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Supplemental Figures S1-S6

Figure S1. The Sin1-PH domain binds the mTOR kinase domain to inhibit mTOR kinase activity, Related to Figure 1. Figure S2. The PH domain is a unique and physiological functional PH domain, Related to Figure 2. Figure S3. PI(3,4,5)P3 binds the Sin1-PH motif to activate mTORC2, Related to Figure 3. Figure S4. The presence of PIP3 species leads to attenuated Sin1-PH domain interaction with mTOR-KD, Related to Figure 4. Figure S5. The Sin1-PH motif mainly interacts with PI(3,4,5)P3 through three critical residues including R393, K428 and K464, Related to Figure 5. Figure S6. The cancerous Sin1-PH mutant lose binding with the mTOR-kinase domain, leading to elevated Akt-S473 phosphorylation and enhanced oncogenic activities, Related to Figure 6. Figure S7. C-terminal tagging of KRas-CAAX to Sin1 could partially rescue the deficiency of the Sin1-CAA mutant in membrane recruitment and mTORC2 activation, Related to Figure 7.

BRD4 functions as an epigenetic reader and plays a crucial role in regulating transcription and genome stability. Dysregulation of BRD4 is frequently observed in various human cancers. However, the molecular details of BRD4 regulation remain largely unknown. Here, we report that PRMT2- and PRMT4-mediated arginine methylation is pivotal for BRD4 functions on transcription, DNA repair, and tumor growth. Specifically, PRMT2/4 interacts with and methylates BRD4 at R179, R181, and R183. This arginine methylation selectively controls a transcriptional program by promoting BRD4 recruitment to acetylated histones/chromatin. Moreover, BRD4 arginine methylation is induced by DNA damage and thereby promotes its binding to chromatin for DNA repair. Deficiency in BRD4 arginine methylation significantly suppresses tumor growth and sensitizes cells to BET inhibitors and DNA damaging agents. Therefore, our findings reveal an arginine methylation–dependent regulatory mechanism of BRD4 and highlight targeting PRMT2/4 for better antitumor effect of BET inhibitors and DNA damaging agents.

Chromosomal rearrangements can generate genetic fusions composed of two distinct gene sequences, many of which have been implicated in tumorigenesis and progression. Our study proposes a model whereby oncogenic gene fusions frequently alter the protein stability of the resulting fusion products, via exchanging protein degradation signal (degron) between gene sequences. Computational analyses of The Cancer Genome Atlas (TCGA) identify 2,406 cases of degron exchange events and reveal an enrichment of oncogene stabilization due to loss of degrons from fusion. Furthermore, we identify and experimentally validate that some recurrent fusions, such as BCR-ABL, CCDC6-RET and PML-RARA fusions, perturb protein stability by exchanging internal degrons. Likewise, we also validate that EGFR or RAF1 fusions can be stabilized by losing a computationally-predicted C-terminal degron. Thus, complementary to enhanced oncogene transcription via promoter swapping, our model of degron loss illustrates another general mechanism for recurrent fusion proteins in driving tumorigenesis., The impact of genetic fusions on degrons, which are motifs for ubiquitin-mediated protein degradation, has not been fully explored. Here, the authors analyse fusion genes affecting degrons in pan-cancer genomics data, validate their functional impact and find enrichment for both internal and C-terminal degron losses.

AKT is involved in a number of key cellular processes including cell proliferation, apoptosis and metabolism. Hyperactivation of AKT is associated with many pathological conditions, particularly cancers. Emerging evidence indicates that arginine methylation is involved in modulating AKT signaling pathway. However, whether and how arginine methylation directly regulates AKT kinase activity remain unknown. Here we report that protein arginine methyltransferase 5 (PRMT5), but not other PRMTs, promotes AKT activation by catalyzing symmetric dimethylation of AKT1 at arginine 391 (R391). Mechanistically, AKT1-R391 methylation cooperates with phosphatidylinositol 3,4,5 trisphosphate (PIP3) to relieve the pleckstrin homology (PH)-in conformation, leading to AKT1 membrane translocation and subsequent activation by phosphoinositide-dependent kinase-1 (PDK1) and the mechanistic target of rapamycin complex 2 (mTORC2). As a result, deficiency in AKT1-R391 methylation significantly suppresses AKT1 kinase activity and tumorigenesis. Lastly, we show that PRMT5 inhibitor synergizes with AKT inhibitor or chemotherapeutic drugs to enhance cell death. Altogether, our study suggests that R391 methylation is an important step for AKT activation and its oncogenic function., Arginine methylation has been reported to regulate AKT-associated signalling. Here, the authors show that PRMT5-mediated arginine methylation promotes AKT activity and induces tumourigenesis.

