ß-TrCP Restricts Lipopolysaccharide (LPS)-Induced Activation of TRAF6-IKK Pathway Upstream of IκBα Signaling.

ß transducin repeat-containing protein (ß-TrCP) is a Skp1-Cul1-F-box ubiquitin ligase, which plays important roles in controlling numerous signaling pathways. Notably, ß-TrCP induces ubiquitination and degradation of inhibitor of NF-κB (IκBα), thus triggering activation of NF-κB signaling. Here, we unexpectedly find that ß-TrCP restricts TRAF6-IKK signaling upstream of IκBα induced by lipopolysaccharide (LPS). In LPS-Toll-like receptor 4 (TLR4) pathway, protein kinase D1 (PKD1) is essential for activation of TRAF6-IKK-IκBα signaling including TRAF6 ubiquitination, IKK phosphorylation and subsequent IκBα degradation. We found that LPS promotes binding of ß-TrCP to PKD1, and results in downregulation of PKD1 and recovery of IκBα protein level. Knockdown of ß-TrCP blocks LPS-induced downregulation of PKD1. Supplement of enough PKD1 in cells inhibits recovery of IκBα protein levels during LPS stimulation. Furthermore, we demonstrate that ß-TrCP inhibits LPS-induced TRAF6 ubiquitination and IKK phosphorylation. Taken together, our findings identify ß-TrCP as an important negative regulator for upstream signaling of IκBα in LPS pathway, and therefore renew the understanding of the roles of ß-TrCP in regulating TLRs inflammatory signaling.

Amphiregulin Confers Regulatory T Cell Suppressive Function and Tumor Invasion via the EGFR/GSK-3ß/Foxp3 Axis.

Previous studies mainly focused on the role of the epidermal growth factor receptor (EGFR) in tumor cells, whereas the effects of the EGFR on immune responses has not been determined. Our study shows that the EGFR signaling pathway play a role in the regulation of regulatory T cells (Treg cells) in cancer patients. The EGF-like growth factor Amphiregulin (AREG) protein was frequently up-regulated in a tissue microarray, which was associated with worse overall survival. Additionally, in sera, tissue specimens, and effusions of lung or gastric cancer patients, up-regulated AREG protein enhanced the suppressive function of Treg cells. AREG maintained the Treg cell suppressive function via the EGFR/GSK-3ß/Foxp3 axis in vitro and in vivo Furthermore, inhibition of EGFR by the tyrosine kinase inhibitor gefitinib restored the activity of GSK-3ß and attenuated Treg cell function. ß-TrCP was involved in GSK-3ß-mediated Foxp3 degradation, and mass spectrometry identified Lys356 as the ubiquitination site of Foxp3 by ß-TrCP. These findings demonstrate the posttranslational regulation of Foxp3 expression by AREG in cancer patients through AREG/EGFR/GSK-3ß signaling, which could lead to Foxp3 protein degradation in Treg cells and a potential therapeutic target for cancer treatment.

Opposing activities of the Ras and Hippo pathways converge on regulation of YAP protein turnover.

Cancer genomes accumulate numerous genetic and epigenetic modifications. Yet, human cellular transformation can be accomplished by a few genetically defined elements. These elements activate key pathways required to support replicative immortality and anchorage independent growth, a predictor of tumorigenesis in vivo. Here, we provide evidence that the Hippo tumor suppressor pathway is a key barrier to Ras-mediated cellular transformation. The Hippo pathway targets YAP1 for degradation via the ßTrCP-SCF ubiquitin ligase complex. In contrast, the Ras pathway acts oppositely, to promote YAP1 stability through downregulation of the ubiquitin ligase complex substrate recognition factors SOCS5/6. Depletion of SOCS5/6 or upregulation of YAP1 can bypass the requirement for oncogenic Ras in anchorage independent growth in vitro and tumor formation in vivo. Through the YAP1 target, Amphiregulin, Ras activates the endogenous EGFR pathway, which is required for transformation. Thus, the oncogenic activity of Ras(V12) depends on its ability to counteract Hippo pathway activity, creating a positive feedback loop, which depends on stabilization of YAP1.

ß-TrCP-mediated IRAK1 degradation releases TAK1-TRAF6 from the membrane to the cytosol for TAK1-dependent NF-κB activation.

Interleukin-1 (IL-1) receptor-associated kinase (IRAK1) is phosphorylated, ubiquitinated, and degraded upon IL-1 stimulation. IRAK1 can be ubiquitinated through both K48- and K63-linked polyubiquitin chains upon IL-1 stimulation. While the Pellino proteins have been shown to meditate K63-linked polyubiquitination on IRAK1, the E3 ligase for K48-linked ubiquitination of IRAK1 has not been identified. In this study, we report that the SCF (Skp1-Cullin1-F-box)-ß-TrCP complex functions as the K48-linked ubiquitination E3 ligase for IRAK1. IL-1 stimulation induced the interaction of IRAK1 with Cullin1 and ß-TrCP. Knockdown of ß-TrCP1 and ß-TrCP2 attenuated the K48-linked ubiquitination and degradation of IRAK1. Importantly, ß-TrCP deficiency abolished the translocation TAK1-TRAF6 complex from the membrane to the cytosol, resulting in a diminishment of the IL-1-induced TAK1-dependent pathway. Taken together, these results implicate a positive role of ß-TrCP-mediated IRAK1 degradation in IL-1-induced TAK1 activation.

Rotavirus nonstructural protein 1 antagonizes innate immune response by interacting with retinoic acid inducible gene I.

BACKGROUND: The nonstructural protein 1 (NSP1) of rotavirus has been reported to block interferon (IFN) signaling by mediating proteasome-dependent degradation of IFN-regulatory factors (IRFs) and (or) the ß-transducin repeat containing protein (ß-TrCP). However, in addition to these targets, NSP1 may subvert innate immune responses via other mechanisms. RESULTS: The NSP1 of rotavirus OSU strain as well as the IRF3 binding domain truncated NSP1 of rotavirus SA11 strain are unable to degrade IRFs, but can still inhibit host IFN response, indicating that NSP1 may target alternative host factor(s) other than IRFs. Overexpression of NSP1 can block IFN-ß promoter activation induced by the retinoic acid inducible gene I (RIG-I), but does not inhibit IFN-ß activation induced by the mitochondrial antiviral-signaling protein (MAVS), indicating that NSP1 may target RIG-I. Immunoprecipitation experiments show that NSP1 interacts with RIG-I independent of IRF3 binding domain. In addition, NSP1 induces down-regulation of RIG-I in a proteasome-independent way. CONCLUSIONS: Our findings demonstrate that inhibition of RIG-I mediated type I IFN responses by NSP1 may contribute to the immune evasion of rotavirus.