Classification of Cushing's syndrome PKAc mutants based upon their ability to bind PKI
Classification of Cushing's syndrome PKAc mutants based upon their ability to bind PKI
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DOI：
10.1042/bcj20230183
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发表时间：
2023-06-01
影响因子：
4.1
通讯作者：
Scott，John D.
中科院分区：
文献类型：
--
作者：
Omar，Mitchell H.;Kihiu，Maryanne;Scott，John D.
库欣综合征是一种由应激激素皮质醇分泌过多引起的内分泌紊乱。精准医学策略已经确定了驱动肾上腺库欣综合征的prkacagene中的单个等位基因突变。这些突变促进了蛋白激酶A （PKAc）催化核心的扰动，从而损害了调节亚基的自抑制作用和通过募集到AKAP信号岛的区隔化。PKAcL205Ris存在于约45%的患者中，而pkac31v、PKAcW196R、L198insW和C199insV插入突变体则不那么普遍。质谱，细胞和生化数据表明库欣的PKAc变异分为两类：那些与热稳定的蛋白激酶抑制剂PKI相互作用的，以及那些不相互作用的。体外活性测定表明，野生型ppkac和W196R活性受到PKI的强烈抑制（IC50< 1 nM）。相比之下，pkacl205的活性不被抑制剂阻断。免疫荧光分析显示，结合PKAc、E31V和W196R的野生型PKAc、E31V和W196R被排除在细胞核之外，免受蛋白水解加工的影响。热稳定性测量表明，与PKI和金属结合核苷酸共孵育后，W196R变体比PKAcL205耐熔化温度高10°C。结构建模将PKI干扰突变映射到与PKI假底物接口的催化结构域活性位点上一个直径为~ 20 Å的区域。因此，库欣激酶通过与PKI的不同关联而被单独控制、划分和处理。
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 thePRKACAgene 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. PKAcL205Ris found in ∼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 vitroactivity measurements show that wild-type PKAc and W196R activities are strongly inhibited by PKI (IC50< 1 nM). In contrast, PKAcL205Ractivity 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 10°C 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.