Protein arginine methyltransferase 5 (PRMT5) is the primary enzyme generating symmetric dimethylarginine (sDMA) on numerous substrates, through which it regulates many cellular processes, such as transcription and DNA repair. Aberrant expression and activation of PRMT5 is frequently observed in various human cancers and associated with poor prognosis and survival. However, the regulatory mechanisms of PRMT5 remain poorly understood. Here, we report that TRAF6 serves as an upstream E3 ubiquitin ligase to promote PRMT5 ubiquitination and activation. We find that TRAF6 catalyzes K63-linked ubiquitination of PRMT5 and interacts with PRMT5 in a TRAF6-binding-motif-dependent manner. Moreover, we identify six lysine residues located at the N-terminus as the primarily ubiquitinated sites. Disruption of TRAF6-mediated ubiquitination decreases PRMT5 methyltransferase activity towards H4R3 in part by impairing PRMT5 interaction with its co-factor MEP50. As a result, mutating the TRAF6-binding motifs or the six lysine residues significantly suppresses cell proliferation and tumor growth. Lastly, we show that TRAF6 inhibitor enhances cellular sensitivity to PRMT5 inhibitor. Therefore, our study reveals a critical regulatory mechanism of PRMT5 in cancers.

The kinase AKT (also known as protein kinase B) is a key regulator of cell proliferation, survival, and metabolism. In addition to being activated by growth factors, AKT is activated in response to DNA damage. Here, we found that the DNA damage response kinase DNA-PK sustains cell survival through a phosphorylation event that leads to increased AKT activity. In various cancer and noncancer cells in culture, DNA damage caused by ionizing radiation or topoisomerase inhibitors triggered DNA-PK–dependent phosphorylation of the mTOR complex 2 (mTORC2) subunit Sin1, which enabled its interaction with the guanine nucleotide exchange factor ECT2. Depleting Sin1 or ECT2 or disrupting the protein interaction or catalytic function of ECT2 attenuated DNA damage–induced AKT activation, thereby enhancing cellular sensitivity to DNA-damaging agents. Our findings elucidate a mechanism mediating DNA damage–induced AKT activation and cell survival.

The Hippo and mammalian target of rapamycin complex 1 (mTORC1) pathways are the two predominant growth-control pathways that dictate proper organ development. We therefore explored potential crosstalk between these two functionally relevant pathways to coordinate their growth-control functions. We found that the LATS1 and LATS2 kinases, the core components of the Hippo pathway, phosphorylate S606 of Raptor, an essential component of mTORC1, to attenuate mTORC1 activation by impairing the interaction of Raptor with Rheb. The phosphomimetic Raptor-S606D knock-in mutant led to a reduction in cell size and proliferation. Compared with Raptor+/+ mice, RaptorD/D knock-in mice exhibited smaller livers and hearts, and a significant inhibition of elevation in mTORC1 signalling induced by Nf2 or Lats1 and Lats2 loss. Thus, our study reveals a direct link between the Hippo and mTORC1 pathways to fine-tune organ growth.

The mechanistic target of rapamycin (mTOR) is a master regulator of cell growth, proliferation, and metabolism by integrating various environmental inputs including growth factors, nutrients, and energy, among others. mTOR signaling has been demonstrated to control almost all fundamental cellular processes, such as nucleotide, protein and lipid synthesis, autophagy, and apoptosis. Over the past fifteen years, mapping the network of the mTOR pathway has dramatically advanced our understanding of its upstream and downstream signaling. Dysregulation of the mTOR pathway is frequently associated with a variety of human diseases, such as cancers, metabolic diseases, and cardiovascular and neurodegenerative disorders. Besides genetic alterations, aberrancies in post-translational modifications (PTMs) of the mTOR components are the major causes of the aberrant mTOR signaling in a number of pathologies. In this review, we summarize current understanding of PTMs-mediated regulation of mTOR signaling, and also update the progress on targeting the mTOR pathway and PTM-related enzymes for treatment of human diseases.

