Ubiquitination was originally described as a mechanism by which cells disposed of short-lived, damaged or abnormal proteins. However, its involvement in diverse cellular processes is coming to light and considered to rival phosphorylation. Ubiquitination is an ATP-requiring process and at the center of this modification is ubiquitin a 76-amino acid (~9 kDa) protein (Figure 1), which is highly conserved across eukaryotes and is synthesized as a fusion protein either to itself or to one of two ribosomal proteins (Schlesinger et al., 1987). Conjugation involves attachment of C-terminal glycine of ubiquitin (Ub) to the ε-amino group in lysine residues of the targeted protein. The conserved conjugation reaction is achieved by sequential actions of three enzymes (Hershko et al., 1998). The reaction commences with the formation of a thiol-ester linkage between the glycine residue at the C terminus of Ub and the active cysteine (Cys) residue of the first enzyme of the system, Ub activating enzyme (commonly referred to as E1). The ubiquitin molecule is then subsequently transferred to the cysteinyl group of the second enzyme called Ub-conjugating enzyme (E2). Lastly, through the action of an Ub ligase (E3), ubiquitin and the marked substrate are linked together via an amide (isopeptide) bond. This ability of an E3 to recognize and bind both the target substrate and the Ub-E2 enzyme suggests this enzyme provides specificity to the Ub reaction. At this point, the ubiquitination reaction may result in the addition of a single Ub molecule to a single target site, monoubiquitination (Figure 2). Alternatively, ubiquitination may result in the addition of single molecules of ubiquitin to other Lys in the target protein giving rise to multi-ubiquitination. After the initial ubiquitin is conjugated to a substrate, it can also be conjugated to another molecule of ubiquitin through one of its seven lysines. An isopeptide bond is formed between Gly76 of one ubiquitin to the ε-NH2 group of one of the seven potential lysines (K6, K11, K27, K29, K33, K48 or K63) of the preceding ubiquitin, giving rise to many different types of poly-ubiquitinated proteins (Adhikari and Chen, 2009). These poly-ubiquitin chains can vary in length with respect to the number of ubiquitin molecules, resulting in different topologies and, ultimately different functional consequences. For example, Lys48-linked polyubiquitination primes proteins for proteolytic destruction by the proteasome (Chau et al., 1989), whereas Lys63-linked polyubiquitination plays a key role in regulating processes such as DNA repair (Spence et al., 1995; Hofmann and Pickart, 1999), stress responses (Arnason and Ellison, 1994), signal transduction (Sun and Chen, 2004; Mukhopadhyay and Riezman, 2007), and intracellular trafficking of membrane proteins (Hicke, 1999; Geetha et al., 2005; Mukhopadhyay and Riezman, 2007). Proteins tagged with ubiquitin are most often destined for degradation by the proteasome. Recent studies reveal that all non-K63 linkages may target proteins for degradation (Xu et al., 2009). However this is still a matter of debate since K63-chains have also been shown to serve as a targeting signal for the 26S proteasome (Seibenhener et al., 2004; Saeki et al., 2009). Both, mono-ubiquitination and poly-ubiquitination also possess non-proteasomal regulatory functions like targeting proteins to nucleus, cytoskeleton and endocytic machinery, or modulating enzymatic activity and protein-protein interactions (Hershko et al., 1998; Pickart, 2001). Recent reports have indicated non lysine moieties can serve as ubiquitin acceptor sites. Ubiquitination occurring at noncanonical site —the N terminus— has been reported for transcription factor MyoD, the latent membrane protein-1 of Epstein-Barr virus, and p21, lead to proteasomemediated degradation (Aviel et al., 2000; Breitschopf et al., 1998; Bloom et al., 2003). Moreover, studies have shown the cysteine residue is required for ubiquitination of major histocompatibility complex class I proteins by the viral E3 ligases (Cadwell and Coscoy, 2005).

Figure 1

Figure 2

Like other posttranslational modifications (e.g. phosphorylation) ubiquitination is highly regulated and reversible process. It is controlled by the opposing activities of the E3 protein ubiquitin ligases which attach Ub molecules covalently to target proteins and de-ubiquitinating enzymes (DUBs) which remove the ubiquitin from target proteins (Wilkinson et al., 1997). Reversible covalent modification allows cells to rapidly and efficiently convey signals across different sub-cellular locations. It has been predicted that the human genome encodes three Ub-protein E1 enzymes, about fifty Ub-protein E2 conjugating complexes, over 600 ubiquitin ligases and about 100 DUBs (Kaiser and Huang, 2005).

