The Target of rapamycin (TOR) protein kinase forms part of TOR complex 1 (TORC1) and TOR complex 2 (TORC2), two multi-subunit protein complexes that regulate growth, proliferation, survival and developmental processes by phosphorylation and activation of AGC-family kinases. In the fission yeast, Schizosaccharomyces pombe, TORC2 and its target, the AGC kinase Gad8 (an orthologue of human AKT or SGK1) are required for viability under stress conditions and for developmental processes in response to starvation cues. In this study, we describe the isolation of gad8 mutant alleles that bypass the requirement for TORC2 and reveal a separation of function of TORC2 and Gad8 under stress conditions. In particular, osmotic and nutritional stress responses appear to form a separate branch from genotoxic stress responses downstream of TORC2-Gad8. Interestingly, TORC2-independent mutations map into the regulatory PIF pocket of Gad8, a highly conserved motif in AGC kinases that regulates substrate binding in PDK1 (phosphoinositide dependent kinase-1) and kinase activity in several AGC kinases. Gad8 activation is thought to require a two-step mechanism, in which phosphorylation by TORC2 allows further phosphorylation and activation by Ksg1 (an orthologue of PDK1). We focus on the Gad8-K263C mutation and demonstrate that it renders the Gad8 kinase activity independent of TORC2 in vitro and independent of the phosphorylation sites of TORC2 in vivo. Molecular dynamics simulations of Gad8-K263C revealed abnormal high flexibility at T387, the phosphorylation site for Ksg1, suggesting a mechanism for the TORC2-independent Gad8 activity. Significantly, the K263 residue is highly conserved in the family of AGC-kinases, which may suggest a general way of keeping their activity in check when acting downstream of TOR complexes.

雷帕霉素（TOR）蛋白激酶的靶标构成TOR复合物1（TORC1）和TOR复合物2（TORC2）的一部分，这两种多亚基蛋白复合物通过AGC家族的磷酸化和激活来调节生长，增殖，存活和发育过程激酶。在裂变酵母中，裂殖酵母，TORC2及其靶标，AGC激酶Gad8（人AKT或SGK1的直系同源物）在应激条件下的生存能力以及响应饥饿信号的发育过程是必需的。在这项研究中，我们描述了gad8的分离突变等位基因绕过TORC2的需求，并揭示了在压力条件下TORC2和Gad8的功能分离。特别是，渗透和营养应激反应似乎与TORC2-Gad8下游的遗传毒性应激反应形成了单独的分支。有趣的是，不依赖TORC2的突变映射到Gad8的PIF口袋中，而后者是AGC激酶中高度保守的基序，它调节PDK1（磷酸肌醇依赖性激酶1）中的底物结合以及几种AGC激酶中的激酶活性。Gad8激活被认为需要一个两步机制，其中TORC2的磷酸化作用允许Ksg1（PDK1的直向同源物）进一步磷酸化和激活。我们专注于Gad8-K263C突变，并证明它使Gad8激酶活性在体外独立于TORC2和独立的TORC2的磷酸化位点在体内。Gad8-K263C的分子动力学模拟显示，在T387（Ksg1的磷酸化位点）处异常高的柔性，提示了不依赖TORC2的Gad8活性的机制。值得注意的是，K263残基在AGC激酶家族中高度保守，这可能是控制TOR复合物下游作用时保持其活性的一般方法。