Cancer stem cells (CSCs) are a small subset of stem-like cells inside tumors, which possess abilities of unlimited self-renewal, differentiation and proliferation. Extensive studies have suggested that CSCs are one of the major drivers of tumor initiation, metastasis, relapse and therapeutic resistance. Several regulatory networks including transcriptional programs and various signaling pathways tightly control the stemness, proliferation and differentiation of CSCs. Emerging evidence has indicated that post-translational modifications, especially ubiquitination, play a critical role in maintenance of CSC properties. In this review, we summarize current understandings on E3 ubiquitin ligase-mediated regulation of transcription factors and key signaling pathways involved in the regulation of CSCs, and discuss the strategy to target CSCs and E3 ubiquitin ligases for combating cancers.

EglN prolyl hydroxylases, a family of oxygen-sensing enzymes, hydroxylate distinct proteins to modulate diverse physiopathological signals. Aberrant regulations of EglNs result in multiple human diseases, including cancer. Different from EglN1 which function largely depends on the role of hypoxia-induce factor alpha (HIFα) in tumors, the functional significance and the upstream regulatory mechanisms of EglN2, especially in prostate cancer setting, remain largely unclear. Here, we demonstrated that dysregulation of EglN2 facilitated prostate cancer growth both in cells and in vivo. Notably, EglN2 was identified highly expressed in human prostate cancer tissues. Mechanically, Cullin 3-based E3 ubiquitin ligase SPOP, a well-characterized tumor suppressor in prostate cancer, could recognize and destruct EglN2. Meanwhile, androgen receptor (AR), playing a pivotal role in progression and development of prostate cancer, could transcriptionally up-regulate EglN2. Pathologically, SPOP loss-of-function mutations or AR amplification, frequently occurring in prostate cancers, could significantly accumulate EglN2 abundance. Therefore, our study not only underlines an oncogenic role of EglN2 in prostate cancer, but also highlights SPOP as a tumor suppressor to down-regulate EglN2 in prostate cancer.

The function of PTEN in the cytoplasm largely depends on its lipid-phosphatase activity, though which it antagonizes the PI3K–AKT oncogenic pathway. However, molecular mechanisms underlying the role of PTEN in the nucleus remain largely elusive. Here, we report that DNA double-strand breaks (DSB) promote PTEN interaction with MDC1 upon ATM-dependent phosphorylation of T/S398-PTEN. Importantly, DNA DSBs enhance NSD2 (MMSET/WHSC1)-mediated dimethylation of PTEN at K349, which is recognized by the tudor domain of 53BP1 to recruit PTEN to DNA-damage sites, governing efficient repair of DSBs partly through dephosphorylation of γH2AX. Of note, inhibiting NSD2-mediated methylation of PTEN, either through expressing methylation-deficient PTEN mutants or through inhibiting NSD2, sensitizes cancer cells to combinatorial treatment with a PI3K inhibitor and DNA-damaging agents in both cell culture and in vivo xenograft models. Therefore, our study provides a novel molecular mechanism for PTEN regulation of DSB repair in a methylation- and protein phosphatase–dependent manner. Significance: NSD2-mediated dimethylation of PTEN is recognized by the 53BP1 tudor domain to facilitate PTEN recruitment into DNA-damage sites, governing efficient repair of DNA DSBs. Importantly, inhibiting PTEN methylation sensitizes cancer cells to combinatorial treatment with a PI3K inhibitor combined with DNA-damaging agents in both cell culture and in vivo xenograft models. This article is highlighted in the In This Issue feature, p. 1143

The Hippo-YAP/TAZ signaling pathway plays a pivotal role in growth control during development and regeneration and its dysregulation is widely implicated in various cancers. To further understand the cellular and molecular mechanisms underlying Hippo signaling regulation, we have found that activities of core Hippo signaling components, large tumor suppressor (LATS) kinases and YAP/TAZ transcription factors, oscillate during mitotic cell cycle. We further identified that the anaphase-promoting complex/cyclosome (APC/C) Cdh1 E3 ubiquitin ligase complex, which plays a key role governing eukaryotic cell cycle progression, intrinsically regulates Hippo signaling activities. CDH1 recognizes LATS kinases to promote their degradation and, hence, YAP/TAZ regulation by LATS phosphorylation is under cell cycle control. As a result, YAP/TAZ activities peak in G1 phase. Furthermore, we show in Drosophila eye and wing development that Cdh1 is required in vivo to regulate the LATS homolog Warts with a conserved mechanism. Cdh1 reduction increased Warts levels, which resulted in reduction of the eye and wing sizes in a Yorkie dependent manner. Therefore, LATS degradation by APC/C Cdh1 represents a previously unappreciated and evolutionarily conserved layer of Hippo signaling regulation.

