With eight possible linkages, and chains of different types and lengths including single, mixed and branched chains possible the conjugation of ubiquitin to cellular proteins may denote many things; degradation via the proteasome, DNA repair, cellular trafficking, or the activation of transcription factors. In addition to all this, ubiquitin itself can also be modified with phosphate and acetyl groups. So what can all this mean for cell signalling and to what end does the cell utilise all this complex cross-talk in post-translational modifications?
Ubiquitination is one of the most prevalent post?translational modifications. It is increasingly seen to engage in extensive crosstalk, with the potential to either positively or negatively modulate signalling networks. Acetylation competes with ubiquitination for the lysine residues in a substrate and can prevent the attachment of ubiquitin groups, thus inhibiting protein degradation. On the other hand, acetylation may trigger proteasome-mediated degradation by promoting the ubiquitination of another lysine residue within the substrate. Similarly, the phosphorylation of a protein typically acts as a signal for an E3-ubiquitin ligase to attach ubiquitin to available lysine residues within the substrate. But in addition to competing for sites of modification on protein substrates, ubiquitin itself can also be modified (link is external) as the ubiquitin surface is theoretically amenable to most post-translational modifications.
Acetylation of ubiquitinated proteins can occur on either the K6 or K48 residues within the ubiquitin structure. This modification of ubiquitin can be regulated by all three different classes of histone deacetylases and appears to block chain elongation as it blocks the positive charge on the lysine residue. Although only a very small amount of in vivo intracellular protein has been shown to be susceptible to acetylation (0.03% of K6 and 0.01% of K48), it may operate as a method to regulate the relative amounts of mono- vs. poly-ubiquitinated proteins.
Phosphorylation of ubiquitin has been shown to hinder ubiquitin chain elongation as it interferes with the interaction between some E2-E3 combinations. However, the most extensive study of the interplay between phosphorylation and ubiquitination has been in the case of mitophagy (link is external). Two proteins associated with Parkinson’s disease, PINK1 and Parkin, are responsible for the surveillance and clearance of damaged mitochondria.
PINK1 is a serine/threonine kinase targeted to the mitochondria. In the event of depolarisation of the mitochondrial membrane, which signifies damage, PINK1 accumulates on the mitochondrial surface and sets to work phosphorylating ubiquitinated proteins on the outer surface of the mitochondria. The phosphorylation of ubiquitinated proteins acts as a signal to recruit Parkin from the cytosol to the mitochondria. Parkin is an E3-ubiquitin ligase, which resides within the cytosol and is natively autoinhibited, thus predominantly inactive. Upon translocation to the mitochondria it becomes activated both by binding to phosphorylated ubiquitin on the mitochondrial surface and through phosphorylation of Parkin itself by PINK1. Activation of Parkin results in a feed-forward loop of ubiquitinated proteins created by Parkin activity being phosphorylated by PINK1, which recruits more Parkin to the mitochondria. In effect PINK1 mediated protein ubiquitination acts as a mitophagy signal (link is external), and Parkin acts to amplify this signal within the cell.
Ubiquitinated proteins on the mitochondrial surface act as a concurrent signal to both degrade these proteins through the proteasome, and to recruit ubiquitin-binding autophagy receptors, such as optineurin and NDP52. These receptors target the clearance of the mitochondria via the lysosome. Although mass spec analysis has shown that K6, K11, K48 and K63 ubiquitin linkages are all associated with mitophagy, it seems that Parkin preferentially forms the non-canonical K6 chains. K6 ubiquitin linkages are formed by Parkin at a faster rate than any of the other species, except K48 linkages.
Deubiquitinases (DUBs) remove ubiquitin from substrate proteins to discontinue this cellular signal and allow control over the signalling pathway. A number of mitophagy focused DUBs have been identified that can act to inhibit mitophagy by removing the ubiquitin tag signals from both Parkin and its ubiquitinated protein substrates. Yet interestingly the phosphorylation of ubiquitin appears to inhibit the activity of DUBs. It is suspected that dephosphorylation of ubiquitin, leaving the ubiquitin chains open to DUBs, must first occur to switch of this intracellular signal, and the serine/threonine phosphatase PGAM5 is one potential candidate for this role.
Taken together, the cross-talk between ubiquitination, acetylation and phosphorylation offers a complex network of interwoven post-translational modifications able to regulate different processes within the cell, and opens up the function of some of the non-canonical ubiquitin linkages, such as K6. While many of the interactions that control these processes are not yet elucidated it is clear that this expands the ubiquitin repertoire. Further cross-talk between ubiquitin signalling and other pathways, such as SUMO (link is external) has also been identified and is able to increase the function of these signalling components in health and disease.
To further explore this signalling network we offer a range of Affimer® reagents that can recognise components of the ubiquitin (link is external) system. These are both untagged and biotinylated for use in a range of applications and include the K6 (link is external) and K33 (link is external) diubiquitin linkage targets, to which there is no antibody equivalent.