Ubiquitination of the transcription factor c-MAF is mediated by multiple lysine residues
Introduction
The Maf oncoproteins belong to the family of basic leucine zipper transcription factors including c-fos and c-jun (Vinson et al., 2002). Eight MAF proteins have been reported, of which seven are expressed in humans, including MafA (Benkhelifa et al., 1998), MafB (Kataoka et al., 1994), c-MAF (Nishizawa et al., 1989), Nrl (Liu et al., 1996), MafF (Fujiwara et al., 1993), MafG (Kataoka et al., 1995), and MafK (Fujiwara et al., 1993). These proteins can be classified into two groups, the large MAF and the small MAF subfamilies. c-MAF is the cellular form of the first identified viral Maf transcription factor and belongs to the large MAF family (Nishizawa et al., 1989). c-MAF contains a DNA binding domain and a transactivation domain, and therefore it can form a hetero- or homo-dimer to modulate transactivation of several important genes, including cyclin D2, integrin β7, CCR1, and ARK5 (Hurt et al., 2004, Suzuki et al., 2005). These genes contain the Maf-recognition element (MARE) in their promoter region (Hurt et al., 2004, Suzuki et al., 2005, Morito et al., 2006, Mao et al., 2007, Robbiani et al., 2007). c-MAF is usually found in the embryonic stage where it regulates several cellular processes, including embryonic lens fiber cell development (Ring et al., 2000), chondrocyte terminal differentiation (MacLean et al., 2003), erythropoiesis in fetal liver (Kusakabe et al., 2011), cell differentiation (Ho et al., 1998), as well as touch receptor development (Wende et al., 2012). In adult humans, c-MAF is frequently seen in multiple myeloma (MM) and T-cell lymphoma, especially in cells with chromosomal translocation t(14:16) (Chesi et al., 1998), and dysregulation of c-MAF is associated with poor prognosis of these diseases.
As a transcription factor, c-MAF function can be modified by several important chemical modifications, including phosphorylation (Rocques et al., 2007), SUMOylation (Leavenworth et al., 2009), and ubiquitination (Mao et al., 2007). Phosphorylation and SUMOylation regulate c-MAF function (Rocques et al., 2007, Leavenworth et al., 2009), whereas ubiquitination leads to c-MAF degradation in the proteasome (Mao et al., 2007). Ubiquitination typically occurs at one or more among various functionally redundant lysine residues in target proteins. However, the molecular details of c-MAF ubiquitination remain unknown. In the present study, we performed a lysine mutation screen and found that not a single but multiple lysine residues are required for c-MAF ubiquitination.
Section snippets
Cell culture
Human embryonic kidney cell line (HEK293T) and cervical cancer cell line HeLa were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% of fetal bovine serum, glutamine and antibiotics. MM cells RPMI-8226 and LP1 were generously provided from Dr. Aaron Schimmer, University of Toronto. MM cells were cultured in Iscove's modified Dulbecco's media (IMDM).
Lysine mutation constructs
The human transcription factor c-MAF cDNA was cloned into pcDNA3.1 vector with an HA tag generously provided by Dr. Sudan He,
c-MAF is degraded via the ubiquitin–proteasome system (UPS)
Our previous study found that anti-myeloma drugs dexamethasone and prednisolone can induce c-MAF degradation in proteasomes (Mao et al., 2007, Mao et al., 2008). To confirm this finding, endogenous c-MAF catabolism was studied in c-MAF expressing MM cell lines RPMI-8226 and LP1 cells by treatment with cycloheximide (CHX), a pan-inhibitor of protein synthesis, alone or in combination with bortezomib (BZ), a proteasome inhibitor approved for MM treatment. Immunoblotting assays revealed that c-MAF
Discussion
In this report, we demonstrated that c-MAF can be modified by ubiquitination and this ubiquitination is mediated by multiple lysine residues, of which K85 and K350 are probably dominant but not the only two acceptor sites for c-MAF ubiquitination.
As a transcription factor, the function of c-MAF is strictly regulated by multiple post-translational modifications, such as phosphorylation and SUMOylation. It has shown that phosphorylation of c-MAF by GSK3 kinase enhances its transforming activity (
Conflict of interest disclosure
All authors disclosed no relevant financial conflict of interest.
Acknowledgements
The authors thanked Dr. Shi-Chuen Miaw from National Taiwan University for his kind gift of mouse c-MAF plasmid which was used in our preliminary study. The authors thanked Dr. Sudan He, Cyrus Tang Hematology Center, Soochow University, for her critical discussion and comments.
This study was partly supported by the National Natural Science Foundation of China (81071935, 81320108023, 81101795, 81272632), the National College Student's Innovative and Experimental Project (5731503011), The
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These authors contributed equally to this study.