In the version of this Article originally published, the labels for Rictor and mTOR in the whole cell lysate (WCL) blots were swapped in Fig. 3b and the mTOR blot was placed upside down. Unprocessed blots of mTOR were also missing from Supplementary Fig. 9. The corrected Figs are shown below. In addition, control blots for the mTOR antibody (Cell Signalling Technology #2972) were also missing. These are now provided below, as Fig. 9, and show that the lower band is likely non-specific.

Polyubiquitylation is canonically viewed as a posttranslational modification that governs protein stability or protein-protein interactions, in which distinct polyubiquitin linkages ultimately determine the fate of modified protein(s). We explored whether polyubiquitin chains have any nonprotein-related function. Using in vitro pull-down assays with synthetic materials, we found that polyubiquitin chains with the Lys(63) (K63) linkage bound to DNA through a motif we called the “DNA-interacting patch” (DIP), which is composed of the adjacent residues Thr(9), Lys(11), and Glu(34). Upon DNA damage, the binding of K63-linked polyubiquitin chains to DNA enhanced the recruitment of repair factors through their interaction with an Ile(44) patch in ubiquitin to facilitate DNA repair. Furthermore, experimental or cancer patient–derived mutations within the DIP impaired the DNA binding capacity of ubiquitin and subsequently attenuated K63-linked polyubiquitin chain accumulation at sites of DNA damage, thereby resulting in defective DNA repair and increased cellular sensitivity to DNA-damaging agents. Our results therefore highlight a critical physiological role for K63-linked polyubiquitin chains in binding to DNA to facilitate DNA damage repair.

Cancer cells can remodel surrounding microenvironments to facilitate cell growth, invasion, and migration by secreting proteins that educate surrounding stromal cells. We report that p85(S6K1), the longest isoform of S6K (ribosomal protein S6 kinase), but not the shorter isoform p70(S6K1) or p56(S6K2), was secreted from cancer cells through its HIV TAT-like, N-terminal six-arginine motif. The exogenously produced p85(S6K1) protein entered cultured transformed and nontransformed cells to promote or confer malignant behaviors, leading to increased cell growth and migration. When injected into mice, the p85(S6K1) protein enhanced the growth of xenografted breast cancer cells and lung metastasis. Hence, our findings reveal a role for p85(S6K1) as a secreted oncogenic kinase and provide a mechanism by which cancer cells remodel their microenvironment by transforming the surrounding cells to drive tumorigenesis.

The ERG gene is fused to TMPRSS2 in approximately 50% of prostate cancers (PrCa), resulting in its overexpression. However, whether this is the sole mechanism underlying ERG elevation in PrCa is currently unclear. Here we report that ERG ubiquitination and degradation are governed by the Cullin 3-based ubiquitin ligase SPOP and that deficiency in this pathway leads to aberrant elevation of the ERG oncoprotein. Specifically, we find that truncated ERG (ΔERG), encoded by the ERG fusion gene, is stabilized by evading SPOP-mediated destruction, whereas prostate cancer-associated SPOP mutants are also deficient in promoting ERG ubiquitination. Furthermore, we show that the SPOP/ERG interaction is modulated by CKI-mediated phosphorylation. Importantly, we demonstrate that DNA damage drugs, topoisomerase inhibitors, can trigger CKI activation to restore the SPOP/ΔERG interaction and its consequent degradation. Therefore, SPOP functions as a tumor suppressor to negatively regulate the stability of the ERG oncoprotein in prostate cancer.

Aberrant activation of AKT disturbs the proliferation, survival and metabolic homeostasis of various human cancers. Thus, it is critical to understand the upstream signalling pathways governing AKT activation. Here, we report that AKT undergoes SETDB1-mediated lysine methylation to promote its activation, which is antagonized by the Jumonji-family demethylase KDM4B. Notably, compared with wild-type mice, mice harbouring non-methylated mutant Akt1 not only exhibited reduced body size but were also less prone to carcinogen-induced skin tumours, in part due to reduced AKT activation. Mechanistically, the interaction of phosphatidylinositol (3,4,5)-trisphosphate with AKT facilitates its interaction with SETDB1 for subsequent AKT methylation, which in turn sustains AKT phosphorylation. Pathologically, genetic alterations, including SETDB1 amplification, aberrantly promote AKT methylation to facilitate its activation and oncogenic functions. Thus, AKT methylation is an important step, synergizing with PI3K signalling to control AKT activation. This suggests that targeting SETDB1 signalling could be a potential therapeutic strategy for combatting hyperactive AKT-driven cancers. Guo et al. identify SETDB1 and KDM4B as the methyltransferase and demethylase, respectively, for AKT. AKT methylation promotes its kinase activity and the subsequent tumorigenesis.

