Biased AKAP signaling mechanisms

ABSTRACT Enzymes do not drift aimlessly within a cytoplasmic soup. Rather cell regulatory events occur within the confines of highly organized intracellular signaling nanodomains. Protein kinase compartmentalization enhances the fidelity of signal transduction and permits the parallel processing of chemical signals within the same cell. Our

lab discovered and defined A-Kinase Anchoring Proteins (AKAPs), a prototypic class of signal organizing proteins. The physiological significance of this mechanism is validated in several diseases. This proposal explores new PKAc mutants that locally drive cortisol secretion in adrenal Cushing’s syndrome. This is an

endocrine disorder where adrenal cells release excess stress hormone cortisol. This disease afflicts about fifteen per million people annually and is 3-5 times more prevalent in women than men. Symptoms include adrenal hyperplasia, midsection weight gain and comorbidities such as hypertension, hyperglycemia, and psychiatric

disorders. Adrenal (also called non-ACTH) Cushing’s is driven by somatic mutations in the catalytic subunit of protein kinase A (PKAc). Most mutations occur within regions of PKAc that bind regulatory subunits. This protein- protein interaction is not only necessary for autoinhibition of kinase activity but directs compartmentalization of

PKA holoenzymes through association with AKAPs. The most prevalent mutant PKAc-L205R is found in ~45% of Cushing’s patients. Other PKAc mutations occur at frequencies of less than 1%. Using an innovative personalized medicine workflow to screen clinical samples we have discovered a new PKAc-W196G variant in

~20% of adrenal Cushing's patients. This single amino acid change promotes structural perturbations in PKAc. Preliminary analyses of patient tissues harboring PKAcW196G detect elevated levels of type I regulatory subunit (RIa). Proximity proteomic screening of adrenal cell lines reveals that PKAcW196G interfaces with RI, dual-function

AKAPs, and the steroidogenic acute regulatory protein. StAR is a PKA substrate that controls the rate-limiting step in cortisol biosynthesis. CRISPR/Cas knockout of dual-function AKAP220 in adrenal cells attenuates stress hormone release. This exciting new data has forged an experimental plan of two specific aims.

AIM 1: EVALUATES THE MUTATIONAL FREQUENCY AND IMPACT OF PKACW196G? Genetic, biochemical, and cellular approaches will monitor a) mutational frequencies of PKAcW196G and its localization in patient tissues, b) the physiochemical and activity profile of PKAcW196G and c) how this Cushing's kinase alters endocrine responses.

AIM 2: TESTS IF ANCHORED TYPE I PKA SIGNALING PREDOMINATES IN ADRENAL CUSHING’S SYNDROME? PKAcW196G and the less common PKAcW196R analog are incorporated into AKAP220 signaling islands via association with RIa. Whole animal physiology studies and a novel precision pharmacology strategy will establish if a) PKAcW196G

(& W196R) signal through AKAP220 and if b) organellar targeting enhances the efficacy of PKA drugs to reduce hypersecretion or biosynthesis of stress hormone.