Lysine residues are a target for diverse posttranslational modification enzymes which either attach methyl, acetyl, hydroxyl, ubiquitin or SUMO moieties to it. Except for hydroxylation, all of these attachments are reversible. In addition to ubiquitin, several ubiquitin-like proteins (Ubls) can also be conjugated to alter the function of the substrate proteins at lysine residues. These small molecular modifiers include NEDD8 (neural precursor cell expressed, developmentally down-regulated 8), ISG15 (interferon-stimulated gene 15), FAT10, FUB1 (FBR-MuSV associated ubiquitously expressed gene), UBL5 (ubiquitin-like 5), URM1 (ubiquitin-related modifier 1), ATG8 (autophagy associated protein 8), ATG12 (autophagy associated protein 12), and three SUMO isoforms to which ubiquitin bears much resemblance (Kerscher et al., 2006). However, modification of these Ubls requires their own unique combinations of E1, E2 and E3 and addition of these tags to the target protein likely serves a different function compared ubiquitination. These protein tags have been implicated in numerous cellular activities including DNA synthesis and repair, transcription, translation, organelle biogenesis, cell cycle control, signal transduction, protein quality control in the endoplasmic reticulum, immune system etc (Kerscher et al., 2006). These different Ubls are activated and conjugated to their substrates by a process very similar to the biochemical reactions of ubiquitination. All the structurally characterized Ubls share the ubiquitin or β-grasp fold, even when their primary sequences have little similarity (Kerscher et al., 2006).

Like several other posttranslational modifications, ubiquitination changes the molecular conformation of a protein, thereby influencing protein-protein interactions. Ubiquitin modification is known to alter protein localization, activity and/or stability through interaction with various proteins. These modifications on the target protein (either through monoubiquitination or polyubiquitination) act as attachment sites for proteins with ubiquitin-binding domains (UBDs) (Bertolaet et al., 2001; Wilkinson et al., 2001). The first UBD was characterized in a proteasome subunit, the S5A/RPN10 protein11. Similarity searches of a short sequence of S5a bound to ubiquitin led to the identification of a sequence pattern known as the ubiquitin-interacting motif (UIM) (Hofmann and Falquet, 2001). The ubiquitin-associated domain (UBA) was identified as a common sequence motif present in multiple proteins participating in ubiquitin-dependent signaling pathways (Hofmann and Bucher, 1996). Of the total sixteen UBDs reported to date, discovery of UIM and UBA domains, was the most important as it propelled the study of ubiquitination. Both UBA and UIM are known to bind poly- and mono- ubiquitin chains. The other ubiquitinbinding domains include a diverse family of structurally dissimilar protein domains, such as MIU, DUIM, CUE, GAT, NZF, A20 ZnF, UBP ZnF, UBZ, Ubc, Uev, UBM, GLUE, Jab1/MPN, and PFU (Hurley et al., 2006). Of these, many UBA-containing proteins are reported to bind polyubiquitin chains, some serve as shuttling factors for delivery of ubiquitinated proteins to the proteasome (e.g. hHR23A, p62 and Dsk2) (Seibenhener et al., 2004). This function is thought to be achieved by binding of the UBA domain to the ubiquitinated substrates, while simultaneously interacting with the proteasome through another domain (like Ubl domain) (Seibenhener et al., 2004).

