Study on the CUE Domain Containing 2 (CUEDC2) negative regulation of NF-kB activity through inhibition of IkBa kinase phosphorylation
Li Huiyan
ABSTRACT
CUEDC2 is revealed by bioinformatics analysis as a CUE domain-contained protein, whose function is unknown. Identified as ubiquitin binding motifs, the CUE domains are small, moderately-conserved domains of about 40 amino acid residues that are found in a variety of eukaryotic proteins. CUE domains interact with both mono- and poly-ubiquitin, and have a dual role in mono- and poly-ubiquitin recognition as well as in facilitating intramolecular monoubiquitination. Recently, we demonstrate that CUEDC2 interacts with progesterone receptor and promotes progesterone-induced PR degradation by the ubiquitin-proteasome pathway. These provide an important insight into the function of CUEDC2 in breast cancer proliferation. In this study, we performed yeast two-hybrid screens to identify CUEDC2-interacting proteins and preliminary analysis on its biological function in NF-κB signaling, to help us understand its mechanisms.
A key event leading connecting extracellular stimuli to the activation of NF-kB is the regulation of IKK activity. It is believed that IKK activity is controlled by the opposing action of kinases and phosphatases. IKK is composed of two highly homologous catalytic subunits, IKKa and IKKb, and a regulatory subunit NF-kB essential modulator (NEMO, also called IKKg). After TNF or IL-1 treatment, IKK is recruited to receptor signaling complexes, where the two catalytic subunits, IKKa and IKKb are activated by phosphorylation. In the case of TNF or IL-1 treatment, IKK is activated promptly, but this elevated activity is reduced to basal amounts shortly after stimulation. Despite extensive study of the mechanisms of IKK activation, the molecular machinery involved in the deactivation of IKK remains poorly understood.
The transcription factor NF-kB is a critical regulator of diverse cytokine-mediated cellular responses and plays a key role in cell survival through inducing the expression of antiapoptotic genes. In most cells, the NF-kB proteins are normally inactive because they are sequestered in the cytoplasm by a family of inhibitory proteins known as IkBs. Extracellular stimuli, including cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1), lead to the activation of the IkB kinase complex (IKK), which phosphorylates and ubiquitinates IkB proteins and promotes their subsequent degradation by the proteasome. This process allows translocation of NF-kB proteins from the cytoplasm to the nucleus, where they activate expression of NF-kB-regulated genes.
The GADD34 (growth arrest and DNA damage) protein was initially reported to be induced by various types of cellular stress and DNA damage such as UV irradiation and unfolded proteins. It is now known that GADD34 is a regulatory subunit of the protein phosphatase 1 (PP1) holoenzyme. In recent study, the protein phosphatase family includes several isoforms, such as PP2A, PP2B and PP2C. These phosphatases have their regulatory suunits. The regulatory subunit is necessary for the activation of protein phosphatase.
Here, we demonstrate that CUE domain containing 2 (CUEDC2), whose function was previously unknown, interacts with IKKa/b and represses NF-κB signaling by reducing IKK phosphorylation. Cytokine-induced activation of the IkB kinases IKKa and IKKb is a key step in the activation of the NF-kB pathway. Thus, precise control of IKK phosphorylation is a crucial component of NF-kB signaling. Specific knockdown of CUEDC2 by siRNA results in increased TNF-induced NF-kB activation, while enforced expression of CUEDC2 sensitizes cells to apoptotic stimuli such as TNF. Interestingly, we also found that CUEDC2 interacts with GADD34, a regulatory subunit of protein phosphatase 1 (PP1). Moreover, we demonstrated that inhibition of NF-kB activation by CUEDC2 is mediated, at least in part, by an IKK-CUEDC2-PP1 complex that exists endogenously. siRNA silencing of CUEDC2 revealed that it is required for formation of this complex, indicating CUEDC2 acts as an adaptor protein that targets IKK for dephosphorylation. Therefore, we have uncovered a potent inhibitor of NF-kB signaling that controls IKK activity, providing important insight into the function of CUEDC2 in NF-kB signaling.
Key words: CUDEC2, GADD34, PP1, IKK, NF-kB
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