Xin Ge

The Ubiquitination of RagA GTPase by RNF152 Negatively Regulates mTORC1 Activation

mTORC1 is essential for regulating cell growth and metabolism in response to various environmental stimuli. Heterodimeric Rag GTPases are required for amino-acid-mediated mTORC1 activation at the lysosome. However, the mechanism by which amino acids regulate Rag activation remains not fully understood. Here, we identified the lysosome-anchored E3 ubiquitin ligase RNF152 as an essential negative regulator of the mTORC1 pathway by targeting RagA for K63-linked ubiquitination. RNF152 interacts with and ubiquitinates RagA in an amino-acid-sensitive manner. The mutation of RagA ubiquitination sites abolishes this effect of RNF152 and enhances the RagA-mediated activation of mTORC1. Ubiquitination by RNF152 generates an anchor on RagA to recruit its inhibitor GATOR1, a GAP complex for Rag GTPases. RNF152 knockout results in the hyperactivation of mTORC1 and protects cells from amino-acid-starvation-induced autophagy. Thus, this study reveals a mechanism for regulation of mTORC1 signaling by RNF152-mediated K63-linked polyubiquitination of RagA.

The deubiquitinase USP21 maintains the stemness of mouse embryonic stem cells via stabilization of Nanog

Nanog is a master pluripotency factor of embryonic stem cells (ESCs). Stable expression of Nanog is essential to maintain the stemness of ESCs. However, Nanog is a short-lived protein and quickly degraded by the ubiquitin-dependent proteasome system. Here we report that the deubiquitinase USP21 interacts with, deubiquitinates and stabilizes Nanog, and therefore maintains the protein level of Nanog in mouse ESCs (mESCs). Loss of USP21 results in Nanog degradation, mESCs differentiation and reduces somatic cell reprogramming efficiency. USP21 is a transcriptional target of the LIF/STAT3 pathway and is downregulated upon differentiation. Moreover, differentiation cues promote ERK-mediated phosphorylation and dissociation of USP21 from Nanog, thus leading to Nanog degradation. In addition, USP21 is recruited to gene promoters by Nanog to deubiquitinate histone H2A at K119 and thus facilitates Nanog-mediated gene expression. Together, our findings provide a regulatory mechanism by which extrinsic signals regulate mESC fate via deubiquitinating Nanog.

p38 inhibition provides anti-DNA virus immunity by regulation of USP21 phosphorylation and STING activation.

Stimulator of IFN genes (STING) is a central adaptor protein that mediates the innate immune responses to DNA virus infection. Although ubiquitination is essential for STING function, how the ubiquitination/deubiquitination system is regulated by virus infection to control STING activity remains unknown. In this study, we found that USP21 is an important deubiquitinating enzyme for STING and that it negatively regulates the DNA virus–induced production of type I interferons by hydrolyzing K27/63-linked polyubiquitin chain on STING. HSV-1 infection recruited USP21 to STING at late stage by p38-mediated phosphorylation of USP21 at Ser538. Inhibition of p38 MAPK enhanced the production of IFNs in response to virus infection and protected mice from lethal HSV-1 infection. Thus, our study reveals a critical role of p38-mediated USP21 phosphorylation in regulating STING-mediated antiviral functions and identifies p38-USP21 axis as an important pathway that DNA virus adopts to avoid innate immunity responses.

An ultrasensitive system for measuring the USPs and OTULIN activity using Nanoluc as a reporter.

The deubiquitinating enzymes (DUBs) are a family of isopeptidases responsible for removing the ubiquitin from the ubiquitinated proteins. Identification of inhibitors for DUBs is emerging as an efficient way for discovering potential medicines for disease treatment. However, the high throughput screening (HTS) assay is still not available for all USPs, especially OTULIN. Here, we described a novel steadily quantifiable DUBs assay platform using Nanoluc (Nluc) as reporter. We further demonstrated that the Ub-Nluc assay could be used for HTS of DUBs inhibitors. Moreover, we generated a sensitive system for OTULIN inhibitors screening using Nluc as a reporter. In summary, our data indicate that Ub-Nluc and the improved Ub-Ub-GS-Nluc assay are efficient systems for measuring activities and screening inhibitors of USPs and OTULIN.

