Positive feedback regulation of p53 transactivity by DNA damage-induced ISG15 modification.

p53 plays a pivotal role in tumour suppression under stresses, such as DNA damage. ISG15 has been implicated in the control of tumorigenesis. Intriguingly, the expression of ISG15, UBE1L and UBCH8 is induced by DNA-damaging agents, such as ultraviolet and doxorubicin, which are known to induce p53. Here, we show that the genes encoding ISG15, UBE1L, UBCH8 and EFP, have the p53-responsive elements and their expression is induced in a p53-dependent fashion under DNA damage conditions. Furthermore, DNA damage induces ISG15 conjugation to p53 and this modification markedly enhances the binding of p53 to the promoters of its target genes (for example, CDKN1 and BAX) as well as of its own gene by promoting phosphorylation and acetylation, leading to suppression of cell growth and tumorigenesis. These findings establish a novel feedback circuit between p53 and ISG15-conjugating system for positive regulation of the tumour suppressive function of p53 under DNA damage conditions.

Deleterious c-Cbl Exon Skipping Contributes to Human Glioma.

c-Cbl, a RING-type ubiquitin E3 ligase, downregulates various receptor tyrosine kinases (e.g., epidermal growth factor receptor (EGFR)), leading to inhibition of cell proliferation. Moreover, patients with myeloid neoplasm frequently harbor c-Cbl mutations, implicating the role of c-Cbl as a tumor suppressor. Recently, we have shown that c-Cbl downregulates αPix-mediated cell migration and invasion, and the lack of c-Cbl in the rat C6 and human A172 glioma cells is responsible for their malignant behavior. Here, we showed that c-Cbl exon skipping occurs in the glioma cells and the brain tissues from glioblastoma patients lacking c-Cbl. This exon skipping resulted in generation of two types of c-Cbl isoforms: type I lacking exon-9 and type II lacking exon-9 and exon-10. However, the c-Cbl isoforms in the cells and tissues could not be detected as they were rapidly degraded by proteasome. Consequently, C6 and A172 cells showed sustained EGFR activation. However, no splice site mutation was found in the region from exon-7 to exon-11 of the c-Cbl gene in C6 cells and a glioblastoma tissue lacking c-Cbl. In addition, c-Cbl exon skipping could be induced when cells transfected with a c-Cbl mini-gene were grown to high density or under hypoxic stress. These results suggest that unknown alternations (e.g., mutation) of splicing machinery in C6 and A172 cells and the glioblastoma brain tissues are responsible for the deleterious exon skipping. Collectively, these findings indicate that the c-Cbl exon skipping contributes to human glioma and its malignant behavior.

c-Cbl regulates αPix-mediated cell migration and invasion.

c-Cbl, a RING-type ubiquitin E3 ligase, down-regulates receptor tyrosine kinases, including EGF receptor, and inhibits cell proliferation. Moreover, c-Cbl mutations are frequently found in patients with myeloid neoplasm. Therefore, c-Cbl is known as a tumor suppressor. αPix is expressed only in highly proliferative and mobile cells, including immune cells, and up-regulated in certain invasive tumors, such as glioblastoma multiforme. Here, we showed that c-Cbl serves as an ubiquitin E3 ligase for proteasome-mediated degradation of αPix, but not ßPix. Remarkably, the rat C6 and human A172 glioma cells were unable to express c-Cbl, which leads to a dramatic accumulation of αPix. Depletion of αPix by shRNA markedly reduced the ability of the glioma cells to migrate and invade, whereas complementation of shRNA-insensitive αPix promoted it. These results indicate that c-Cbl negatively regulates αPix-mediated cell migration and invasion and the lack of c-Cbl in the C6 and A172 glioma cells is responsible for their malignant behavior.

Modification of DBC1 by SUMO2/3 is crucial for p53-mediated apoptosis in response to DNA damage.

DBC1 is a major inhibitor of SIRT1, which plays critical roles in the control of diverse cellular processes, including stress response and energy metabolism. Therefore, the DBC1-SIRT1 interaction should finely be regulated. Here we report that DBC1 modification by Small Ubiquitin-like Modifier 2/3 (SUMO 2/3), but not by SUMO1, is crucial for p53 transactivation under genotoxic stress. Whereas etoposide treatment reduced the interaction of DBC1 with SENP1, it promoted that with PIAS3, resulting in an increase in DBC1 sumoylation. Remarkably, the switching from SENP1 to PIAS3 for DBC1 binding was achieved by ATM/ATR-mediated phosphorylation of DBC1. Furthermore, DBC1 sumoylation caused an increase in the DBC1-SIRT1 interaction, leading to the release of p53 from SIRT1 for transcriptional activation. Consistently, SENP1 knockdown promoted etoposide-induced apoptosis, whereas knockdown of PIAS3 or SUMO2/3 and overexpression of sumoylation-deficient DBC1 mutant inhibited it. These results establish the role of DBC1 sumoylation in the promotion of p53-mediated apoptosis in response to genotoxic stress.

Modification of ASC1 by UFM1 is crucial for ERα transactivation and breast cancer development.

Biological roles for UFM1, a ubiquitin-like protein, are largely unknown, and therefore we screened for targets of ufmylation. Here we show that ufmylation of the nuclear receptor coactivator ASC1 is a key step for ERα transactivation in response to 17ß-estradiol (E2). In the absence of E2, the UFM1-specific protease UfSP2 was bound to ASC1, which maintains ASC1 in a nonufmylated state. In the presence of E2, ERα bound ASC1 and displaced UfSP2, leading to ASC1 ufmylation. Polyufmylation of ASC1 enhanced association of p300, SRC1, and ASC1 at promoters of ERα target genes. ASC1 overexpression or UfSP2 knockdown promoted ERα-mediated tumor formation in vivo, which could be abrogated by treatment with the anti-breast cancer drug tamoxifen. In contrast, expression of ufmylation-deficient ASC1 mutant or knockdown of the UFM1-activating E1 enzyme UBA5 prevented tumor growth. These findings establish a role for ASC1 ufmylation in breast cancer development by promoting ERα transactivation.

