Negative regulation of NF-κB signaling in T lymphocytes by the ubiquitin-specific protease USP34
© Poalas et al.; licensee BioMed Central Ltd. 2013
Received: 8 February 2013
Accepted: 3 April 2013
Published: 16 April 2013
NF-κB is a master gene regulator involved in plethora of biological processes, including lymphocyte activation and proliferation. Reversible ubiquitinylation of key adaptors is required to convey the optimal activation of NF-κB. However the deubiquitinylases (DUBs), which catalyze the removal of these post-translational modifications and participate to reset the system to basal level following T-Cell receptor (TCR) engagement continue to be elucidated.
Here, we performed an unbiased siRNA library screen targeting the DUBs encoded by the human genome to uncover new regulators of TCR-mediated NF-κB activation. We present evidence that knockdown of Ubiquitin-Specific Protease 34 (USP34) selectively enhanced NF-κB activation driven by TCR engagement, similarly to siRNA against the well-characterized DUB cylindromatosis (CYLD). From a molecular standpoint, USP34 silencing spared upstream signaling but led to a more pronounced degradation of the NF-κB inhibitor IκBα, and culminated with an increased DNA binding activity of the transcription factor.
Collectively, our data unveils USP34 as a new player involved in the fine-tuning of NF-κB upon TCR stimulation.
KeywordsDUBs NF-κB Ubiquitinylation T-Cell receptor
Nuclear factor-κB (NF-κB) transcription factors initiate transcription of genes essential for mounting an adequate immune response . Ubiquitously expressed NF-κB heterodimers of Rel family proteins are normally sequestered in the cytosol of the cells by Inhibitors of NF-κB (IκBs) proteins . In lymphocytes, the ligation of antigen receptors assembles the so-called CBM complex that consists of the scaffold CARMA1 and the heterodimer BCL10/MALT1 . The CBM microenvironment drives oligomerized BCL10 and MALT1 to undergo K63-linked non-degradative ubiquitinylation [4–7]. This authorizes the recruitment and activation of the IκB kinase (IKK) complex that comprises two catalytic subunits (IKKα and IKKβ) and a regulatory subunit (NEMO, also called IKKγ) . IKK phosphorylation of IκBs precipitates their K48-linked ubiquitinylation and proteasomal elimination, and thereby allows NF-κB to translocate to the nucleus where it binds DNA and initiates transcription . NF-κB-dependent neosynthesis of IκBs subsequently drives NF-κB to shuttle back to the cytosol . Although reversible ubiquitinylation processes are central for T-cell receptor-(TCR)-mediated NF-κB activation, the deubiquitinylases (DUBs) in charge of trimming these poly-ubiquitin chains to ensure optimal signaling, as well as to reset the system to basal levels remain poorly defined . Thus far, two DUBs, namely cylindromatosis (CYLD) and A20 (also known as TNFAIP3), were demonstrated to negatively regulate antigen receptor signaling [9, 10]. Herein, we provide evidence that Ubiquitin-Specific Protease 34 (USP34) also contributes to the fine-tuning of NF-κB upon TCR engagement.
Almost 100 DUBs were identified in the human genome and yet, only a few have been ascribed a function . As for TCR signaling, the well studied A20 and CYLD thwart NF-κB at different levels . CYLD targets and inhibits the ubiquitin-dependent IKKβ kinase TAK1 and therefore prevents aberrant lymphocyte activation [18, 19], while A20 dampens NF-κB activity by trimming K63-ubiquitin chains attached to MALT1 [20, 21]. Our study now unveils USP34 as an additional negative regulator of NF-κB in lymphocytes. How USP34 tempers NF-κB activity remains unclear. In contrast to CYLD and A20, which target apical signaling [18, 20], USP34 rather seems to function downstream of the IKK complex. Given that USP34 does not bind to the NF-κB core components (our unpublished results), we favor a model in which USP34 impacts on the activity of a cytosolic co-activator to ensure IκBα fine-tuning [22, 23]. Alternatively, USP34 might also intervene in other checkpoints to control NF-κB signal outcome and intensity such as post-translational modifications, nuclear shift, or DNA three-dimensional structure [22, 24, 25]. Nevertheless, our data illustrates how various layers of control cooperate to ensure the fine-tuning of NF-κB following engagement of the TCR.
Extracellular signal-regulated kinases
Inhibitors of NF-κB
Mitogen-activated protein kinase
Nuclear factor of activated T-cells
phorbol 12-myristate 13-acetate
We thank V. Quesada and C. Lopez-Otín (Universidad de Oviedo, Spain) for providing USP34-CD plasmid, and D. Arnoult (INSERM U1014) for helpful discussions. This work was supported by grants from ANR JCJC, Fondation ARC, Fondation de France, Ligue Nationale contre le Cancer, and by Fondation pour la Recherche Médicale. KP and SMD are supported by fellowships from Université Paris Sud, and EMH by la Ligue contre le Cancer.
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