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 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 The design of peptide-based substrates for the cdc2 protein kinase

1995 ◽  
Vol 309 (3) ◽  
pp. 927-931 ◽  
Author(s):  
J Srinivasan ◽  
M Koszelak ◽  
M Mendelow ◽  
Y G Kwon ◽  
D S Lawrence
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Amino Acid Sequence ◽  
Substrate Specificity ◽  
Bovine Brain ◽  
Sequence Specificity ◽  
Cyclin Dependent Kinase ◽  
Cdc2 Kinase ◽  
Kinase Substrate ◽  
Pisaster Ochraceus

The substrate sequence specificity of the cdc2 protein kinase from Pisaster ochraceus has been evaluated. The peptide, Ac-Ser-Pro-Gly-Arg-Arg-Arg-Arg-Lys-amide, serves as an efficient cdc2 kinase substrate with a Km of 1.50 +/- 0.04 microM and a Vmax. of 12.00 +/- 0.18 mumol/min per mg. The amino acid sequence of this peptide is not based on any sequence in a known protein substrate of the cyclin-dependent kinase, but rather was designed from structural attributes that appear to be important in the majority of cdc2 substrates. The cyclin-dependent enzyme is remarkably indiscriminate in its ability to recognize and phosphorylate peptides that contain an assortment of structurally diverse residues at the P-2, P-1 and P+2 positions. However, peptides that contain a free N-terminal serine or lack an arginine at the P+4 position are relatively poor substrates. These aspects of the substrate specificity of the cdc2 protein kinase are compared and contrasted with the previously reported substrate specificity of a cdc2-like protein kinase from bovine brain [Beaudette, Lew and Wang (1993) J. Biol. Chem. 268, 20825-20830].

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.

Sign in / Sign up

Export Citation Format

Share Document