Classification of Cushing's syndrome PKAc mutants based upon their ability to bind PKI

Abstract

Cushing’s syndrome is an endocrine disorder caused by excess production of the stress hormone cortisol. Precision medicine strategies have identified single allele mutations within the PRKACA gene that drive adrenal Cushing’s syndrome. These mutations promote perturbations in the catalytic core of protein kinase A (PKAc) that impair autoinhibition by regulatory subunits and compartmentalization via recruitment into AKAP signaling islands. PKAcL205R is found in approximately 45% of patients, whereas PKAcE31V, PKAcW196R and L198insW and C199insV insertion mutants are less prevalent. Mass spectrometry, cellular, and biochemical data indicate that Cushing’s PKAc variants fall into two categories: those that interact with the heat-stable protein kinase inhibitor PKI, and those that do not. In vitro activity measurements show that wild-type PKAc and W196R activities are strongly inhibited by PKI (IC50 < 1 nM). In contrast, PKAcL205R activity is not blocked by the inhibitor. Immunofluorescent analyses show that the PKI-binding variants wild-type PKAc, E31V, and W196R are excluded from the nucleus and protected against proteolytic processing. Thermal stability measurements reveal that upon co-incubation with PKI and metal-bound nucleotide, the W196R variant tolerates melting temperatures 10ºC higher than PKAcL205. Structural modeling maps PKI-interfering mutations to a ~20 Å diameter area at the active site of the catalytic domain that interfaces with the pseudosubstrate of PKI. Thus, Cushing’s kinases are individually controlled, compartmentalized, and processed through their differential association with PKI.

Discovery of a Cushing’s syndrome protein kinase A mutant that biases signaling through type I AKAPs

“…The subcellular distribution of PKAc L205R depicted in Fig. 2 (C and E) is consistent with the inability of this Cushing’s variant to interact with R subunits ( 25 , 26 ). PKAc L205R is also unable to interact with the endogenous heat-stable PKA inhibitor PKI ( 26 , 28 ).…”

“…The most prevalent mutant, PKAc-L205R, is found in ~45% of patients with Cushing’s syndrome, while other PKAc mutations have been reported at frequencies of less than 1% ( 24 ). These disease-causing kinase variants diverge in protein stability, levels of autophosphorylation, subcellular distribution, and their engagement with downstream signaling pathways ( 25 , 26 ). Cushing’s PKAc variants can also be classified on the basis of differential association with the endogenous heat-stable inhibitor PKI ( 26 ).…”

“…These disease-causing kinase variants diverge in protein stability, levels of autophosphorylation, subcellular distribution, and their engagement with downstream signaling pathways ( 25 , 26 ). Cushing’s PKAc variants can also be classified on the basis of differential association with the endogenous heat-stable inhibitor PKI ( 26 ). This suggests that the mechanisms of action of individual Cushing’s kinases may be distinctive.…”

“…We previously reported that the added charge of Arg 196 perturbs the topology of PKAc W196R in a manner that mobilizes ERK signaling cascades. Unlike Arg 205 , this mutation does not compromise catalytic efficiency of the kinase or disrupt interactions with PKI ( 25 , 26 ). Here, we report the discovery of a genetic Cushing’s variant that introduces glycine at position 196 in the catalytic core of the kinase.…”

Protein Kinase Structure and Dynamics: Role of the αC-β4 Loop

“…Which of these mechanisms can abolish the synergistic high-affinity binding of ATP and IP20 or can all of them do it? Some of these "uncoupling motifs" such as F327, Y204, and E230 are sites that we have experimentally queried, whereas others such as W196R, L205R, and E31V are known disease mutations that correlate with Cushing's Syndrome (51)(52)(53)(54)(55).…”