Szu Wei Lee

The E3 Ligase TRAF6 Regulates Akt Ubiquitination and Activation

Regulating Akt The protein kinase Akt is activated in response to receptor-activated generation of the signaling second messenger phosphatidylinositol 3,4,5-trisphosphate and has roles in regulation of diverse processes from metabolism and cell survival to transcription and tumorigenesis. Yang et al. (p. 1134; see the Perspective by Restuccia and Hemmings) report a previously unrecognized mode of regulation of Akt: covalent modification of Akt by linkage to lysine 63 of ubiquitin molecules. Such ubiquitination of Akt promotes localization to the cell membrane and consequent activation in cells stimulated with growth factors. TRAF6 (TNF receptor–associated factor 6) was implicated as the E3 ubiquitin ligase that mediates ubiquitination of Akt. Ubiquitination of Akt may influence its role in cancer cells: A mutant form of Akt associated with human cancer showed increased ubiquitination, and depletion of TRAF6 decreased tumorigenicity of a prostate cancer cell line in a mouse cancer model. Localization and activation of signaling proteins in cancer cells are controlled by ubiquitin labeling. Akt signaling plays a central role in many biological functions, such as cell proliferation and apoptosis. Because Akt (also known as protein kinase B) resides primarily in the cytosol, it is not known how these signaling molecules are recruited to the plasma membrane and subsequently activated by growth factor stimuli. We found that the protein kinase Akt undergoes lysine-63 chain ubiquitination, which is important for Akt membrane localization and phosphorylation. TRAF6 was found to be a direct E3 ligase for Akt and was essential for Akt ubiquitination, membrane recruitment, and phosphorylation upon growth-factor stimulation. The human cancer-associated Akt mutant displayed an increase in Akt ubiquitination, in turn contributing to the enhancement of Akt membrane localization and phosphorylation. Thus, Akt ubiquitination is an important step for oncogenic Akt activation.

Skp2 targeting suppresses tumorigenesis by Arf-p53 -independent cellular senescence

Cellular senescence has been recently shown to have an important role in opposing tumour initiation and promotion. Senescence induced by oncogenes or by loss of tumour suppressor genes is thought to critically depend on induction of the p19Arf–p53 pathway. The Skp2 E3-ubiquitin ligase can act as a proto-oncogene and its aberrant overexpression is frequently observed in human cancers. Here we show that although Skp2 inactivation on its own does not induce cellular senescence, aberrant proto-oncogenic signals as well as inactivation of tumour suppressor genes do trigger a potent, tumour-suppressive senescence response in mice and cells devoid of Skp2. Notably, Skp2 inactivation and oncogenic-stress-driven senescence neither elicit activation of the p19Arf–p53 pathway nor DNA damage, but instead depend on Atf4, p27 and p21. We further demonstrate that genetic Skp2 inactivation evokes cellular senescence even in oncogenic conditions in which the p19Arf–p53 response is impaired, whereas a Skp2–SCF complex inhibitor can trigger cellular senescence in p53/Pten-deficient cells and tumour regression in preclinical studies. Our findings therefore provide proof-of-principle evidence that pharmacological inhibition of Skp2 may represent a general approach for cancer prevention and therapy.

The Skp2-SCF E3 ligase regulates Akt ubiquitination, glycolysis, herceptin sensitivity, and tumorigenesis.

Akt kinase plays a central role in cell growth, metabolism, and tumorigenesis. The TRAF6 E3 ligase orchestrates IGF-1-mediated Akt ubiquitination and activation. Here, we show that Akt ubiquitination is also induced by activation of ErbB receptors; unexpectedly, and in contrast to IGF-1 induced activation, the Skp2 SCF complex, not TRAF6, is a critical E3 ligase for ErbB-receptor-mediated Akt ubiquitination and membrane recruitment in response to EGF. Skp2 deficiency impairs Akt activation, Glut1 expression, glucose uptake and glycolysis, and breast cancer progression in various tumor models. Moreover, Skp2 overexpression correlates with Akt activation and breast cancer metastasis and serves as a marker for poor prognosis in Her2-positive patients. Finally, Skp2 silencing sensitizes Her2-overexpressing tumors to Herceptin treatment. Our study suggests that distinct E3 ligases are utilized by diverse growth factors for Akt activation and that targeting glycolysis sensitizes Her2-positive tumors to Herceptin treatment.

