SYT-SSX1 (synovial sarcoma translocated) regulates PIASy ligase activity to cause overexpression of NCOA3 protein

Chromosomal translocations are a major source of genetic abnormalities causally linked to certain malignancies. Synovial sarcoma is an aggressive soft tissue tumor characterized by a chromosomal translocation between chromosome 18 and X, generating oncoproteins such as SYT-SSX1 and SYT-SSX2. The molecular mechanism underlying the oncogenic potential of SYT-SSX1/2 is not clear. Here we show that SYT-SSX1 leads to up-regulation of NCOA3, a protein critical for the formation of various cancers. The increase of NCOA3 is essential for SYT-SSX1-mediated synovial sarcoma formation. SYT-SSX1 does so by increasing the sumoylation of NCOA3 through interaction with a SUMO E3 ligase, PIASy, as well as the sumoylation of NEMO. NEMO has also been shown to physically interact with NCOA3. Increased sumoylation of NCOA3 leads to its increased steady state level and nuclear localization. Our findings represent the first example that an oncoprotein directly regulates substrate modification by a SUMO E3 ligase, and leads to overexpression of a protein essential for tumor formation. Such a mechanistic finding provides an opportunity to design specific therapeutic interventions to treat synovial sarcoma.     

PIDD mediates NF-kappaB activation in response to DNA damage

Activation of NF-kappaB following genotoxic stress allows time for DNA-damage repair and ensures cell survival accounting for acquired chemoresistance, an impediment to effective cancer therapy. Despite this clinical relevance, little is known about pathways that enable genotoxic-stress-induced NF-kappaB induction. Previously, we reported a role for the p53-inducible death-domain-containing protein, PIDD, in caspase-2 activation and apoptosis in response to DNA damage. We now demonstrate that PIDD plays a critical role in DNA-damage-induced NF-kappaB activation. Upon genotoxic stress, a complex between PIDD, the kinase RIP1, and a component of the NF-kappaB-activating kinase complex, NEMO, is formed. PIDD expression enhances genotoxic-stress-induced NF-kappaB activation through augmented sumoylation and ubiquitination of NEMO. Depletion of PIDD and RIP1, but not caspase-2, abrogates DNA-damage-induced NEMO modification and NF-kappaB activation. We propose that PIDD acts as a molecular switch, controlling the balance between life and death upon DNA damage.     

Interleukin-1-induced NF-kappaB activation is NEMO-dependent but does not require IKKbeta

Activation of NF-kappaB by the pro-inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) requires the IkappaB kinase (IKK) complex, which contains two kinases named IKKalpha and IKKbeta and a critical regulatory subunit named NEMO. Although we have previously demonstrated that NEMO associates with both IKKs, genetic studies reveal that only its interaction with IKKbeta is required for TNF-induced NF-kappaB activation. To determine whether NEMO and IKKalpha can form a functional IKK complex capable of activating the classical NF-kappaB pathway in the absence of IKKbeta, we utilized a panel of mouse embryonic fibroblasts (MEFs) lacking each of the IKK complex subunits. This confirmed that TNF-induced IkappaBalpha degradation absolutely requires NEMO and IKKbeta. In contrast, we consistently observed intact IkappaBalpha degradation and NF-kappaB activation in response to IL-1 in two separate cell lines lacking IKKbeta. Furthermore, exogenously expressed, catalytically inactive IKKbeta blocked TNF- but not IL-1-induced IkappaBalpha degradation in wild-type MEFs, and reconstitution of IKKalpha/beta double knockout cells with IKKalpha rescued IL-1- but not TNF-induced NF-kappaB activation. Finally, we have shown that incubation of IKKbeta-deficient MEFs with a cell-permeable peptide that blocks the interaction of NEMO with the IKKs inhibits IL-1-induced NF-kappaB activation. Our results therefore demonstrate that NEMO and IKKalpha can form a functional IKK complex that activates the classical NF-kappaB pathway in response to IL-1 but not TNF. These findings further suggest NEMO differentially regulates the fidelity of the IKK subunits activated by distinct upstream signaling pathways.     

Mechanism Underlying IκB Kinase Activation Mediated by the Linear Ubiquitin Chain Assembly Complex

The linear ubiquitin chain assembly complex (LUBAC) ligase, consisting of HOIL-1L, HOIP, and SHARPIN, specifically generates linear polyubiquitin chains. LUBAC-mediated linear polyubiquitination has been implicated in NF-κB activation. NEMO, a component of the IκB kinase (IKK) complex, is a substrate of LUBAC, but the precise molecular mechanism underlying linear chain-mediated NF-κB activation has not been fully elucidated. Here, we demonstrate that linearly polyubiquitinated NEMO activates IKK more potently than unanchored linear chains. In mutational analyses based on the crystal structure of the complex between the HOIP NZF1 and NEMO CC2-LZ domains, which are involved in the HOIP-NEMO interaction, NEMO mutations that impaired linear ubiquitin recognition activity and prevented recognition by LUBAC synergistically suppressed signal-induced NF-κB activation. HOIP NZF1 bound to NEMO and ubiquitin simultaneously, and HOIP NZF1 mutants defective in interaction with either NEMO or ubiquitin could not restore signal-induced NF-κB activation. Furthermore, linear chain-mediated activation of IKK2 involved homotypic interaction of the IKK2 kinase domain. Collectively, these results demonstrate that linear polyubiquitination of NEMO plays crucial roles in IKK activation and that this modification involves the HOIP NZF1 domain and recognition of NEMO-conjugated linear ubiquitin chains by NEMO on another IKK complex.     

Protein interactions in the sumoylation cascade: lessons from X-ray structures

Sumoylation is a multi-step protein modification reaction in which SUMO (small ubiquitin-like modifier) proteins are covalently attached to lysine residues of substrate proteins. Here, we compare the sequences and structures of modifiers and enzymes involved in sumoylation with those of the related ubiquitination and neddylation cascades. By using available structural data on modifier/enzyme/substrate interactions, we discuss and model sumoylation complexes that include SUMO-1 and the E1 and E2 enzymes Aos1-uba2 and ubc9, or SUMO-1 and E2 together with the E3 ligase RanBP2 and its substrate RanGAP1. Their comparison provides insight into the protein interactions underlying sumoylation, and suggests how SUMO proteins may be translocated between enzymes during the various steps of the protein modification reaction.