LAUSR

The PKAL205R hotspot mutation has been implicated in Cushing's syndrome through hyperactive gain‐of‐function PKA signaling; however, its influence on substrate specificity has not been investigated. Here, we employ the Proteomic Peptide Library (ProPeL) approach to create high‐resolution models for PKAWT and PKAL205R substrate specificity. We reveal that the L205R mutation reduces canonical hydrophobic preference at the substrate P + 1 position, and increases acidic preference in downstream positions. Using these models, we designed peptide substrates that exhibit altered selectivity for specific PKA variants, and demonstrate the feasibility of selective PKAL205R loss‐of‐function signaling. Through these results, we suggest that substrate rewiring may contribute to Cushing's syndrome disease etiology, and introduce a powerful new paradigm for investigating mutation‐induced kinase substrate rewiring in human disease.