Figure 3

Ubiquitin-protein ligases (E3) are the last (but likely the most important) components in the ubiquitin conjugation system because they play an important role in controlling target specificity. The E3s recruit target proteins, position them for optimal transfer of the Ub moiety from the E2 to a lysine residue in the target protein, and initiate the conjugation. Ubiquitin E3 ligases can be either monomeric proteins or multimeric complexes with the most common type of Ub ligases grouped into two classes depending on their modular architecture and catalytic mechanism. Typically E3s containing a HECT domain (Homologous to E6-AP C Terminus) forms a direct thioester bond with ubiquitin. Their approximately 350 amino acid HECT domains contain a conserved Cys residue that participates in the direct transfer of activated ubiquitin from the E2 to a target protein (Hershko et al., 1998; Pickart, 2001). On the other hand, RING (Really Interesting New Gene) finger domain ligase consists of Cys and His residues that coordinate two Zn⁺⁺ ions. The globular architecture of the domain primarily functions as a scaffold for the interaction of E2s with their target proteins (Hershko et al., 1998; Pickart, 2001). These ligases require a structural and/or catalytic motif that facilitates ubiquitination without directly forming a bond with ubiquitin. RING finger domain containing E3s comprise the largest ligase family, and contain both monomeric and multimeric ubiquitin ligases. There are three types of multisubunit E3s —SCF (Skp1-Cullin-F-box protein), the APC, and the VHL (von Hippel Lindau protein) E3(s)— where a small RING finger protein is an essential component. A lesser known family of Ub E3 ligases includes an E2-binding domain called the U-box adaptor E3 ligases. The U-box ligase was first identified in yeast Ufd2 acting as an accessory protein (E4) promoting polyubiquitination of another E3's substrate (Kuhlbrodt et al., 2005). Bioinformatics studies placed them under conventional RING E3 ligases, as the U-box ligases adopt a RING domain-like conformation via electrostatic interactions (Aravind and Koonin et al., 2000). Genome-wide annotation of the human E3 superfamily genes (Li et al., 2008) had revealed the number of putative E3 genes, 617, to be greater than the number of human genes for protein kinases, 518, suggesting the extent of biological targets of ubiquitination.

One salient question is what determines whether or not a protein is tagged by Ub? While as of yet this cannot fully be answered, recent research has uncovered some interesting clues. It has been proposed that proteins contain an "embedded code" that is recognized by the Ub machinery (Figure 3). For example, E3 ubiquitin ligases recognize their corresponding protein substrates via a variety of structural determinants, including primary sequence, post-translational modifications and protein folding state. Herein, we consider some of the other examples discovered thus far for directing target specificity.

Particular amino acid sequences within the polypeptide act as proteolytic recognition signals. Analysis of sequence motifs in rapidly degraded proteins, lead Roberts and Rechsteiner to identify PEST sequences. Stretches of PEST sequences which are rich in proline (P), glutamate (E), serine (S), and threonine (T) (along with a lesser extent, aspartic acid) serve as a destruction signal (so called "PEST sequences") (Rogers et al., 1986). Ubiquitination of proteins by multi- subunit ligases, consisting of Ubc3/Cdc34, Skp1, cullin/Cdc53 and F-box proteins, has been shown to be preceded by phosphorylation within the PEST motif (Feldmann et al., 1997). Furthermore, phosphorylation of Ser or Thr residues in the PEST regions of proteins has been shown to activate their recognition and processing by the ubiquitinproteasome pathway (Yaglom et al., 1995; Lanker et al., 1996; Willems et al., 1996; Won and Reed, 1996).

There is a need for novel techniques designed to identify and characterize protein modifications on a large or global scale. For example, there are more than 500 E3s in the human genome, yet functional information is available for only a small fraction. Linking an E3 with its substrates is difficult and is generally dependent on either a functional connection or a physical association between the proteins. Given the large number of potentially ubiquitinated substrates and E3s, new strategies to deduce E3-substrate pairs are needed since performing biochemical screens for E3 substrates is labor-intensive, is hampered by low substrate levels, as well as, the intrinsically weak interactions between E3s and their substrates.

