Cushing’s syndrome driver mutation disrupts protein kinase A allosteric network, altering both regulation and substrate specificity

Abstract

Genetic alterations in the PRKACA gene coding for the catalytic α subunit of the cAMP-dependent protein kinase A (PKA-C) are linked to cortisol-secreting adrenocortical adenomas, resulting in Cushing’s syndrome. Among those, a single mutation (L205R) has been found in up to 67% of patients. Because the x-ray structures of the wild-type and mutant kinases are essentially identical, the mechanism explaining aberrant function of this mutant remains under active debate. Using NMR spectroscopy, thermodynamics, kinetic assays, and molecular dynamics simulations, we found that this single mutation causes global changes in the enzyme, disrupting the intramolecular allosteric network and eliciting losses in nucleotide/pseudo-substrate binding cooperativity. Remarkably, by rewiring its internal allosteric network, PKA-CL205R is able to bind and phosphorylate non-canonical substrates, explaining its changes in substrate specificity. Both the lack of regulation and change in substrate specificity reveal the complex role of this mutated kinase in the formation of cortisol-secreting adrenocortical adenomas.

Defective internal allosteric network imparts dysfunctional ATP/substrate-binding cooperativity in oncogenic chimera of protein kinase A

“…Previous studies from our group have shown that PKA-C WT behaves like a K-type enzyme, exhibiting positive binding cooperativity between nucleotide (ATPγN) and pseudo-substrate (PKI 5–24 ). The latter property can be reduced by mutations, while maintaining a virtually constant maximal rate 20 , 23 , 24 . Thus, we sought to understand how this canonical positive binding cooperativity is altered in PKA-C DNAJB1 , using isothermal titration calorimetry (ITC).…”

“…3a ). These correlations constitute central allosteric nodes necessary for binding cooperativity 18 , 20 , 31 . In addition, allosteric cross-talk exists between residues in the C-terminal tail and residues in the αA-helix (K21, K28), αE-helix (R144, A147, L152), activation loop (R190, V191, G193), and αF-helix (G214, G225).…”

“…We found that the J-domain fused to the N-terminus of PKA-C dramatically attenuates the canonical positive nucleotide/pseudo-substrate-binding cooperativity through alterations in the intramolecular network of communication. Analogous to other pathologic mutants of PKA-C 20 , the concomitant loss in binding cooperativity and intramolecular communication may render highly cooperative events, such as regulation, dysfunctional, contributing to disease progression.…”

Molecular Basis for Ser/Thr Specificity in PKA Signaling

“…Another mutation leading to Cushing’s syndrome in the Cα subunit of PKA is L205R [ 56 ], which disrupts the intramolecular allosteric network [ 65 ]. Consequently, PKA C L205R has a decreased catalytic efficiency for Kemptide and phosphorylates non-canonical substrates leading to a dysregulated signaling network in tumor cells [ 65 , 66 , 67 ].…”

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“…In addition, we could subsequently show that PRKACA mutations alter PKA substrate specificity leading to hyperphosphorylation of proteins like histone 1.4, which is implicated in the control of cell proliferation 50 . Furthermore, it has been shown by NMR spectroscopy that the Leu206Arg substitution changes the intramolecular allosteric network in PKA interfering with the normal cooperativity between cAMP and substrate binding 51 . These findings provide a mechanistic explanation for the aberrant activation of PKA caused by PRKACA mutations, and, thus, for the development of cortisol-secreting adrenocortical adenomas in the presence of these mutations.…”