Defining an Embedded Code for Protein Ubiquitination

Abstract

It has been more than 30 years since the initial report of the discovery of ubiquitin as an 8.5 kDa protein of unknown function expressed universally in living cells. And still, protein modification by covalent conjugation of the ubiquitin molecule is one of the most dynamic posttranslational modifications studied in terms of biochemistry and cell physiology. Ubiquitination plays a central regulatory role in number of eukaryotic cellular processes such as receptor endocytosis, growth-factor signaling, cell-cycle control, transcription, DNA repair, gene silencing, and stress response. Ubiquitin conjugation is a three step concerted action of the E1-E2-E3 enzymes that produces a modified protein. In this review we investigate studies undertaken to identify both ubiquitin and SUMO (small ubiquitin-related modifier) substrates with the goal of understanding how lysine selectivity is achieved. The SUMOylation pathway though distinct from that of ubiquitination, draws many parallels. Based upon the recent findings, we present a model to explain how an individual ubiquitin ligase may target specific lysine residue(s) with the co-operation from a scaffold protein.

Figure 1

Ubiquitination reaction. The protein substrate is ubiquitinated in a reaction involving three types of ubiquitinating enzymes: the ubiquitin activating protein E1, an ubiquitin carrier protein E2, and an ubiquitin-protein ligase E3. Following addition of a single ubiquitin molecule to a protein substrate (monoubiquitination), further ubiquitin molecules can be added to the first, yielding a polyubiquitin chain. The fate of the protein depends on the type of ubiquitin chain formed on the protein substrate.

Figure 2

Ubiquitin modifications. A) Mono-ubiquitination is involved in transcription, histone function, endocytosis and membrane trafficking. B) Multi-monoubiquitination is involved in protein regulation. C) Polyubiquitination is involved in signal transduction, endocytosis, DNA repair, stress response, and targeting proteins to the proteasome.

Figure 3

Presence of an “embedded code” within the substrate protein sequence. Multiple lysines may be present in the primary protein sequence. However, typically a one or more select lysine residues are selected for ubiquitination.

Figure 4

Model for substrate selection mechanism for Ub E3 ligase/scaffold complex. The target lysine site can either be masked or buried inside the hydrophobic pocket of the globular protein structure or be exposed to the exterior surface on the substrate. A) The scaffold protein interacts with the E3/E2 complex providing specificity for ubiquitination. Employing an embedded code the complex, with the assistance of the scaffold, directs ubiquitination of the target substrate on one or more specific lysine residues. This model is supported by studies with p62/TRAF6 complex (Geetha et al., 2005). B) Alternatively, the interaction of the E3 with the putative substrate changes the conformation of the substrate and allows it to recruit scaffold protein which in turn provides a platform for the ubiquitination reaction to take place.