Noncanonical PDZ Interaction between PKA RIα R368X Acrodysostosis Mutant and P‐Rex1 PDZ Domain

Abstract

Protein Kinase A (PKA), a tetrameric enzyme comprised of two catalytic (C) and two regulatory (R) subunits, is the prototypical kinase that responds to intracellular cAMP. Hindering the capacity of the RIα subunit isoform to bind cAMP, results in overregulation of the C subunit, a rare condition called Acrodysostosis (ACRDYS). Patients with ACRDYS typically have mutations in either RIα or PDE4D genes, and display stunted growth, mental retardation, and in some cases, hormonal resistance. Recently, a new interaction between RIα and the tandem PDZ domains of Phosphatidylinositol 3,4,5‐Triphosphate‐Dependent Rac Exchanger 1 (P‐Rex1) was discovered. P‐Rex1, a multi‐domain scaffolding protein with Rac specific guanine nucleotide exchange factor (GEF) activity, is highly expressed in certain leukocyte populations, and is intimately involved with regulation of cell migration. Using peptide blots, we scanned the Tandem PDZ domains of P‐Rex1 (residues 607–783) for interactions with RIα. We found that RIα bound to the α2 helix and the linker region between the tandem PDZ domains. Pulldown assays from HEK293 cells further confiirmed the interaction of RIα and P‐Rex1 PDZ domains. Interestingly, an ACRDYS mutation of RIα (R368X), deleting the last 14 amino acids from the C‐terminus of RIα, bound stronger to the PDZ domains of P‐Rex1 in contrast to the canonically defined mechanism of PDZ domain interactions, which bind to and recognize the C‐terminus via a β‐strand addition mechanism. Having solved the structure of this R368X ACRDYS mutant allowed us to model this noncanonical complex in silico using ZDock. We found that the PDZ domain may bind in the pocket of the second cyclic nucleotide binding domain (CNB‐B) of RIα, in agreement with our peptide blot data. This suggest that Acrodysostosis may not only alter regulation of the C subunit, but also change the PKA signalosome. We hope to validate this interaction by X‐ray crystallography.Support or Funding InformationThis Work was supported by NIH Grant GM34921 to Susan S. Taylor.Jason C. Del Rio was supported in part by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology through an institutional training grant from the National Institute of General Medical Sciences, T32 GM007752.