Cushing's syndrome mutant PKA^L205R exhibits altered substrate specificity

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.

Evolution of protein kinase substrate recognition at the active site

“…This suggests that this approach can successfully identify residues that are of relevance for the functional divergence of kinases. Of these residues, 8 have been implicated in determining differences in kinase specificity: domain position 84 [32–34], 86 [35], 144 [16,36,37], 157 [37,38], 158 [35,37], 162 [35], 164 [39,40], and 205 [35]. The number of substitutions for ‘proximal’ residues is generally higher than that for residues without an assigned function (Mann-Whitney, one-tailed, p = 3.2 × 10 −6 ).…”

Molecular Basis for Ser/Thr Specificity in PKA Signaling

“…Another mutation leading to Cushing’s syndrome in the Cα subunit of PKA is L205R [ 56 ], which disrupts the intramolecular allosteric network [ 65 ]. Consequently, PKA C L205R has a decreased catalytic efficiency for Kemptide and phosphorylates non-canonical substrates leading to a dysregulated signaling network in tumor cells [ 65 , 66 , 67 ].…”

Characterizing Protein Kinase Substrate Specificity Using the Proteomic Peptide Library (ProPeL) Approach

“…We have curated a master negative control list (Lubner et al, 2017), which was generated by pooling phosphopeptides previously identified in negative control experiments (Chou et al, 2012), previously identified endogenous E. coli phosphorylation sites (Macek et al, 2008; Soares et al, 2013), and phosphorylation sites identified in empty vector and kinase dead negative control experiments. This list is available as Supplementary Table S1.…”

“…Using our lab’s suite of computational tools, we can extract and visualize kinase specificity motifs, and make high-confidence predictions of downstream targets. ProPeL can also be used to evaluate the influence of disease-associated mutations on kinase substrate specificity (Lubner et al, 2017). …”

“… Key References Chou et al, 2012 Original proof-of-concept publication for ProPeL Lubner et al, 2017 Publication using the current ProPeL workflow …”