Growth signals, such as extracellular nutrients and growth factors, have substantial effects on genome integrity; however, the direct underlying link remains unclear. Here, we show that the mechanistic target of rapamycin (mTOR)-ribosomal S6 kinase (S6K) pathway, a central regulator of growth signalling, phosphorylates RNF168 at Ser60 to inhibit its E3 ligase activity, accelerate its proteolysis and impair its function in the DNA damage response, leading to accumulated unrepaired DNA and genome instability. Moreover, loss of the tumour suppressor liver kinase B1 (LKB1; also known as STK11) hyperactivates mTOR complex 1 (mTORC1)-S6K signalling and decreases RNF168 expression, resulting in defects in the DNA damage response. Expression of a phospho-deficient RNF168-S60A mutant rescues the DNA damage repair defects and suppresses tumorigenesis caused by Lkb1 loss. These results reveal an important function of mTORC1-S6K signalling in the DNA damage response and suggest a general mechanism that connects cell growth signalling to genome stability control.

The bromodomain and extraterminal (BET) family of proteins comprises four members-BRD2, BRD3, BRD4 and the testis-specific isoform BRDT-that largely function as transcriptional coactivators and play critical roles in various cellular processes, including the cell cycle, apoptosis, migration and invasion. BET proteins enhance the oncogenic functions of major cancer drivers by elevating the expression of these drivers, such as c-Myc in leukemia, or by promoting the transcriptional activities of oncogenic factors, such as AR and ERG in prostate cancer. Pathologically, BET proteins are frequently overexpressed and are clinically linked to various types of human cancer; they are therefore being pursued as attractive therapeutic targets for selective inhibition in patients with cancer. To this end, a number of bromodomain inhibitors, including JQ1 and I-BET, have been developed and have shown promising outcomes in early clinical trials. Although resistance to BET inhibitors has been documented in preclinical models, the molecular mechanisms underlying acquired resistance are largely unknown. Here we report that cullin-3SPOP earmarks BET proteins, including BRD2, BRD3 and BRD4, for ubiquitination-mediated degradation. Pathologically, prostate cancer-associated SPOP mutants fail to interact with and promote the degradation of BET proteins, leading to their elevated abundance in SPOP-mutant prostate cancer. As a result, prostate cancer cell lines and organoids derived from individuals harboring SPOP mutations are more resistant to BET-inhibitor-induced cell growth arrest and apoptosis. Therefore, our results elucidate the tumor-suppressor role of SPOP in prostate cancer in which it acts as a negative regulator of BET protein stability and also provide a molecular mechanism for resistance to BET inhibitors in individuals with prostate cancer bearing SPOP mutations.

Mammalian target of rapamycin (mTOR) plays essential roles in cell proliferation, survival and metabolism by forming at least two functional distinct multi-protein complexes, mTORC1 and mTORC2. External growth signals can be received and interpreted by mTORC2 and further transduced to mTORC1. On the other hand, mTORC1 can sense inner-cellular physiological cues such as amino acids and energy states and can indirectly suppress mTORC2 activity in part through phosphorylation of its upstream adaptors, IRS-1 or Grb10, under insulin or IGF-1 stimulation conditions. To date, upstream signaling pathways governing mTORC1 activation have been studied extensively, while the mechanisms modulating mTORC2 activity remain largely elusive. We recently reported that Sin1, an essential mTORC2 subunit, was phosphorylated by either Akt or S6K in a cellular context-dependent manner. More importantly, phosphorylation of Sin1 at T86 and T398 led to a dissociation of Sin1 from the functional mTORC2 holo-enzyme, resulting in reduced Akt activity and sensitizing cells to various apoptotic challenges. Notably, an ovarian cancer patient-derived Sin1-R81T mutation abolished Sin1-T86 phosphorylation by disrupting the canonical S6K-phoshorylation motif, thereby bypassing Sin1-phosphorylation-mediated suppression of mTORC2 and leading to sustained Akt signaling to promote tumorigenesis. Our work therefore provided physiological and pathological evidence to reveal the biological significance of Sin1 phosphorylation-mediated suppression of the mTOR/Akt oncogenic signaling, and further suggested that misregulation of this process might contribute to Akt hyper-activation that is frequently observed in human cancers.

