Ubiquitin (Ub) is a highly conserved 76 amino-acid protein found throughout eukaryotic cells. A vast number of cellular processes, including targeted protein degradation, cell cycle progression, DNA repair, protein trafficking, inflammatory response, virus budding, and receptor endocytosis, are regulated by Ub-mediated signalling; where the target protein is tagged by single or multi-monomeric Ub (monomeric Ub attached to multiple sites on the substrate) or a polymeric chain of Ubs (Fushman et al., 2010). This post-translational modification is tightly controlled by an enzymatic cascade involving several enzymes (E1, E2, and E3) and occurs through either an isopeptide bond between the C-terminal Glycyl residue of Ub and the epsilon amino group of a Lysyl residue on a target protein or through a peptide bond between the C-terminal Glycyl residue of Ub and the N-terminal amine on a further Ub. In the former (isopeptide bond-linked) case the substrate protein may either be ubiquitin itself - thus leading to the generation of poly-ubiquitin chains - or another target protein (Fushman et al., 2010). Thus, ubiquitin can be attached to a substrate either as a monomer or as a poly-ubiquitin chain. Further - depending on their linkage type (M1, K6, K11, K27, K29, K33, K48 and K63 linked) - the Ub chains can take different structural forms. Chains containing all eight possible Ub linkages have been found in living cells and different ubiquitin chain types may encode different biological signals, allowing this single protein to mediate many diverse functions (Komander 2009; Weeks et al., 2009; Walczak et al., 2012). The functionality of Ub chains is most commonly associated with their attachment to substrate proteins but there is also evidence that they may also play a role in cellular signalling as free chains (Braten et al., 2012). A mass spectrometry-based study found that K33 linkages account for just 4% of all yeast ubiquitin-ubiquitin linkages. The relative abundance of the other linkages were K6 (11%), K11 (28%), K27 (9%), K29 (3%), K48 (29%) and K63 (16%) (Xu et al., 2009). AMPK (AMP-activated protein kinase)-related kinases, NUAK1 and MARK4 have been shown to be polyubiquitylated in vivo and interact with the deubiquitylase enzyme USP9x. These AMPK-related kinases regulate cell polarity as well as proliferation and are known to be activated by the LKB1-tumour suppressor kinase. NUAK1 and MARK4 have been shown to be conjugated to K29 and/or K33 ubiquitin chains rather than the more common Lys48/Lys63. Ubiquitylation of NUAK1 and MARK4 does not control their stability, but instead inhibits their phosphorylation and activation by LKB1 (Al-Hakim et al., 2008). A recent study characterizes K33-linked polyubiquitylation of T cell receptor-zeta (TCR-ζ) and indicates the important role of unconventional cell surface receptor ubiquitylation in regulating T cell activation in a proteolysis-independent manner (Huang et al., 2010).
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