USP47 and C terminus of Hsp70-interacting protein (CHIP) antagonistically regulate katanin-p60-mediated axonal growth.

Katanin is a heterodimeric enzyme that severs and disassembles microtubules. While the p60 subunit has the enzyme activity, the p80 subunit regulates the p60 activity. The microtubule-severing activity of katanin plays an essential role in axonal growth. However, the mechanisms by which neuronal cells regulate the expression of katanin-p60 remains unknown. Here we showed that USP47 and C terminus of Hsp70-interacting protein (CHIP) antagonistically regulate the stability of katanin-p60 and thereby axonal growth. USP47 was identified as a katanin-p60-specific deubiquitinating enzyme for its stabilization. We also identified CHIP as a ubiquitin E3 ligase that promotes proteasome-mediated degradation of katanin-p60. Moreover, USP47 promoted axonal growth of cultured rat hippocampal neurons, whereas CHIP inhibited it. Significantly, treatment with basic fibroblast growth factor (bFGF), an inducer of axonal growth, increased the levels of USP47 and katanin-p60, but not CHIP. Consistently, bFGF treatment resulted in a marked decrease in the level of ubiquitinated katanin-p60 and thereby in the promotion of axonal growth. On the other hand, the level of USP47, but not CHIP, decreased concurrently with that of katanin-p60 as axons reached their target cells. These results indicate that USP47 plays a crucial role in the control of axonal growth during neuronal development by antagonizing CHIP-mediated katanin-p60 degradation.

Chemosensitivity is controlled by p63 modification with ubiquitin-like protein ISG15.

Identification of the cellular mechanisms that mediate cancer cell chemosensitivity is important for developing new cancer treatment strategies. Several chemotherapeutic drugs increase levels of the posttranslational modifier ISG15, which suggests that ISGylation could suppress oncogenesis. However, how ISGylation of specific target proteins controls tumorigenesis is unknown. Here, we identified proteins that are ISGylated in response to chemotherapy. Treatment of a human mammary epithelial cell line with doxorubicin resulted in ISGylation of the p53 family protein p63. An alternative splice variant of p63, ΔNp63α, suppressed the transactivity of other p53 family members, and its expression was abnormally elevated in various human epithelial tumors, suggestive of an oncogenic role for this variant. We showed that ISGylation played an essential role in the downregulation of ΔNp63α. Anticancer drugs, including doxorubicin, induced ΔNp63α ISGylation and caspase-2 activation, leading to cleavage of ISGylated ΔNp63α in the nucleus and subsequent release of its inhibitory domain to the cytoplasm. ISGylation ablated the ability of ΔNp63α to promote anchorage-independent cell growth and tumor formation in vivo as well to suppress the transactivities of proapoptotic p53 family members. These findings establish ISG15 as a tumor suppressor via its conjugation to ΔNp63α and provide a molecular rationale for therapeutic use of doxorubicin against ΔNp63α-mediated cancers.

ISG15 modification of filamin B negatively regulates the type I interferon-induced JNK signalling pathway.

Interferon (IFN)-induced signalling pathways have essential functions in innate immune responses. In response to type I IFNs, filamin B tethers RAC1 and a Jun N-terminal kinase (JNK)-specific mitogen-activated protein kinase (MAPK) module--MEKK1, MKK4 and JNK--and thereby promotes the activation of JNK and JNK-mediated apoptosis. Here, we show that type I IFNs induce the conjugation of filamin B by interferon-stimulated gene 15 (ISG15). ISGylation of filamin B led to the release of RAC1, MEKK1 and MKK4 from the scaffold protein and thus to the prevention of sequential activation of the JNK cascade. By contrast, blockade of filamin B ISGylation by substitution of Lys 2467 with arginine or by knockdown of ubiquitin-activating enzyme E1-like (UBEL1) prevented the release of the signalling molecules from filamin B, resulting in persistent promotion of JNK activation and JNK-mediated apoptosis. These results indicate that filamin B ISGylation acts as a negative feedback regulatory gate for the desensitization of type I IFN-induced JNK signalling.

Filamin B serves as a molecular scaffold for type I interferon-induced c-Jun NH2-terminal kinase signaling pathway.

Type I interferons (IFNs) activate Janus tyrosine kinase-signal transducer and activator of transcription pathway for exerting pleiotropic biological effects, including antiviral, antiproliferative, and immunomodulatory responses. Here, we demonstrate that filamin B functions as a scaffold that links between activated Rac1 and a c-Jun NH(2)-terminal kinase (JNK) cascade module for mediating type I IFN signaling. Filamin B interacted with Rac1, mitogen-activated protein kinase kinase kinase 1, mitogen-activated protein kinase kinase 4, and JNK. Filamin B markedly enhanced IFNalpha-dependent Rac1 activation and the sequential activation of the JNK cascade members. Complementation assays using M2 melanoma cells revealed that filamin B, but not filamin A, is required for IFNalpha-dependent activation of JNK. Furthermore, filamin B promoted IFNalpha-induced apoptosis, whereas short hairpin RNA-mediated knockdown of filamin B prevented it. These results establish a novel function of filamin B as a molecular scaffold in the JNK signaling pathway for type I IFN-induced apoptosis, thus providing the biological basis for antitumor and antiviral functions of type I IFNs.