Background
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 and Walker, 2010). More recently the demonstration that ubiquitin itself can be modified through phosphorylation by the kinase PTEN Induced putative Kinase1 (PINK1) provides a major breakthrough linking the two most important signalling pathways in cells; phosphorylation and ubiquitylation (Kane et al., 2014; Kazlauskaite et al., 2014; Koyano et al., 2014). Parkin and PINK1, the two main proteins associated with Parkinson's Disease (PD) comprise a mitochondrial quality control pathway that promotes neuronal survival through autophagy of damaged mitochondria in a process known as mitophagy (Sauve and Gehring, 2014). The accumulation of PINK1 on depolarised or damaged mitochondria leads to the activation and translocation of Parkin to the outer mitochondrial membrane (OMM). Phosphorylation of Parkin by PINK1 at Ser65 located in its Ubl domain markedly increases the E3 ligase activity of Parkin resulting in ubiquitylation of proteins on the OMM, triggering selective mitophagy (Kondapalli et al., 2012; Spratt et al., 2013; Trempe et al., 2013; Wauer and Komander, 2013).
Several studies have revealed that ubiquitin is also a PINK1 substrate in this pathway where PINK1 directly phosphorylates ubiquitin on Ser65, a residue that is also shared by the Parkin Ubl domain (Kane et al., 2014; Kazlauskaite et al., 2014; Koyano et al., 2014). Parkin is activated by Ser65 phosphorylated ubiquitin in a manner which is independent of ubiquitin's ability to be conjugated to lysine residues on target proteins. The mechanism of Parkin priming and activation is thought to occur through a conformational change induced by PINK1 phosphorylation of Ser65 on Parkin followed by the binding of PINK1 Ser65 phosphorylated ubiquitin on the RING1 domain which optimises the ubiquitylation activity of Parkin Kazlauskaite et al., 2014; Koyano et al., 2014). Studies have also identified the presence of at least five phosphorylation sites in Parkin including Ser378, shown to be phosphorylated by Casein kinase 1 (CK 1) suggesting that further phosphorylation of Parkin may also act to regulate its ubiquitin ligase activity (Yamamoto et al., 2005). Phospho-ubiquitin may play other roles in regulating Parkin but more generally the identification of phospho-ubiquitin as a second messenger in signalling pathways could reveal the existence of ubiquitin phosphatases and lead to the discovery of additional kinase and ubiquitin related substrates and signalling functions (Sauve and Gehring, 2014).
Ubiquitin (pSer65) (Cat# 60-0202-050) is a phosphorylated synthetically made ubiquitin which may be used alongside Biotin-Ahx Ubiquitin (pSer65) (Cat# 60-0207-050) and the non-phosphorylated control Ubiquitin (synthetic) (Cat# 60-0200-050).
References
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Kane LA, Lazarou M, Fogel AI, Li Y, Yamano K, Sarraf SA, et al.(2014) PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity. J Cell Biol, 205, 143-153.
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Kondapalli C, Kazlauskaite A, Zhang N, Woodroof HI, Campbell DG, Gourlay R, et al. (2012) PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65. Open Biol, 2, 120080.
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