Of the several new Ubl modifiers that have been discovered in the past few years, the SUMO pathway has received the most intense scrutiny. SUMO was identified in 1996 as a peptide conjugated to the nucleocytoplasmic-transport protein RanGAP1, resulting in a change in its cellular localization (Matunis et al., 1996). Since the discovery of SUMO as a post-translational protein modifier over 10 years ago, more than 200 proteins targets have been reported, with the majority being nuclear proteins. SUMOylation is known to cause either alteration in protein localization, a change in protein activity, or differences in interaction with binding partners (Geiss-Friedlander and Melchior, 2007). SUMO is about 20% similar to ubiquitin in its primary sequence and contains ∼15 additional N-terminal amino acid residues (Bayer et al. , 1998). Like, ubiquitination, SUMOylation is achieved by sequential action of three enzymes; the activating (E1), conjugating (E2), and ligating (E3) enzymes. Nevertheless, SUMO E1, E2, and E3s are very distinct from the E1, E2 and E3 of the ubiquitination system (Yeh et al., 2000). Despite the similarities in structure and conjugation mechanism, they both have distinct physiological effects in the cell. To date, there is only one reported example of both E1 (SAE1/SAE2 heterodimer) and E2 (UBC9) for SUMOylation, in contrast to the large number of E1s and E2s reported for the ubiquitination pathway. Like the ubiquitination system several SUMO E3 ligases have been identified, most of which have a SiYz/ PIAS (SP)-ring motif required for their function. There are three types of known SUMO E3 ligases – PIAS proteins, RanBP2, and Pc2 each conferring substrate specificity to the SUMOylation reaction. As additional SUMO targets and pathways influenced by SUMO regulation are recognized, the significance of this pathway is beginning to be appreciated. SUMOylation is known to participate in diverse cellular events, including chromosome segregation and cell division, DNA replication and repair, transcriptional regulation, nuclear transport and signal transduction (Müller et al., 2001). Four different type of SUMO isoforms (SUMO1 - 4) are reported in mammals. SUMO-1 is the most commonly found conjugated isoform under normal conditions. SUMO-2 and SUMO-3 have very similar sequence identity and appear to be conjugated in response to stress signals. SUMO-4 is more tissue-specific, as it is identified in human kidney, suggesting its involvement in more tissuedependent functions. Both SUMO2/3 and SUMO-4 contain an internal consensus motif ΨKXE (where Ψ represents a large hydrophobic amino acid, and X represents any amino acid) that is required for SUMO modification both in vivo and in vitro (Rodriguez et al., 2001), which is missing in SUMO-1. Exploiting the fact that Ubc9 binds to this motif directly (Sampson et al., 2001), a number of SUMO targets have been identified via their interaction with Ubc9 in the yeast two-hybrid screen. Not all ΨKXE motif found in proteins are modified, as SUMO E3s are presumed to enhance specificity by interacting with other features of the substrate. In addition, to the consensus sequence amino acids upstream or downstream of the acceptor lysine may help to insure accessibility of the substrate for the conjugation apparatus. For some SUMO substrates, additional interactions occur outside the consensus sequence (Anckar and Sistonen, 2007; Bernier-Villamor et al., 2002), demonstrating the involvement of multiple, co-operating interactions in regulating the target selection process. In this regard, the consensus sequence can be seen as a local mediator of substrateconjugation apparatus interaction, fine-tuning the SUMO conjugation event by facilitating the correct positioning of the target lysine residue to the active site of Ubc9.

Approaches similar to the identification of ubiquitinated substrates have been utilized in identifying novel SUMO targets and/or total SUMOylated substrates in the cell. These methods rely upon purification of SUMOylated proteins from cell lysates via affinity tags, followed by MS analysis (Li et al., 2004; Zhao et al., 2004; Zhou et al., 2004; Vertegaal et al., 2004; Wohlschlegel et al., 2004; Panse et al., 2004). A variety of affinity-tagged SUMOs have been described that have been overexpressed to overcome low levels of SUMOylated proteins in the cells, a major barrier to MS sensitivity. Moreover, at a given time only a small fraction of proteins in the cells are SUMOylated, since it is a dynamic process in which conjugation and de-conjugation work in concert. It has been suggested that <1% of the proteins in a cell are SUMO modified at any given time (Johnson, 2004), thus making efforts at detecting these modified proteins difficult. The use of several genomic/ proteomic and in silico combinatorial approaches to idenidentify global pool of 'Sumo-tome' has lead to identification of ∼500 potential SUMO substrates (Wohlschlegel at el., 2004; Gocke et al., 2005; Zhou et al., 2005). However, bona fide SUMOylation sites may still remain to be identified or confirmed in vivo. Thus, as experimental proteomics approaches become more and more-labor intensive and timeconsuming, there is a growing need to develop prediction tools that would aid in successfully predicting the target substrate. In this regard, computational techniques have presented a promising approach toward identifying SUMOylation sites. Given this, the first computational prediction tool SUMOplot, was developed which predicted the probability for a SUMO attachment. The SUMOplot prediction heavily depended on identification of the SUMO consensus motif. This limited the prediction results as many non-consensus true positives were missed. SUMOsp was developed based on a manually curated 239 experimentverified SUMOylation sites from the literature (Xue et al., 2006). GPS and MotifX, two earlier described strategies, were applied to the dataset, yielding good (89.12%) prediction platform for SUMOylation sites. Another bioinformatic study accurately predicted SUMO modified sites employing a statistical method based on properties of individual amino acid surrounding the SUMO site (Xu et al., 2008).