Wei and colleagues report that phosphorylation of Sin1 by S6K or Akt results in its dissociation from mTORC2, thus suppressing mTORC2 activity. A cancer-patient-derived Sin1 mutation that impairs this phosphorylation leads to mTORC2 hyperactivation and increased tumour formation in mice.

Recent studies revealed that mutations in SPOP (Speckle-type POZ protein) occur in up to 15% of patients with prostate cancer. However, the physiological role of SPOP in regulating prostate tumorigenesis remains elusive. Here, we identified the Cdc20 oncoprotein as a novel ubiquitin substrate of SPOP. As such, pharmacological inhibition of Cullin-based E3 ligases by MLN4924 could stabilize endogenous Cdc20 in cells. Furthermore, we found that Cullin 3, and, to a less extent, Cullin 1, specifically interacted with Cdc20. Depletion of Cullin 3, but not Cullin 1, could upregulate the abudance of Cdc20 largely via prolonging Cdc20 half-life. Moreover, SPOP, the adaptor protein of Cullin 3 family E3 ligase, specifically interacted with Cdc20, and promoted the poly-ubiquitination and subsequent degradation of Cdc20 in a degron-dependent manner. Importantly, prostate cancer-derived SPOP mutants failed to interact with Cdc20 to promote its degradation. As a result, SPOP-deficient prostate cancer cells with elevated Cdc20 expression became resistant to a pharmacological Cdc20 inhibitor. Therefore, our results revealed a novel role of SPOP in tumorigenesis in part by promoting the degradation of the Cdc20 oncoprotein.

Transcriptional R loops are anomalous RNA:DNA hybrids that have been detected in organisms from bacteria to humans. These structures have been shown in eukaryotes to result in DNA damage and rearrangements; however, the mechanisms underlying these effects have remained largely unknown. To investigate this, we first show that R-loop formation induces chromosomal DNA rearrangements and recombination in Escherichia coli, just as it does in eukaryotes. More importantly, we then show that R-loop formation causes DNA replication fork stalling, and that this in fact underlies the effects of R loops on genomic stability. Strikingly, we found that attenuation of replication strongly suppresses R-loop-mediated DNA rearrangements in both E. coli and HeLa cells. Our findings thus provide a direct demonstration that R-loop formation impairs DNA replication and that this is responsible for the deleterious effects of R loops on genome stability from bacteria to humans.

The mechanistic target of rapamycin (mTOR) has a key role in the integration of various physiological stimuli to regulate several cell growth and metabolic pathways. mTOR primarily functions as a catalytic subunit in two structurally related but functionally distinct multi-component kinase complexes, mTOR complex 1 (mTORC1) and mTORC2 (refs 1, 2). Dysregulation of mTOR signalling is associated with a variety of human diseases, including metabolic disorders and cancer. Thus, both mTORC1 and mTORC2 kinase activity is tightly controlled in cells. mTORC1 is activated by both nutrients and growth factors, whereas mTORC2 responds primarily to extracellular cues such as growth-factor-triggered activation of PI3K signalling. Although both mTOR and GβL (also known as MLST8) assemble into mTORC1 and mTORC2 (refs 11, 12, 13, 14, 15), it remains largely unclear what drives the dynamic assembly of these two functionally distinct complexes. Here we show, in humans and mice, that the K63-linked polyubiquitination status of GβL dictates the homeostasis of mTORC2 formation and activation. Mechanistically, the TRAF2 E3 ubiquitin ligase promotes K63-linked polyubiquitination of GβL, which disrupts its interaction with the unique mTORC2 component SIN1 (refs 12, 13, 14) to favour mTORC1 formation. By contrast, the OTUD7B deubiquitinase removes polyubiquitin chains from GβL to promote GβL interaction with SIN1, facilitating mTORC2 formation in response to various growth signals. Moreover, loss of critical ubiquitination residues in GβL, by either K305R/K313R mutations or a melanoma-associated GβL(ΔW297) truncation, leads to elevated mTORC2 formation, which facilitates tumorigenesis, in part by activating AKT oncogenic signalling. In support of a physiologically pivotal role for OTUD7B in the activation of mTORC2/AKT signalling, genetic deletion of Otud7b in mice suppresses Akt activation and Kras-driven lung tumorigenesis in vivo. Collectively, our study reveals a GβL-ubiquitination-dependent switch that fine-tunes the dynamic organization and activation of the mTORC2 kinase under both physiological and pathological conditions.

