Neddylation pathway regulates T-cell function by targeting an adaptor protein Shc and a protein kinase Erk signaling

Abstract

NEDD8 (neural precursor cell expressed, developmentally downregulated 8) is a ubiquitin-like molecule whose action on modifying protein substrates is critical in various cellular functions but whose importance in the immune system is not well understood. Here we investigated the role of protein neddylation in regulating T-cell function using an in vivo knockdown technique. We found that reduced expression of Ubc12 in CD4 + T cells led to impaired T-cell receptor/CD28-induced proliferation and cytokine production both in vitro and in vivo, accompanied by reduced Erk activation. These findings were recapitulated by treatment with MLN4924, an inhibitor of NEDD8-activating enzyme. Furthermore, Shc, an adaptor molecule between antigen receptors and the Ras/Erk pathway, was identified as a target for neddylation. Importantly, mice adoptively transferred with Ubc12 knockdown CD4 + T cells showed markedly ameliorated allergic responses. This study thus identifies an important role for protein neddylation in T-cell function, which may serve as a therapeutic target for inflammatory diseases.allergy | signal transduction | posttranslational modification E ngagement of T-cell antigen receptor (TCR) and costimulatory molecules leads to the activation of CD4 + T cells through several signaling pathways, which ultimately induces proliferation, cytokine production, and differentiation into different subsets of T helper type (Th) cells and memory cells (1). Notably, posttranslational modification by ubiquitin and ubiquitin-like proteins (UBLs) has emerged as a critical mechanism regulating T-cell function (2, 3). UBLs compose a diverse group of evolutionarily conserved small proteins, and attachment of different UBLs to a target has different biological consequences (4). Among the UBLs, NEDD8 (neural precursor cell expressed, developmentally down-regulated 8) is covalently attached to lysines in substrate proteins by a series of enzymatic reactions similar to those involved in ubiquitination, including the E1 (Nae1/Uba3) and E2 (Ubc12) reactions (5). The well-characterized substrates of NEDD8 modification are the cullin subunits of cullin-RING ubiquitin E3 ligases, and this modification of cullins is critical for the transfer of ubiquitin from recruited E2-ubiquitin to the substrate (6, 7). The importance of this pathway was further underscored by the recent development of a pharmacologic inhibitor of Nae1, MLN4924, which inhibits cullin neddylation, resulting in substrate accumulation and cancer cell apoptosis by the deregulation of S-phase DNA synthesis, and offers great promise for the treatment of cancer (8-10).In the present study, we investigated whether the NEDD8 pathway is involved in CD4 + T-cell function. Using siRNAmediated depletion of the NEDD8 system in vivo and the NEDD8-activating enzyme inhibitor MLN4924, we found that the NEDD8 pathway is required for TCR-induced proliferation and activation. Interestingly, our results demonstrate that Erk activation by TCR stimulation requires an intact neddylation system. Dif...

Inhibition of atherogenesis by the COP9 signalosome subunit 5 in vivo

“…Also, MLN4924 reduced MAPK signaling in inflammatory-elicited macrophages. These data are in accord with reports of MLN4924 effects on HIF-1α stabilization in microvascular endothelial cells (26) and MAPK activation in T cells, the latter linked to MLN-induced stabilization of SHC, a negative regulator of ERK signaling that was identified as a target for NEDDylation (67). Interestingly, the pathways identified in our study to be affected by MLN are linked with each other, e.g., reduced NF-κB activity, as observed upon MLN4924, was inversely linked with M2 polarization (68), and Arginase-1 has been reported to be regulated by HIF-2α (69), which is stabilized by NEDDylation (58).…”

MLN4924, an NAE inhibitor, suppresses AKT and mTOR signaling via upregulation of REDD1 in human myeloma cells

“…12,13 Through its effects on protein neddylation, MLN4924 is known to have many effects including induction of DNA rereplication, apoptosis, autophagy, cell growth inhibition through p21-dependent senescence, and regulation of T-cell-mediated inflammatory response. [14][15][16][17][18] However, from a plasma cell biology perspective, MLN4924 can selectively inhibit the turnover of specific proteins which are targeted by CRLs.…”

SAG/Rbx2-Dependent Neddylation Regulates T-Cell Responses

“…Recently, Jin et al 20 have shown that the global inhibition of all CRL complexes through knocking down of Ubc12 inhibits neddylation and regulates T-cell functions. Our datadwhich used the pharmacological small-molecule inhibitor of neddylation, MLN4924, and a more targeted genetic approach of SAG À/À T cellsdconfirm and extend these observations.…”

“…8,18 Recent data have suggested that targeting neddylation with MLN4924 regulated the inflammatory functions of dendritic cells and macrophages, whereas inhibition of neddylation by knocking down Ubc12 mitigated CD4 T-cell responses. 1,19,20 Collectively, these studies point to an emerging role for neddylation in the regulation of immune cell subsets. However, the exclusive utilization of either the small molecule or the shRNA knockdown approaches does not rule out the possibility of potential off-target effects.…”