Deciphering the transcriptional complex critical for RhoA gene expression and cancer metastasis

The RhoA GTPase is crucial in numerous biological functions and is linked to cancer metastasis. However, the understanding of the molecular mechanism responsible for RhoA transcription is still very limited. Here we show that RhoA transcription is orchestrated by the Myc–Skp2–Miz1–p300 transcriptional complex. Skp2 cooperates with Myc to induce RhoA transcription by recruiting Miz1 and p300 to the RhoA promoter independently of Skp1-Cullin-F-box protein containing complex (SCF)–Skp2 E3 ligase activity. Deficiency of this complex results in impairment in RhoA expression, cell migration, invasion, and breast cancer metastasis, recapitulating the phenotypes observed in RhoA knockdown, and RhoA restoration rescues the defect in cell invasion. Overexpression of the Myc–Skp2–Miz1 complex is found in metastatic human cancers and is correlated with RhoA expression. Our study provides insight into how oncogenic Skp2 and Myc coordinate to induce RhoA transcription and establishes a novel SCF–Skp2 E3-ligase-independent function for oncogenic Skp2 in transcription and cancer metastasis.

LncRNA NBR2 engages a metabolic checkpoint by regulating AMPK under energy stress

Long non-coding RNAs (lncRNAs) have emerged as critical regulators in various cellular processes. However, the potential involvement of lncRNAs in kinase signalling remains largely unknown. AMP-activated protein kinase (AMPK) acts as a critical sensor of cellular energy status. Here we show that the lncRNA NBR2 (neighbour of BRCA1 gene 2) is induced by the LKB1–AMPK pathway under energy stress. On energy stress, NBR2 in turn interacts with AMPK and promotes AMPK kinase activity, thus forming a feed-forward loop to potentiate AMPK activation during energy stress. Depletion of NBR2 attenuates energy-stress-induced AMPK activation, resulting in unchecked cell cycling, altered apoptosis/autophagy response, and increased tumour development in vivo. NBR2 is downregulated and its low expression correlates with poor clinical outcomes in some human cancers. Together, the results of our study uncover a mechanism coupling lncRNAs with metabolic stress response, and provides a broad framework to understand further the regulation of kinase signalling by lncRNAs.

ER stress inhibits mTORC2 and Akt signaling through GSK-3β-mediated phosphorylation of rictor

Cellular stress attenuates growth factor signaling through a phosphorylation event that blocks substrate access to the kinase complex mTORC2. No Access During Stressful Times Under conditions of cellular stress, cells tend to halt anabolic processes, such as cell growth and proliferation, to conserve resources. mTORC2 (mammalian target of rapamycin complex 2), which mediates its effects through activation of the kinase Akt, is a key signaling complex that promotes anabolic processes. Chen et al. investigated the mechanisms by which mTORC2 activity is inhibited by endoplasmic reticulum (ER) stress. They found that glycogen synthase kinase–3β (GSK-3β), which is activated by ER stress, phosphorylated rictor, a component of mTORC2 that helps to determine substrate specificity for the complex. This phosphorylation event decreased binding of Akt to mTORC2, resulting in reduced activation of Akt and cell proliferation. Furthermore, transformed cells expressing a mutant form of rictor lacking the GSK-3β phosphorylation site formed larger tumors in mice than did those expressing wild-type rictor or a rictor mutant that mimicked a constitutively phosphorylated form. These results define a pathway by which mTORC2 and Akt signaling can be attenuated by cellular stress and provide a potential therapeutic target for limiting cell proliferation (such as in cancer). In response to environmental cues, cells coordinate a balance between anabolic and catabolic pathways. In eukaryotes, growth factors promote anabolic processes and stimulate cell growth, proliferation, and survival through activation of the phosphoinositide 3-kinase (PI3K)–Akt pathway. Akt-mediated phosphorylation of glycogen synthase kinase–3β (GSK-3β) inhibits its enzymatic activity, thereby stimulating glycogen synthesis. We show that GSK-3β itself inhibits Akt by controlling the mammalian target of rapamycin complex 2 (mTORC2), a key activating kinase for Akt. We found that during cellular stress, GSK-3β phosphorylated the mTORC2 component rictor at serine-1235, a modification that interfered with the binding of Akt to mTORC2. The inhibitory effect of GSK-3β on mTORC2-Akt signaling and cell proliferation was eliminated by blocking phosphorylation of rictor at serine-1235. Thus, in response to cellular stress, GSK-3β restrains mTORC2-Akt signaling by specifically phosphorylating rictor, thereby balancing the activities of GSK-3β and Akt, two opposing players in glucose metabolism.