To better understand lysine selectivity within a protein destined for ubiquitination (Figure 3), it is first important to survey the literature for reported proteins and their ubiquination sites. The first report exploring the preferences for a specific ubiquitination site was conducted on human red blood cell protein a-spectrin (Galluzzi et al., 2001). The investigators demonstrated that the leucine zipper was a potential ubiquitin recognition motif by site-directed mutagenesis. Moreover, in addition to the primary sequence it has been suggested that secondary folding also plays a role in directing the lysine selected for ubiquitination. The leucine zipper described in multi-ubiquitination of c-Jun (Treir et al., 1994) is observed in a number of other gene regulatory proteins with 75% similarity to the flanking regions of ubiquitinated α-spectrin lysine (Murantani and Tansey, 2003). This suggests a conformational recognition mechanism in which positioning of the Lys plays an important role in directing specificity. In another study, K187 (out of the possible six available lysines) was found to be a preferred ubiquitin target site in the transcription activator Rpn4 (Ju and Xie, 2006). Primary sequence analysis revealed the close proximity of K187 to the N-terminal acidic domain, which acts as ubiquitination signal for transcription activators. Additionally, surface hydrophobic residues are known to be required for ubiquitination of several proteins for proteasomal degradation (Bogusz et al., 2006; Johnson et al., 1998). The neurotrophin receptor TrkA was one of the first receptors to be identified as a K63-polyubiquitin tagged at K485 (Geetha et al., 2005). Recently, ubiquitination of a lysine within the membrane proximal region of granulocyte colonystimulating factor receptor (G-CSFR) was reported (Wolfler et al., 2009) and K63-ubiquitination of K338 was reported for the Jen1 Transporter (Paiva et al., 2009) Altogether, a picture is emerging where K63-chains may play a role in regulating internalization and sorting of receptors.

Studies conducted on both the Huntingtin and Androgen receptors support the importance of conserved pentapeptide pattern (FQXL(L/F)) as determinants in their degradation by the proteasome (Chandra et al., 2008). Another report on the E3 substrate selection process analyzed the ubiquitinized-yeast proteome based on subcellular localization (Catic et al., 2004). This study revealed the presence of compartment-specific sequence patterns for ubiquitinated substrates. Structural analysis of ubiquitinated proteins demonstrate a preference for an exposed lysine residue on the surface of the molecule. Additionally, a survey of 40 ubiquination sites from 23 proteins showed clear secondary structure preference for lysine ubiquitination. Modifications were prominent at the lysines occurring in loop regions (26/ 40) followed by lysines in α-helices (10/40) (Catic et al., 2004). This investigation also reported the presence of compartment- specific motifs within the dataset. For example, nuclear proteins had preference for ubiquitination of lysines near the phosphorylatable residues. Similar bias was observed for ubiquitinated plasma membrane proteins that had either Glu or Asp at -1 or -2 positions from the acceptor lysine (Catic et al., 2004). Thus, investigating the overall primary and secondary structure as well as the proteins’ subcellular localization could yield important information regarding the targeting of the substrates.

Many E3 ligases are known to interact with specific substrates either directly or through scaffold proteins. Scaffold proteins facilitate interaction between the E3 enzymes and their substrates through their multi-domain architecture. One such scaffold is p62, a highly conserved and transcriptionally regulated protein that plays important roles in ubiquitination, receptor trafficking, protein aggregation, and inclusion formation (Seibenhener et al., 2004). P62 acts as a scaffold by interacting with the RING E3, TRAF6, through a TRAF-binding site (TBS) as well as other proteins through one of its many protein-protein interaction domains. Interaction between p62 and TRAF6 has been shown to autoactivate TRAF6 (Wooten et al., 2001; 2006). Functional domains in p62 include a Phox and Bem1p (PB1) domain, a TRAF6-binding region, and an UBA domain (Geetha et al., 2002). The C-terminal UBA domain of p62 has been shown to non-covalently bind ubiquitin (Mueller et al., 2002). Moreover, p62 functions as a shuttling factor for polyubiquitinated substrates by binding the ubiquitinated proteins through its UBA domain and the 26S proteasome through its N-terminal PB1 domain (Wooten et al., 2005). The tyrosine kinase receptor A (TrkA) (Geetha et al., 2005) and the neurotrophin receptor interacting factor (NRIF) (Geetha et al., 2005), both have been shown to be K63- polyubiquitinated by the TRAF6/p62 complex. In a recent study, in a attempt to understand the lysine selection process employed by TRAF6/ p62 the primary sequences of the lysines that were targeted for ubiquitination in both TrkA and NRIF were examined for a possible consensus motif (Jadhav et al., 2008). A close look at these two substrates revealed the presence of a conserved consensus pattern for ubiquitination by the TRAF6/p62 complex. This consensus pattern has also been observed in others members of the Trk receptor family, TrkB and TrkC (Jadhav et al., 2008). Interestingly a consensus pattern identified in these proteins was a 10-amino acid long stretch {[–(hydrophobic)–K–(hydrophobic)–X–X–(hydrophobic)–( polar)–(hydrophobic)–(polar)–(hydrophobic)] where K was the ubiquitinated lysine residue and X any other amino acid} required to successfully target the primary lysine residue (Jadhav et al., 2008). These studies further suggest the possibility that an "embedded code" that exists whereby an E3 ligase targets a specific lysine residues for modification over others. Therefore, to better understand the lysine selection process during ubiquitination, it is important to examine the enzyme-specific selection process. The development of an algorithm to search a training dataset of p62/TRAF6 interactors could be employed as a first step in development of a computational tool to aid in discovery of TRAF6 targets.

