scholarly journals Cushing’s Syndrome mutant PKAL205R exhibits altered substrate specificity

2016 ◽  
Author(s):  
Joshua M. Lubner ◽  
Kimberly L. Dodge-Kafka ◽  
Cathrine R. Carlson ◽  
George M. Church ◽  
Michael F. Chou ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Cushing’S Syndrome ◽  
Cushing's Syndrome ◽  
Peptide Library ◽  
Loss Of Function ◽  
New Paradigm ◽  
Disease Etiology ◽  
Peptide Substrates ◽  
Kinase Substrate ◽  
High Resolution Models

AbstractThe 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.

scholarly journals Cushing's syndrome mutant PKAL 205R exhibits altered substrate specificity

FEBS Letters ◽  
2017 ◽  
Vol 591 (3) ◽  
pp. 459-467 ◽  
Author(s):  
Joshua M. Lubner ◽  
Kimberly L. Dodge-Kafka ◽  
Cathrine R. Carlson ◽  
George M. Church ◽  
Michael F. Chou ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Cushing’S Syndrome ◽  
Cushing's Syndrome

scholarly journals Reprogramming protein kinase substrate specificity through synthetic mutations

2016 ◽  
Author(s):  
Joshua M. Lubner ◽  
George M. Church ◽  
Michael F. Chou ◽  
Daniel Schwartz
Keyword(s):  
Protein Kinase ◽  
Substrate Specificity ◽  
Structure Function ◽  
Binding Pocket ◽  
Missense Mutations ◽  
New Paradigm ◽  
Substrate Binding Pocket ◽  
Kinase Substrate ◽  
Substrate Preferences ◽  
Kinase Specificity

Protein kinase specificity is largely imparted through substrate binding pocket motifs. Missense mutations in these regions are frequently associated with human disease, and in some cases can alter substrate specificity. However, current efforts at decoding the influence of mutations on substrate specificity have been focused on disease-associated mutations. Here, we adapted the Proteomic Peptide Library (ProPeL) approach for determining kinase specificity to the task of exploring structure-function relationships in kinase specificity by interrogating the effects of synthetic mutation. We established a specificity model for the wild-type DYRK1A kinase with unprecedented resolution. Using existing crystallographic and sequence homology data, we rationally designed mutations that precisely reprogrammed the DYRK1A kinase at the P+1 position to mimic the substrate preferences of a related kinase, CK II. This study illustrates a new synthetic biological approach to reprogram kinase specificity by design, and a powerful new paradigm to investigate structure-function relationships underpinning kinase substrate specificity.

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

2018 ◽  
Vol 10 (2) ◽  
pp. e38 ◽  
Author(s):  
Joshua M. Lubner ◽  
Jeremy L. Balsbaugh ◽  
George M. Church ◽  
Michael F. Chou ◽  
Daniel Schwartz
Keyword(s):  
Protein Kinase ◽  
Substrate Specificity ◽  
Peptide Library ◽  
Kinase Substrate

scholarly journals Cushing’s syndrome driver mutation disrupts protein kinase A allosteric network, altering both regulation and substrate specificity

2019 ◽  
Vol 5 (8) ◽  
pp. eaaw9298 ◽  
Author(s):  
Caitlin Walker ◽  
Yingjie Wang ◽  
Cristina Olivieri ◽  
Adak Karamafrooz ◽  
Jordan Casby ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Substrate Specificity ◽  
Protein Kinase A ◽  
Cushing’S Syndrome ◽  
Genetic Alterations ◽  
Cushing's Syndrome ◽  
Single Mutation ◽  
Α Subunit ◽  
Adrenocortical Adenomas ◽  
Kinase A

Genetic alterations in the PRKACA gene coding for the catalytic α subunit of the cAMP-dependent protein kinase A (PKA-C) are linked to cortisol-secreting adrenocortical adenomas, resulting in Cushing’s syndrome. Among those, a single mutation (L205R) has been found in up to 67% of patients. Because the x-ray structures of the wild-type and mutant kinases are essentially identical, the mechanism explaining aberrant function of this mutant remains under active debate. Using NMR spectroscopy, thermodynamics, kinetic assays, and molecular dynamics simulations, we found that this single mutation causes global changes in the enzyme, disrupting the intramolecular allosteric network and eliciting losses in nucleotide/pseudo-substrate binding cooperativity. Remarkably, by rewiring its internal allosteric network, PKA-CL205R is able to bind and phosphorylate non-canonical substrates, explaining its changes in substrate specificity. Both the lack of regulation and change in substrate specificity reveal the complex role of this mutated kinase in the formation of cortisol-secreting adrenocortical adenomas.

p38 Map Kinase Substrate Specificity Differs Greatly for Protein and Peptide Substrates

2000 ◽  
Vol 382 (2) ◽  
pp. 310-313 ◽  
Author(s):  
Julio Hawkins ◽  
Song Zheng ◽  
Betsy Frantz ◽  
Philip LoGrasso
Keyword(s):  
Substrate Specificity ◽  
Map Kinase ◽  
P38 Map Kinase ◽  
Peptide Substrates ◽  
Kinase Substrate
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