Activation of the serine-threonine kinase Akt promotes the survival and proliferation of various cancers. Hypoxia promotes the resistance of tumor cells to specific therapies. We therefore explored a possible link between hypoxia and Akt activity. We found that Akt was prolyl-hydroxylated by the oxygen-dependent hydroxylase EglN1. The von Hippel–Lindau protein (pVHL) bound directly to hydroxylated Akt and inhibited Akt activity. In cells lacking oxygen or functional pVHL, Akt was activated to promote cell survival and tumorigenesis. We also identified cancer-associated Akt mutations that impair Akt hydroxylation and subsequent recognition by pVHL, thus leading to Akt hyperactivation. Our results show that microenvironmental changes, such as hypoxia, can affect tumor behaviors by altering Akt activation, which has a critical role in tumor growth and therapeutic resistance.

The retinoblastoma (Rb) protein exerts its tumor suppressor function primarily by inhibiting the E2F family of transcription factors that govern cell cycle progression. However, it remains largely elusive whether hyper-phosphorylated, non-E2F1-interacting form, of Rb has any physiological role. Here, we report that hyper-phosphorylated Rb directly binds to, and suppresses the function of mTORC2, but not mTORC1. Mechanistically, Rb, but not p107 nor p130, interacts with Sin1 and blocks the access of Akt to mTORC2, leading to attenuated Akt activation and increased sensitivity to chemotherapeutic drugs. As such, inhibition of Rb phosphorylation by depleting cyclin D, or using CDK4/6 inhibitors, releases Rb-mediated mTORC2 suppression. This, in turn, leads to elevated Akt activation to confer resistance to chemotherapeutic drugs in Rb-proficient cells, which can be attenuated with Akt inhibitors. Therefore, our work provides a molecular basis for the synergistic usage of CDK4/6 and Akt inhibitors in treating Rb-proficient cancer.

To maintain genome stability, mammalian cells have developed a delicate, yet efficient, system to sense and repair damaged DNA, including two evolutionarily conserved DNA damage repair (DDR) pathways: homologous recombination (HR) and non-homologous-end-joining (NHEJ). Deregulation in these repair pathways may lead to genomic instability and subsequent human diseases, including cancer. On the other hand, hyper-activation of the oncogenic Akt signaling pathway has been observed in almost all solid tumors. Emerging evidence has begun to reveal a possible role of active Akt in regulating DDR, possibly through suppression of HR. However, whether and how Akt regulates NHEJ remains largely undefined. To this end, we recently reported that Akt impairs NHEJ by phosphorylating XLF at T181, to trigger its dissociation from the functional DNA ligase IV (LIG4)/XRCC4 complex. Here, we provide an additional perspective discussing how Akt is activated upon DNA damage to regulate DNA repair pathways as well as the cellular apoptotic responses.

Deficiency in repair of damaged DNA leads to genomic instability and is closely associated with tumorigenesis. Most DNA double-strand-breaks (DSBs) are repaired by two major mechanisms, homologous-recombination (HR) and non-homologous-end-joining (NHEJ). Although Akt has been reported to suppress HR, its role in NHEJ remains elusive. Here, we report that Akt phosphorylates XLF at Thr181 to trigger its dissociation from the DNA ligase IV/XRCC4 complex, and promotes its interaction with 14-3-3β leading to XLF cytoplasmic retention, where cytosolic XLF is subsequently degraded by SCF(β-TRCP) in a CKI-dependent manner. Physiologically, upon DNA damage, XLF-T181E expressing cells display impaired NHEJ and elevated cell death. Whereas a cancer-patient-derived XLF-R178Q mutant, deficient in XLF-T181 phosphorylation, exhibits an elevated tolerance of DNA damage. Together, our results reveal a pivotal role for Akt in suppressing NHEJ and highlight the tight connection between aberrant Akt hyper-activation and deficiency in timely DSB repair, leading to genomic instability and tumorigenesis.

The mammalian target of rapamycin (mTOR) is a central regulator of cell growth, proliferation and metabolism by forming at least 2 functionally distinct complexes, mTOR complex-1 (mTORC1) and mTOR ...

