**5. RNF152**

In terms of studies on RNF152, it was first reported as having decreased expression in breast and prostate cancer cell lines [45]. Next, RNF152 was cloned and characterized as a ubiquitin ligase [40]. It is localized in lysosomes and its overexpression can mediate self-polyubiquitylation through K48 linkage and induce apoptosis. However, its substrates and function initially remained unknown.

For the first time among members of the RNF183 family, RNF152 has been reported to be involved in mTOR signaling [43]. The ubiquitylation of RagA, which belongs to the Rag family of small GTPases and recruits mTORC1 to the lysosomal membrane [62], is markedly increased in response to amino acid starvation. RNF152 was identified among some lysosomal ubiquitin ligases and ubiquitylated RagA in a K63-linked manner. Increased amino acid levels facilitate the interaction of RNF152 with RagA and RNF152-mediated RagA ubiquitylation. RNF152 selectively ubiquitylates the inactive form of RagA (RagA-GDP/RagC-GTP), but not the active form (RagA-GTP/RagC-GDP), suggesting that the interaction between RNF152 and RagA is regulated by the nucleotide-bound status of RagA. Furthermore, the authors carefully demonstrated that RNF152 acts as a negative regulator of amino-acid-induced mTORC1 activation. RNF152-mediated RagA ubiquitylation promotes the binding between RagA and GATOR1, a GAP complex for Rag GTPases [62], resulting in the inactivation of RagA. USP17L2/DUB3 (ubiquitin carboxyl-terminal hydrolase 17), a deubiquitylating enzyme, was identified for RagA ubiquitylated by RNF152. Moreover, RNF152 knockout revealed that the deficiency of RNF152 causes the hyperactivation of mTORC1 and inhibits amino-acid-starvation-induced autophagy.

Four years later, the same group demonstrated that RNF152 is involved in the ubiquitylation of Rheb, another mTORC1 signal molecule, in growth factor-induced mTORC1 activation, not amino acids [62]. Rheb acts as a small GTPase like RagA and activates mTORC1 in its GTP-bound form. RNF152 induces Rheb monoubiquitylation that enhances binding to TSC2, which is a core subunit of the TSC complex and functions as a GAP [62], resulting in mTORC1 inactivation. On the other hand, USP4 was identified as a DUB for Rheb [64]. USP4 promotes the activation of Rheb by removing ubiquitin from it. USP4 activity is regulated by the EGF-Akt-mediated phosphorylation at S445. Therefore, RNF152 and USP4 can regulate Rheb activity negatively and positively, respectively, downstream of the EGF pathway. Since Rheb ubiquitylation negatively regulates mTORC1 activation, RNF152 can regulate cellular autophagy positively and cell proliferation negatively. Studies of USP4 knockout mice revealed that USP4 upregulates tumor growth in an mTORC1-dependent manner. In contrast, TCGA database indicates that RNF152 expression is downregulated in various types of cancer, including colon, lung, kidney, and liver cancers.

RNF152 in addition to RNF183 is also associated with CRC. 1,2-Dimethylhydrazine (DMH) is a potent carcinogen that acts as a DNA methylating agent. Microarray gene expression analysis showed that Rnf152 was upregulated in DMH-injected CRC model mice provided with high-calcium feed, compared with that in those provided with normal feed [46]. An additional relationship of RNF152 with CRC was reported in mTORC1 signaling [47]. RNF152 expression is significantly reduced in CRC tissues compared with that in normal tissues. The expression levels of RNF152 were reported to be correlated with prognosis in patients with CRC. Using cell lines and xenografts, it was demonstrated that RNF152 overexpression significantly decreased CRC cell growth in vitro and in vivo. RNF152 inhibits CRC cell proliferation by suppressing mTORC1, resulting in the induction of autophagy and apoptotic cell death. Although the ubiquitylation of RagA by RNF152 was not demonstrated in CRC cells, this finding is consistent with RNF152-mediated mTORC1 downregulation.

Rnf152 was first revealed to play a physiological role using zebrafish embryos [41]. The rnf152 transcript is now known to be expressed from the one-cell stage (maternally) to 48 h post-fertilization (hpf) (zygotically). Rnf152 is ubiquitously expressed in the brain at 24 hpf, whereas its expression is restricted to the eyes, midbrain–hindbrain boundary (MHB), and rhombomeres at 48 hpf. Since Rnf152 knockdown in zebrafish embryos leads to morphological defects in the eyes, MHB, and rhombomeres at 24 hpf, Rnf152 is required for appropriate development of the eyes and neural tube later than 18 hpf during embryogenesis. NeuroD is a marker for the inner and outer layers of the eyes at 48 hpf and

plays a crucial role in regulating cell cycle exit and cell fate determination in mitotic cells [65,66]. NeuroD expression in rnf152-deficient embryos disappears in the marginal zone, outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL) of the eyes at 27 hpf. Furthermore, the expression of deltaD and notch1a in rnf152 morphants is remarkably reduced in the ONL, INL, subpallium, tectum, and cerebellum [67]. Knockdown of rnf152 was found to cause decreases of the expression of her4 and ascl1a, Notch target genes [68], in specific regions at 24 hpf. Taking these findings together, Rnf152 may play essential roles in the development of the eyes, midbrain, and hindbrain, as well as the activation of Delta-Notch signaling. However, Deng et al. have already reported that Rnf152 knockout mice exhibited no difference in birth rates from the expected Mendelian ratios, suggesting that RNF152 is not required for embryonic development, at least in mouse [43]. Although these findings sugges<sup>t</sup> that ubiquitin ligases other than Rnf152 can work in the neural development of mammals, it remains unknown whether Rnf182, which is also expressed in the nervous system, works compensatorily.

One study reported on the relationship between the RNF152–mTORC1 axis and neural development [42]. The cell proliferation rate of the floor plate in the ventral region of the neural tube remains low [69]. Forkhead-type transcription factor FoxA2 was demonstrated to be a negative regulator of mTORC1 signaling in the floor plate [70]. Furthermore, RNF152 was identified as a target gene for FoxA2 among mTORC1 signaling genes. The silencing of RNF152 causes aberrant mTOR activation and aberrant cell division in the floor plate. Therefore, RNF152 may function as a negative regulator for the mTORC1 signaling in the floor plate downstream of FoxA2.
