AKT/PKB is downregulated by the ubiquitin-proteasome system (UPS), which plays a key role in cell survival and tumor progression in various types of cancer. The objective of this study was to determine the relationship between the sequential ubiquitination of lysine residues K284 to K214 in AKT and R-HSPA5 (the arginylated form of HSPA5), which contribute to the autophagic/lysosomal degradation of AKT when impaired proteasomal activity induces cellular stress. Results show that proteasome inhibitors (PIs) increased ATE1 (arginyltransferase 1)-mediated R-HSPA5 levels in a reactive oxygen species (ROS)-dependent manner. Further, binding of fully ubiquitinated AKT with R-HSPA5 induced AKT degradation via the autophagy-lysosome pathway. Specifically, the K48 (Lys48)-linked ubiquitinated form of AKT was selectively degraded in the lysosome with R-HSPA5. The deubiquitinase, USP7 (ubiquitin specific peptidase 7), prevented AKT degradation by inhibiting AKT ubiquitination via interaction with AKT. MUL1 (mitochondrial ubiquitin ligase activator of NFKB 1) also played a vital role in the lysosomal degradation of AKT by sequentially ubiquitinating AKT residues K284 to K214 for R-HSPA5-mediated autophagy. Consistent with this finding, despite HSPA5 arginylation, AKT was not degraded in mul1 KO cells. These results suggest that MUL1-mediated sequential ubiquitination of K284 to K214 may serve as a novel mechanism by which AKT is designated for lysosomal degradation. Moreover, binding of R-HSPA5 with fully ubiquitinated AKT is required for the autophagic/lysosomal degradation of AKT. Thus, modulating the MUL1-mediated non-proteasomal proteolysis mechanisms, such as sequential ubiquitination, may prove to be a novel therapeutic approach for cancer treatment.

Abbreviations: AKT1: thymoma viral proto-oncogene 1; ATE1: arginyltransferase 1; ATG5: autophagy related 5; CASP3: caspase 3; EGFP: enhanced green fluorescent protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSK3B; glycogen synthase kinase 3 beta; HA: hemagglutinin; HSPA5/GRP78/BIP: heat shock protein 5; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; MUL1: mitochondrial ubiquitin ligase activator of NFKB1; NAC: N-acetylcysteine; NEK2: NIMA (never in mitosis gene a)-related expressed kinase 2; NH₄Cl: ammonium chloride; PARP1: poly(ADP-ribose) polymerase family, member 1; PI: proteasome inhibitor; R-HSPA5: arginylated HSPA5; ROS: reactive oxygen species; SQSTM1: sequestome 1; Ub: ubiquitin; USP7: ubiquitin specific peptidase 7.

AKT/PKB 被泛素-蛋白酶体系统 (UPS) 下调，该系统在各种癌症的细胞存活和肿瘤进展中起关键作用。本研究的目的是确定 AKT 中赖氨酸残基 K284 至 K214 的顺序泛素化与 R-HSPA5（HSPA5 的精氨酸化形式）之间的关系，当蛋白酶体活性受损时，这有助于 AKT 的自噬/溶酶体降解。压力。结果表明蛋白酶体抑制剂 (PI) 以活性氧 (ROS) 依赖性方式增加了 ATE1（精氨酸转移酶 1）介导的 R-HSPA5 水平。此外，完全泛素化的 AKT 与 R-HSPA5 的结合通过自噬-溶酶体途径诱导 AKT 降解。具体而言，K48 (Lys48) 连接的泛素化形式的 AKT 在溶酶体中被 R-HSPA5 选择性降解。去泛素化酶 USP7（泛素特异性肽酶 7）通过与 AKT 相互作用抑制 AKT 泛素化来阻止 AKT 降解。MUL1（NFKB 1 的线粒体泛素连接酶激活剂）通过依次泛素化 AKT 残基 K284 至 K214 以进行 R-HSPA5 介导的自噬，在 AKT 的溶酶体降解中也发挥了重要作用。与这一发现一致，尽管 HSPA5 精氨酸化，AKT 在mul1 KO 细胞。这些结果表明 MUL1 介导的 K284 到 K214 的顺序泛素化可能作为一种新机制，通过该机制，AKT 被指定用于溶酶体降解。此外，AKT 的自噬/溶酶体降解需要 R-HSPA5 与完全泛素化的 AKT 结合。因此，调节 MUL1 介导的非蛋白酶体蛋白水解机制，如顺序泛素化，可能被证明是一种新的癌症治疗方法。

缩写：AKT1：胸腺瘤病毒原癌基因1；ATE1：精氨酸转移酶 1；ATG5：自噬相关5；CASP3：半胱天冬酶3；EGFP：增强型绿色荧光蛋白；GAPDH：3-磷酸甘油醛脱氢酶；GSK3B; 糖原合酶激酶 3 β；HA：血凝素；HSPA5/GRP78/BIP：热休克蛋白5；LAMP1：溶酶体相关膜蛋白 1；MAP1LC3B：微管相关蛋白 1 轻链 3 β；MEF：小鼠胚胎成纤维细胞；MUL1：NFKB1的线粒体泛素连接酶激活剂；NAC：N-乙酰半胱氨酸；NEK2：NIMA（从未在有丝分裂基因a中）相关表达的激酶2；NH ₄Cl：氯化铵；PARP1：聚（ADP-核糖）聚合酶家族，成员 1；PI：蛋白酶体抑制剂；R-HSPA5：精氨酸化的HSPA5；ROS：活性氧；SQSTM1：sequestome 1; Ub：泛素；USP7：泛素特异性肽酶 7。