Akt, also known as protein kinase B, plays key roles in cell proliferation, survival and metabolism. Akt hyperactivation contributes to many pathophysiological conditions, including human cancers1–3, and is closely associated with poor prognosis and chemo- or radio-therapeutic resistance4. Phosphorylation of Akt at S473 (ref. 5) and T308 (ref. 6) activates Akt. However, it remains unclear whether further mechanisms account for full Akt activation, and whether Akt hyperactivation is linked to misregulated cell cycle progression, another cancer hallmark7. Here we report that Akt activity fluctuates across the cell cycle, mirroring cyclin A expression. Mechanistically, phosphorylation of S477 and T479 at the Akt extreme carboxy terminus by cyclin-dependent kinase 2 (Cdk2)/cyclin A or mTORC2, under distinct physiological conditions, promotes Akt activation through facilitating, or functionally compensating for, S473 phosphorylation. Furthermore, deletion of the cyclin A2 allele in the mouse olfactory bulb leads to reduced S477/T479 phosphorylation and elevated cellular apoptosis. Notably, cyclin A2-deletion-induced cellular apoptosis in mouse embryonic stem cells is partly rescued by S477D/T479E-Akt1, supporting a physiological role for cyclin A2 in governing Akt activation. Together, the results of our study show Akt S477/T479 phosphorylation to be an essential layer of the Akt activation mechanism to regulate its physiological functions, thereby providing a new mechanistic link between aberrant cell cycle progression and Akt hyperactivation in cancer.

mTOR serves as a central regulator of cell growth and metabolism by forming two distinct complexes, mTORC1 and mTORC2. Although mechanisms of mTORC1 activation by growth factors and amino acids have been extensively studied, the upstream regulatory mechanisms leading to mTORC2 activation remain largely elusive. Here, we report that the pleckstrin homology (PH) domain of SIN1, an essential and unique component of mTORC2, interacts with the mTOR kinase domain to suppress mTOR activity. More importantly, PtdIns(3,4,5)P3, but not other PtdInsPn species, interacts with SIN1-PH to release its inhibition on the mTOR kinase domain, thereby triggering mTORC2 activation. Mutating critical SIN1 residues that mediate PtdIns(3,4,5)P3 interaction inactivates mTORC2, whereas mTORC2 activity is pathologically increased by patient-derived mutations in the SIN1-PH domain, promoting cell growth and tumor formation. Together, our study unravels a PI3K-dependent mechanism for mTORC2 activation, allowing mTORC2 to activate AKT in a manner that is regulated temporally and spatially by PtdIns(3,4,5)P3. Significance: The SIN1-PH domain interacts with the mTOR kinase domain to suppress mTOR activity, and PtdIns(3,4,5)P3 binds the SIN1-PH domain to release its inhibition on the mTOR kinase domain, leading to mTORC2 activation. Cancer patient–derived SIN1-PH domain mutations gain oncogenicity by loss of suppressing mTOR activity as a means to facilitate tumorigenesis. Cancer Discov; 5(11); 1194–209. ©2015 AACR. See related commentary by Yuan and Guan, p. 1127. This article is highlighted in the In This Issue feature, p. 1111

Somatic hypermutation (SHM) of Ig variable region ( IgV ) genes requires both IgV transcription and the enzyme activation-induced cytidine deaminase (AID). Identification of a cofactor responsible for the fact that IgV genes are much more sensitive to AID-induced mutagenesis than other genes is a key question in immunology. Here, we describe an essential role for a splice isoform of the prototypical serine/arginine-rich (SR) protein SRSF1, termed SRSF1-3, in AID-induced SHM in a DT40 chicken B-cell line. Unexpectedly, we found that SHM does not occur in a DT40 line lacking SRSF1-3 (DT40-ASF), although it is readily detectable in parental DT40 cells. Strikingly, overexpression of AID in DT40-ASF cells led to a large increase in nonspecific (off-target) mutations. In contrast, introduction of SRSF1-3, but not SRSF1, into these cells specifically restored SHM without increasing off-target mutations. Furthermore, we found that SRSF1-3 binds preferentially to the IgV gene and inhibits processing of the Ig transcript, providing a mechanism by which SRSF1-3 makes the IgV gene available for AID-dependent SHM. SRSF1 not only acts as an essential splicing factor but also regulates diverse aspects of mRNA metabolism and maintains genome stability. Our findings, thus, define an unexpected and important role for SRSF1, particularly for its splice variant, in enabling AID to function specifically on its natural substrate during SHM.