Abstract
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.
Footnotes
Abbreviations: PKA, cAMP-dependent protein kinase A; ProPeL, Proteomic Peptide Library; P-site, phosphoacceptor-site; RIα, cAMP-dependent protein kinase type I-alpha regulatory subunit; PKI, cAMP-dependent protein kinase inhibitor alpha; CDK16, Cyclin-dependent kinase 16; ACN, acetonitrile; FA, formic acid