血清和糖皮质激素诱导的蛋白激酶1(SGK1)是一种参与多种生理和病理过程的丝/苏氨酸激酶。它在结构，功能以及活化方式上都与同为AGC蛋白激酶家族的AKT非常相近。SGK1的激酶活性主要由其序列中的两个丝/苏氨酸位点的磷酸化决定，一个是位于疏水基序中的丝氨酸422位点，被mTORC2磷酸化；另一个是位于激活环中的苏氨酸256位点，被PDK1磷酸化。目前尚未有研究阐明这两个关键位点是如何被去磷酸化调控的。 为了深入探究SGK1磷酸化水平及活性的调控机制，我制备了针对小鼠SGK1丝氨酸422位点以及苏氨酸256位点的特异性兔多克隆磷酸化抗体。我们发现，即使在上游磷酸化刺激（生长因子等）存在的情况下，SGK1的磷酸化水平仍然非常低，而短时间的磷酸酶抑制剂处理却能极为显著的提升细胞内SGK1磷酸化水平，说明磷酸酶介导的去磷酸化过程对于调控SGK1活性极为重要。本研究发现多种磷酸酶，包括PP1、PP2A和PP5，都参与了对SGK1的去磷酸化调控。SGK1蛋白的低稳定性使其严重依赖Hsp90，Hsp70等蛋白质分子伴侣。我们发现磷酸酶PP5与Hsp90/CDC37/SGK1复合物结合并不断的去磷酸化SGK1。当在细胞中过表达PP2A内源抑制蛋白FAM122A时，SGK1的磷酸化水平和活性都得到显著提升。SGK1作为PP2A底物，在细胞内与PP2A B55γ/δ, B56β亚基存在相互作用，而PP2A B55γ/δ亚基同时也与FAM122A存在相互作用。其中值得注意的是，将FAM122A的丝氨酸37位点突变为丙氨酸或天冬氨酸后，FAM122A无法对SGK1的磷酸化水平造成的影响，说明FAM122A S37位点的磷酸化对其功能至关重要。 SGK1的低磷酸化水平并不是稳定不变的。本研究发现，在细胞DNA损伤药物刺激下，SGK1磷酸化水平显著提高。DNA损伤激活DNA依赖性蛋白激酶（DNA-PK），增强了SGK1上游激酶mTORC2的活性，进而使SGK1磷酸化水平和活性得到显著提升。值得注意的是，DNA损伤情况下SGK1活性的增加显著提高了人胚胎肾细胞和几种癌细胞系的细胞活力。鉴于目前主流癌症治疗方案中诱导癌细胞DNA损伤的相关疗法被广泛应用，本项研究发现了一种新的机制，即DNA损伤情况下SGK1激酶活性被显著提升，促进了癌细胞的应激生存，可能降低诱导DNA损伤药物的治疗效果。
Serum and glucocorticoid-induced protein kinase 1 (SGK1) is a serine and threonine kinase involved in a number of physiological and pathological processes. As an AGC protein kinase closely related to AKT, the activity of SGK1 is strictly regulated by sequential phosphorylation of serine 422 in the hydrophobic motif by mTORC2 and threonine 256 in the activation loop by the constitutively active protein kinase PDK1. The underlying mechanisms of how active SGK1 is dephosphorylated under various cellular contexts remain unclear. In order to explore regulation mechanisms of SGK1 phosphorylation state and activity, I developed specific rabbit polyclonal antibodies against mouse SGK1 S422 and T256 and discovered that SGK1 undergoes relentless dephosphorylation that keeps its activity low even in the presence of upstream stimulation for phosphorylation, implicating cellular phosphatases and their regulation are major determinants of SGK1 activity. Multiple phosphatases, PP1, PP2A and PP5, are involved in SGK1 dephosphorylation processes. SGK1 is heavily addicted to protein chaperons Hsp90 and Hsp70 due to its low intrinsic stability. Here I found that phosphatase PP5 associates with Hsp90/CDC37/SGK1 and constantly dephosphorylates SGK1 as a component of the tetra-complex. An endogenous phosphatase inhibitor for PP2A, FAM122A, when co-overexpressed with SGK1 in cells significantly increases SGK1 phosphorylation state and activity. SGK1 is a substrate of PP2A and the association of these two proteins is mediated by a few regulatory B subunits of PP2A, among which B55γ/δ also bind to FAM122A. Substitution of S37 in FAM122A by either alanine or aspartic acid abolishes the impact of FAM122A on SGK1 phosphorylation, indicating that phosphorylation of S37 site is crucial for FAM122A function. The low phosphorylation state of SGK1 is not fixed. I show that the dominant impact of phosphatases over kinases is reversed in cells under genotoxic stress by a mechanism wherein DNA breaks activate the DNA-dependent protein kinase (DNA-PK) that leads to mTORC2 enhanced phosphorylation directed to SGK1. Increased SGK1 activity during the DDR improves the viability of 293T and several cancer cells lines. Given that the DNA damage response operates in many cancer cells and is further induced by genotoxic drugs, our results provide a mechanism wherein increased phosphorylation of SGK1 promotes stress survival of cancer cells and potentially diminishes the efficacy of cancer treatments.
