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

ABSTRACT: 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, ?Np63?, 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 ?Np63?. Anticancer drugs, including doxorubicin, induced ?Np63? ISGylation and caspase-2 activation, leading to cleavage of ISGylated ?Np63? in the nucleus and subsequent release of its inhibitory domain to the cytoplasm. ISGylation ablated the ability of ?Np63? 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 ?Np63? and provide a molecular rationale for therapeutic use of doxorubicin against ?Np63?-mediated cancers.

SUBMITTER: Jeon YJ

PROVIDER: S-EPMC3386819 | biostudies-other | 2012 Jul

REPOSITORIES: biostudies-other

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

Jeon Young Joo YJ Jo Mi Gyeong MG Yoo Hee Min HM Hong Se-Hoon SH Park Jung-Mi JM Ka Seung Hyeun SH Oh Kyu Hee KH Seol Jae Hong JH Jung Yong Keun YK Chung Chin Ha CH

The Journal of clinical investigation 20120618 7

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 ...[more]

PMID: 22706304

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Project description:The ubiquitin-like protein ISG15 (interferon-stimulated gene of 15 kDa) is strongly induced by type I interferons and displays antiviral activity. As other ubiquitin-like proteins (Ubls), ISG15 is post-translationally conjugated to substrate proteins by an isopeptide bond between the C-terminal glycine of ISG15 and the side chains of lysine residues in the substrates (ISGylation). ISG15 consists of two ubiquitin-like domains that are separated by a hinge region. In many orthologs, this region contains a single highly reactive cysteine residue. Several hundred potential substrates for ISGylation have been identified but only a few of them have been rigorously verified. In order to investigate the modification of several ISG15 substrates, we have purified ISG15 conjugates from cell extracts by metal-chelate affinity purification and immunoprecipitations. We found that the levels of proteins modified by human ISG15 can be decreased by the addition of reducing agents. With the help of thiol blocking reagents, a mutational analysis and miRNA mediated knock-down of ISG15 expression, we revealed that this modification occurs in living cells via a disulphide bridge between the substrates and Cys78 in the hinge region of ISG15. While the ISG15 activating enzyme UBE1L is conjugated by ISG15 in the classical way, we show that the ubiquitin conjugating enzyme Ubc13 can either be classically conjugated by ISG15 or can form a disulphide bridge with ISG15 at the active site cysteine 87. The latter modification would interfere with its function as ubiquitin conjugating enzyme. However, we found no evidence for an ISG15 modification of the dynamin-like GTPases MxA and hGBP1. These findings indicate that the analysis of potential substrates for ISG15 conjugation must be performed with great care to distinguish between the two types of modification since many assays such as immunoprecipitation or metal-chelate affinity purification are performed with little or no reducing agent present.

Dataset Information

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

Publications

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

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