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. 2017 Feb;591(3):459-467.
doi: 10.1002/1873-3468.12562. Epub 2017 Feb 3.

Cushing's syndrome mutant PKAL205R exhibits altered substrate specificity

Joshua M Lubner  1 , Kimberly L Dodge-Kafka  2 , Cathrine R Carlson  3 , George M Church  4   5 , Michael F Chou  4   5 , Daniel Schwartz  1
Affiliations

Cushing's syndrome mutant PKAL205R exhibits altered substrate specificity

Joshua M Lubner et al. FEBS Lett. 2017 Feb.

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.

Keywords: Cushing's syndrome; protein kinase A; substrate specificity.

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Conflict of interest statement

Conflict of Interest

The authors declare no conflict of interest with this work. However, GMCs complete list of potential conflicts of interest are available at http://arep.med.harvard.edu/gmc/tech.html.

Figures

Figure 1
Figure 1. Experimental rationale and workflow
(A) Structural visualization of the substrate peptide binding site for PKAWT (PDB Model 4WB5 [13]) indicating putative hydrophobic interaction between PKA residues L198/P202/L205 and PKI substrate I22 at P+1. In the Cushing’s syndrome mutant PKAL205R (PDB Model 4WB6 [13]), this pocket has been destabilized. PKA is colored in cyan, with peptide substrate in magenta. Expanded views of both structures are available in Fig. S1. (B) Schematic overview of the experimental ProPeL workflow. A kinase of interest is cloned, and expressed in E. coli. Resulting bacterial phosphorylation is evaluated by SDS-PAGE with Pro-Q Diamond and coomassie staining. Lysate is digested, phosphoenriched and identified by tandem mass spectrometry. Data sets are visualized with pLogo [19].
Figure 2
Figure 2. PKAL205R exhibits altered downstream substrate specificity
(A to B) pLogos [19] illustrate substrate preferences for serine substrates of (A) PKAWT and (B) PKAL205R. Zoom (A,B): zoomed in view of P+1 to P+7 positions. Overrepresented residues are displayed above the x-axis, underrepresented residues are below the x-axis. The n(fg) and n(bg) values at the bottom left of the pLogo indicate the number of aligned foreground and background sequences used to generate the image, respectively. The red horizontal bars correspond to p = 0.05 (corrected for multiple hypothesis testing), and y-axis is logarithmic scale. The grey box indicates a “fixed” residue. Additional pLogos for threonine-centered substrates are available in Fig. S3.
Figure 3
Figure 3. In vitro kinase assays confirm pLogo visualizations
(A) Peptide arrays were used to evaluate PKAWT phosphorylation of different candidate peptides by incubating membranes with recombinant kinase and [γ-32P]ATP in an in vitro kinase assay. Bars indicate adjusted counts (see Table S3) across technical replicates (n = 6–8), and error bars are ±SEM. A One-way ANOVA found the means are not significantly different [F(2,14) = 0.56, p = 0.58349]. (B) Peptide arrays were used to evaluate the phosphorylation activity of either PKAWT or PKAL205R. Data points are color coded to indicate related candidate peptide pairs: pLogo-derived peptides (dark grey), Kemptide variants (light grey), and RIα variants (white). The dashed line indicates no preference (i.e. peptide is phosphorylated with equal efficiency by both PKAWT and PKAL205R). Candidate peptides that fall below the line are phosphorylated at a higher efficiency by PKAWT, while candidate peptides that fall above the line are phosphorylated at a higher efficiency by PKAL205R. n = 6–8 technical replicates, and error bars are ±SEM. (C) Bars indicate the log2 ratio of PKAWT activity to PKAL205R activity. Candidate peptides that are phosphorylated at a higher efficiency by PKAWT extend to the right, while candidate peptides that are phosphorylated at a higher efficiency by PKAL205R extend to the left, and x = 0 indicates no preference. Error bars represent the propagated ±SEM. Peptide pairs are color coded as in the previous panel. (D) Phosphorylation of the known human PKAWT substrate CDK16S119 is significantly reduced for PKAL205R compared with PKAWT (n = 8, t-test, p = 3.04 x 10−6). Error bars are ±SEM. Full blots and quantification are available in the Supporting information (Fig. S4 and Table S3). Peptides are: PKAWT-derived pLogo - RKRRRRKSFIENDRR, PKAL205R-derived pLogo - KRRRRRGSLDEDDQG, Kemptide - LRRASLG, Kemptide +1V - LRRASVG, Observed Peptide 1 - PLRMRRGSIPALVNN, Observed Peptide 2 - IVLPRRLSDEVADRV, RIα (P0 sub.) - KGRRRRGSISAEVYT, RIα (acidic) - KGRRRRGSDEAEVYT, CDK16S119 - EDINKRLSLPADIRL.
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