ABSTRACT Cushing’s syndrome occurs when the adrenal glands release too much of the stress hormone cortisol. This endocrine disorder is naturally caused by adrenal or pituitary tumors and afflicts women five times more frequently than men. Chronic exposure to cortisol also increases blood glucose and elevates blood pressure. This proposal investigates the molecular pathogenesis of adrenal Cushing’s syndrome. This disease is often an outcome of defective cAMP signaling in the adrenal cortex. Advances in precision medicine have identified point mutations and insertions in protein kinase A catalytic subunits (PKAc) as drivers of this disease. The most prevalent mutant is PKAc-L205R, which is found in ~45% of Cushing’s patients. Other genetic lesions include PKAc-W196R. Both disease-causing mutations reside within a region of the kinase domain that interfaces with its regulatory subunits. This protein-protein interface is necessary for autoinhibition of kinase activity, but also directs the subcellular targeting of PKA holoenzymes through association with A kinase anchoring proteins (AKAPs). Our biochemical, cell-based, and clinical analyses infer that carrying a mutant allele of PKAc-L205R or W196R is sufficient to displace the kinase from normal subcellular sites of action in Cushing’s patients. This has led to a working hypothesis that losing spatiotemporal control of protein kinase A underlies adrenal Cushing’s syndrome. Aim 1 will test if i) restoration of PKA anchoring amends cortisol release, ii) identify which AKAPs coordinate cortisol biosynthesis and iii) considers an opposing premise that “substrate rewiring” of PKAc variants or altered enzyme stability drives Cushing’s syndrome. At first glance, the PKAc-L205R and PKAc-W196R mutants look virtually identical. Yet we have discovered that these are physiochemically distinct kinases that engage different cell signaling pathways. Analyses of patient tissue reveal that PKAc-L205R mobilizes the Hippo signaling pathway to control organ size. Conversely phenotypic profiling of adrenal-specific knock-in mice indicate that PKAc-W196R activates the ERK mitogenic kinase cascade. Aim 2 will evaluate i) if female PKAcWT/W196R mice are more prone to Cushing’s comorbidities, ii) test if PKAc-L205R engages the Hippo signaling cascade to drive cortisol hyperproduction and iii) ascertain if PKAc-W196R coordinates ERK signaling events to promote adrenal hyperplasia. This proposal explores two transformative ideas that will change how we think about Cushing’s syndrome. 1) Losing spatiotemporal control of protein kinase A underlies adrenal Cushing’s pathologies. 2) Adjacent and identical mutations in PKAc drive adrenal hyperplasia and cortisol hypersecretion in Cushing’s patients.