Modification of PCNA by ISG15 plays a crucial role in termination of error-prone translesion DNA synthesis.

In response to DNA damage, PCNA is mono-ubiquitinated and triggers translesion DNA synthesis (TLS) by recruiting polymerase-η. However, it remained unknown how error-prone TLS is turned off after DNA lesion bypass to prevent mutagenesis. Here we showed that ISG15 modification (ISGylation) of PCNA plays a key role in TLS termination. Upon UV irradiation, EFP, an ISG15 E3 ligase, bound to mono-ubiquitinated PCNA and promoted its ISGylation. ISGylated PCNA then tethered USP10 for deubiquitination and in turn the release of polymerase-η from PCNA. Eventually, PCNA was deISGylated by UBP43 for reloading of replicative DNA polymerases and resuming normal DNA replication. However, ISGylation-defective Lys-to-Arg mutations in PCNA or knockdown of any of ISG15, EFP, or USP10 led to persistent recruitment of mono-ubiquitinated PCNA and polymerase-η to nuclear foci, causing an increase in mutation frequency. These findings establish a crucial role of PCNA ISGylation in termination of error-prone TLS for preventing excessive mutagenesis.

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.