Regulation of Skp2 expression and activity and its role in cancer progression

The regulation of cell cycle entry is critical for cell proliferation and tumorigenesis. One of the key players regulating cell cycle progression is the F-box protein Skp2. Skp2 forms a SCF complex with Skp1, Cul-1, and Rbx1 to constitute E3 ligase through its F-box domain. Skp2 protein levels are regulated during the cell cycle, and recent studies reveal that Skp2 stability, subcellular localization, and activity are regulated by its phosphorylation. Overexpression of Skp2 is associated with a variety of human cancers, indicating that Skp2 may contribute to the development of human cancers. The notion is supported by various genetic mouse models that demonstrate an oncogenic activity of Skp2 and its requirement in cancer progression, suggesting that Skp2 may be a novel and attractive therapeutic target for cancers.

Critical Role of Monoubiquitination of Histone H2AX Protein in Histone H2AX Phosphorylation and DNA Damage Response

DNA damage response is an important surveillance mechanism used to maintain the integrity of the human genome in response to genotoxic stress. Histone variant H2AX is a critical sensor that undergoes phosphorylation at serine 139 upon genotoxic stress, which provides a docking site to recruit the mediator of DNA damage checkpoint protein 1 (MDC1) and DNA repair protein complex to sites of DNA breaks for DNA repair. Here, we show that monoubiquitination of H2AX is induced upon DNA double strand breaks and plays a critical role in H2AX Ser-139 phosphorylation (γ-H2AX), in turn facilitating the recruitment of MDC1 to DNA damage foci. Mechanistically, we show that monoubiquitination of H2AX induced by RING finger protein 2 (RNF2) is required for the recruitment of active ataxia telangiectasia mutated to DNA damage foci, thus affecting the formation of γ-H2AX. Importantly, a defect in monoubiquitination of H2AX profoundly enhances ionizing radiation sensitivity. Our study therefore suggests that monoubiquitination of H2AX is an important step for DNA damage response and may have important clinical implications for the treatment of cancers.

Skp2-Mediated RagA Ubiquitination Elicits a Negative Feedback to Prevent Amino-Acid-Dependent mTORC1 Hyperactivation by Recruiting GATOR1

The regulation of RagA(GTP) is important for amino-acid-induced mTORC1 activation. Although GATOR1 complex has been identified as a negative regulator for mTORC1 by hydrolyzing RagA(GTP), how GATOR1 is recruited to RagA to attenuate mTORC1 signaling remains unclear. Moreover, how mTORC1 signaling is terminated upon amino acid stimulation is also unknown. We show that the recruitment of GATOR1 to RagA is induced by amino acids in an mTORC1-dependent manner. Skp2 E3 ligase drives K63-linked ubiquitination of RagA, which facilitates GATOR1 recruitment and RagA(GTP) hydrolysis, thereby providing a negative feedback loop to attenuate mTORC1 lysosomal recruitment and prevent mTORC1 hyperactivation. We further demonstrate that Skp2 promotes autophagy but inhibits cell size and cilia growth through RagA ubiquitination and mTORC1 inhibition. We thereby propose a negative feedback whereby Skp2-mediated RagA ubiquitination recruits GATOR1 to restrict mTORC1 signaling upon sustained amino acid stimulation, which serves a critical mechanism to maintain proper cellular functions.

The role of Skp2 in hematopoietic stem cell quiescence, pool size, and self-renewal

Although the maintenance of HSC quiescence and self-renewal are critical for controlling stem cell pool and transplantation efficiency, the mechanisms by which they are regulated remain largely unknown. Understanding the factors controlling these processes may have important therapeutic potential for BM failure and cancers. Here, we show that Skp2, a component of the Skp2 SCF complex, is an important regulator for HSC quiescence, frequency, and self-renewal capability. Skp2 deficiency displays a marked enhancement of HSC populations through promoting cell cycle entry independently of its role on apoptosis. Surprisingly, Skp2 deficiency in HSCs reduces quiescence and displays increased HSC cycling and proliferation. Importantly, loss of Skp2 not only increases HSC populations and long-term reconstitution ability but also rescues the defect in long-term reconstitution ability of HSCs on PTEN inactivation. Mechanistically, we show that Skp2 deficiency induces Cyclin D1 gene expression, which contributes to an increase in HSC cycling. Finally, we demonstrate that Skp2 deficiency enhances sensitivity of Lin(-) Sca-1(+) c-kit(+) cells and leukemia cells to chemotherapy agents. Our findings show that Skp2 is a novel regulator for HSC quiescence and self-renewal and that targeting Skp2 may have therapeutic implications for BM transplantation and leukemia stem cell treatment.