OTUB1 suppresses mTOR complex 1 (mTORC1) activity by deubiquitinating the mTORC1 inhibitor DEPTOR

Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) integrates various environmental signals to regulate cell growth and metabolism. DEPTOR, also termed DEPDC6, is an endogenous inhibitor of mTORC1 and mTORC2 activities. The abundance of DEPTOR centrally orchestrates the mTOR signaling network. However, the mechanisms by which DEPTOR stability is regulated are still elusive. Here, we report that OTU domain–containing ubiquitin aldehyde-binding protein 1 (OTUB1) specifically deubiquitinates DEPTOR in a deubiquitination assay. We found that OTUB1 directly interacted with DEPTOR via its N-terminal domain, deubiquitinated DEPTOR, and thereby stabilized DEPTOR in a Cys-91–independent but Asp-88–dependent manner, suggesting that OTUB1 targets DEPTOR for deubiquitination via a deubiquitinase activity–independent non-canonical mechanism. The interaction between OTUB1 and DEPTOR was enhanced when the cells were treated with amino acids. Moreover, OTUB1 suppressed amino acid–induced activation of mTORC1 in a DEPTOR-dependent manner and thereby ultimately controlled cellular autophagy, cell proliferation, and size. Our findings reveal a mechanism that stabilizes the mTORC1 inhibitor DEPTOR via OTUB1s deubiquitinase activity. Our insights may inform research into various mTOR activity–related diseases, such as cancer, and may contribute to the identification of new diagnostic markers and therapeutic strategies for cancer treatments.

Palmitoylated SCP1 is targeted to the plasma membrane and negatively regulates angiogenesis

SCP1 as a nuclear transcriptional regulator acts globally to silence neuronal genes and to affect the dephosphorylation of RNA Pol ll. However, we report the first finding and description of SCP1 as a plasma membrane-localized protein in various cancer cells using EGFP- or other epitope-fused SCP1. Membrane-located SCP1 dephosphorylates AKT at serine 473, leading to the abolishment of serine 473 phosphorylation that results in suppressed angiogenesis and a decreased risk of tumorigenesis. Consistently, we observed increased AKT phosphorylation and angiogenesis followed by enhanced tumorigenesis in Ctdsp1 (which encodes SCP1) gene - knockout mice. Importantly, we discovered that the membrane localization of SCP1 is crucial for impeding angiogenesis and tumor growth, and this localization depends on palmitoylation of a conserved cysteine motif within its NH2 terminus. Thus, our study discovers a novel mechanism underlying SCP1 shuttling between the plasma membrane and nucleus, which constitutes a unique pathway in transducing AKT signaling that is closely linked to angiogenesis and tumorigenesis. DOI: http://dx.doi.org/10.7554/eLife.22058.001

EGFR tyrosine kinase inhibitors differentially affect autophagy in head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent cancer worldwide. The majority of HNSCCs overexpress Epidermal Growth Factor Receptor (EGFR), an essential receptor tyrosine kinase (RTK) that promotes HNSCC growth and metastasis. Therefore, EGFR has been used as an important therapeutic target to treat HNSCC. Inhibition of EGFR stimulates autophagy in cancer cells. However, the role of autophagy in EGFR inhibitor-induced cancer suppression is still in a debate. Here, we reveal that the first- and the second-generation EGFR tyrosine kinase inhibitors (TKIs) differentially affect HNSCC autophagy. The second-generation EGFR TKIs have much stronger effects on autophagy than the first-generation TKIs. The second-generation EGFR TKIs not only promote autophagy initiation signaling but also block autophagic flux by disturbing the lysosomes function, indicating a novel mechanism by which EGFR TKIs modulate cancer cell autophagy. Blocking the initiation of autophagy does not affect the second-generation EGFR TKI-induced HNSCC growth suppression. This suggests that the anti-growth effect of the second-generation EGFR TKIs on HNSCC is not dependent on autophagy.

FBW7 targets KLF10 for ubiquitin-dependent degradation

FBW7, a key component of SCFFBW7 E3 ubiquitin ligase, targets various proteins for degradation via the conserved Cdc4 phosphodegron (CPD) in substrates. In this study, we report that KLF10 is degraded by FBW7 via a conserved CPD. Through systematic analysis of the degradation of KLF transcription factors by FBW7, we identified KLF10 as a novel degradation target of FBW7. Ectopic expression of FBW7 markedly promoted the degradation of KLF10 while knockdown of endogenous FBW7 increased the protein levels of KLF10. In addition, simultaneous mutations of both threonine 82 (T82) and serine 86 (S86) significantly reduced the FBW7-mediated KLF10 degradation. Moreover, KLF10 containing a conserved putative CPD (TPPXSP) from amino acids 82 to 87, directly interacted with WD40 domain of FBW7 in a phosphorylation-dependent manner. Importantly, FBW7 could reverse the KLF10-mediated inhibition of Smad7 activity. Thus, our study uncovers a novel regulatory mechanism underlying which KLF10 stability and its biological function are mediated by FBW7.