Substrate selection and site specificity is a multi-step process depending on two types of signals, both primary and secondary. The primary signals are the structural motifs; α-helices or β-sheets that influence the local architecture of the primary sequence. Secondary signals, on the other hand, are inherent primary sequences that are essential for the recognition of the primary ubiquitination site. Of both, secondary signals can vary slightly depending on the localization of proteins in the cell.

What can be learned from the E3 TRAF6? In the case of TrkA site-specific ubiquitination (Geetha et al., 2005), the E3, TRAF6, exists as a complex with the E2, UbcH7, in the cytosol. Post-receptor stimulation, the E2/E3 pair form a transient complex recruited to the scaffold, p62, to mediate the ubiquitination of TrkA (Geetha et al., 2005). The target lysine within a protein can either be buried inside a hydrophobic pocket of the globular protein structure or masked, while the protein is interacting with a different binding partner. Binding of the scaffold protein likely induces a conformational change in the proteins' structure exposing the buried target site (Figure 4A). Thereafter, the scaffold recruits the activated E3/E2 complexes to the substrate protein. The enzyme complex then scans the exposed surface for an acceptor lysine that possesses the appropriate conformation. Once an accessible lysine is recognized and if the nearby flanking residues present an appropriate environment, transfer of the ubiquitin molecule occurs. In other cases, the active enzyme complex E3/E2 first binds to the substrate protein and produces a similar type of conformational change (i.e., exposure of the target site). This binding of substrate to the E3 produces structural changes for accommodating the scaffold protein to the complex, which aids in the enzymatic process (Figure 4B). Our results suggest that the former model is more likely operative for sitespecific ubiquitination of the target (Geetha et al., 2005).

Figure 4

The analysis of the 'ubiquitome' presents one of the most exciting and challenging tasks in current proteomics research. The ultimate limiting factor in studying ubiquitination substrate selection mechanism is the lack of curated data sets of ubiquinated proteins. This makes it difficult to evaluate, and compare target sites to decode selectivity and specificity. With identification of more than 500 or so ubiquitin ligases there exists a need to rapidly and precisely identify enzyme-specific substrates. This task demands that we take multiple novel approaches as well as a combination of techniques to precisely identify target sites for these ligases. With rapid advancement in mass spectrometric analysis and more sophistication in proteomic tools and novel approaches we can expect the number of precisely identified sites to rise. Moreover, use of bioinformatic methods to predict site modification in silico could yield more efficient results. These prediction tools should be closely integrated into the interpretation of proteomic experiments. Also as proteomics methods identify more and more in vivo ubiquitination sites, prediction algorithms can be fine tuned and improved with this information. The model that we propose here can be applied to other E3 Ub ligases that are known to employ scaffold proteins to aid in their substrate selection process (Figure 4). For example, the BTB-domain proteins that were indentified as substrate-specific scaffolds for Ub E3 ligase CUL-3 in C. elegans (Xu et al., 2003). Lysine ubiquitination interplays actively with other post-translational modifications, either agonistically or antagonistically, to form a coded message for intramolecular signaling programs that are crucial for governing cellular functions. Given the intricacy of the ubiquitin system, research into its functions and mechanisms should continue to yield novel insights into cell regulation.