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Discovery of a new Cushing’s syndrome PKAc mutant that is retained within anchored type I holoenzymes

ABSTRACT: Adrenal Cushing’s syndrome is a disease of cortisol hypersecretion often caused by mutations in protein kinase A catalytic subunit (PKAc). Using a personalized medicine screening platform, we discovered a new Cushing’s driver mutation, PKAc-W196G, in ~20% of patient samples analyzed. Proximity proteomics and photokinetic imaging reveal that PKAcW196G is distinct from other Cushing’s variants. This mutant surprisingly retains association with type I regulatory subunits (RI) and their corresponding A kinase anchoring proteins (AKAPs). Molecular dynamics simulations predict that substitution of glycine for tryptophan 196 creates a 653 Å3 cleft between the catalytic core of PKAcW196G and type II regulatory subunits (RII), but only a 395 Å3 cleft with RI. Endocrine measurements show that overexpression of RIα or redistribution of PKAcW196G via AKAP recruitment counteracts stress hormone overproduction. We conclude that a W196G mutation in the kinase catalytic core skews R subunit selectivity and biases AKAP association to drive Cushing’s syndrome.

INSTRUMENT(S): Orbitrap Fusion Lumos, Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human) Escherichia Coli

SUBMITTER: leonard daly

LAB HEAD: Prof Claire E Eyers

PROVIDER: PXD045556 | Pride | 2024-02-10

REPOSITORIES: Pride

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			Action	DRS
	20220525_LD_PKA_W196G_rep1_T_max.raw	Raw
	20220525_LD_PKA_W196G_rep2_T_max.raw	Raw
	20220525_LD_PKA_W196G_rep3_T_max.raw	Raw
	20220525_LD_PKA_W196G_rep4_T_max.raw	Raw
	F1881561.mgf	Mgf

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Displacement of PKA catalytic subunit from AKAP signaling islands drives pathology in Cushing’s syndrome C4PR_LIV

Project description:Mutations in the catalytic subunit of protein kinase A (PKAc) drive the stress hormone disorder adrenal Cushing’s syndrome. We define mechanisms of action for the PKAcL205R and W196R variants. Proximity proteomic techniques demonstrate that both Cushing’s mutants are excluded from A kinase anchoring protein (AKAP) signaling islands, whereas live-cell photoactivation microscopy reveals that these kinase mutants indiscriminately diffuse throughout the cell. Only cAMP analog drugs that displace native PKAc from AKAPs enhance cortisol release. Rescue experiments that incorporate PKAc mutants into AKAP complexes abolish cortisol overproduction, indicating that kinase anchoring restores normal endocrine function. Analyses of adrenal-specific PKAc-W196R knock-in mice and Cushing’s syndrome patient tissue reveal defective signaling mechanisms of the disease. Surprisingly each Cushing’s mutant engages a different mitogenic signaling pathway, with upregulation of YAP/TAZ by PKAc-L205R and ERK kinase activation by PKAc-W196R. Thus, aberrant spatiotemporal regulation of each Cushing’s variant promotes the transmission of distinct downstream pathogenic signals.

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