# Molecular mechanisms of PKA mutations underlying Cushing's syndrome

> **NIH NIH F32** · UNIVERSITY OF WASHINGTON · 2020 · $65,310

## Abstract

Project Summary
Chronically high levels of the stress hormone cortisol are deleterious to many organs systems. ACTH-
independent Cushing's syndrome is an endocrine disorder wherein the adrenal glands constitutively produce
excess cortisol. Symptoms include obesity, thinning of the skin, cognitive and emotional problems, and bone
loss. Recent studies have identified a mutation in the protein kinase A catalytic subunit (PKAc) in
approximately 50% of these cases. This mutation, L205R, is on the face of PKAc that binds to its regulatory
subunit, and is predicted to disrupt holoenzyme formation.
Traditionally, the model for cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling has
relied on evidence using supraphysiological levels of cAMP stimulation, which leads to full dissociation of PKAc
from its regulatory subunit and from A-kinase anchoring proteins (AKAPs). Recent evidence shows that
physiological levels of cAMP do not dissociate PKA holoenzymes, thereby redefining the model of PKA
activation in cells. Based on this new information, the proposed experiments will test if Cushing's syndrome
mutations in PKAc disrupt localization of active PKA and cause ectopic phosphorylation of substrates and
downstream cortisol secretion. The hypothesis will be tested in two aims:
Aim 1: Is the spatiotemporal profile of PKAc L205R altered in Cushing's syndrome? A combined strategy
of CRISPR/Cas9 gene-editing and live-cell imaging with photoactivatable fluorophores and FRET-based
biosensors will ascertain: 1) if mutant PKAc is recruited to AKAP-signaling islands, 2) whether these Cushing's
mutants are more mobile inside cells, and 3) if PKAc activity aberrantly accumulates at subcellular regions.
Aim 2: How does mutant PKAc cause excess cortisol production? I will use chemical biology techniques
combined with cortisol measurement in NCI-H295R adrenal cells 1) to determine if mislocalization of PKAc
activity is necessary and sufficient to boost cortisol release. Next, by combining miniTurbo proximity labeling
with phospho-proteomics, I will establish 2) if the disease-causing L205R PKAc mutant displays altered
substrate selectivity that adversely impacts downstream signaling. Additionally, a mouse model of adrenal
PKAc L205R expression will be generated to evaluate candidate therapeutic intervention strategies resulting
from this aim.
The long-term goals of this research plan are to elucidate molecular mechanisms of the signaling underlying
hypercortisolism in disease and to spur development of new therapeutic tools. The research will be conducted
at the University of Washington in the Department of Pharmacology. This environment provides excellent
training for academic-track postdoctoral researchers. Training benefits include strong collaboration within and
among departments, approachable principal investigators performing innovative work, and frequent seminars
from multiple departments featuring world experts in their respective fields.

## Key facts

- **NIH application ID:** 9904116
- **Project number:** 5F32DK121415-02
- **Recipient organization:** UNIVERSITY OF WASHINGTON
- **Principal Investigator:** Mitchell Hamed Omar
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $65,310
- **Award type:** 5
- **Project period:** 2019-04-01 → 2021-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9904116

## Citation

> US National Institutes of Health, RePORTER application 9904116, Molecular mechanisms of PKA mutations underlying Cushing's syndrome (5F32DK121415-02). Retrieved via AI Analytics 2026-05-28 from https://api.ai-analytics.org/grant/nih/9904116. Licensed CC0.

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