phosphorylation-induced monomerization of 14-3-3 proteins is sufficient to alter cellular protein fate, a question that cannot be addressed by conventional methods. Installation of nhpS at the conserved dimer interface created a proteasome-sensitive, degradation-prone state across multiple paralogs. Although cereblon preferentially associated with monomeric 14-3-3, cereblon knockout did not rescue degradation, and monomeric 14-3-3 also engaged the E3 ligase adaptor SKP1. These findings reveal phosphorylation-induced monomerization as a conserved trigger of 14-3-3 proteostatic control and establish direct phospho-proteoform delivery as a general route to connect single phosphorylation events to cellular phenotypes.