When ubiquitination meets phosphorylation: a systems biology perspective of EGFR/MAPK signalling
© Nguyen et al.; licensee BioMed Central Ltd. 2013
Received: 4 April 2013
Accepted: 26 July 2013
Published: 31 July 2013
Ubiquitination, the covalent attachment of ubiquitin to target proteins, has emerged as a ubiquitous post-translational modification (PTM) whose function extends far beyond its original role as a tag for protein degradation identified three decades ago. Although sharing parallel properties with phosphorylation, ubiquitination distinguishes itself in important ways. Nevertheless, the interplay and crosstalk between ubiquitination and phosphorylation events have become a recurrent theme in cell signalling regulation. Understanding how these two major PTMs intersect to regulate signal transduction is an important research question. In this review, we first discuss the involvement of ubiquitination in the regulation of the EGF-mediated ERK signalling pathway via the EGF receptor, highlighting the interplay between ubiquitination and phosphorylation in this cancer-implicated system and addressing open questions. The roles of ubiquitination in pathways crosstalking to EGFR/MAPK signalling will then be discussed. In the final part of the review, we demonstrate the rich and versatile dynamics of crosstalk between ubiquitination and phosphorylation by using quantitative modelling and analysis of network motifs commonly observed in cellular processes. We argue that given the overwhelming complexity arising from inter-connected PTMs, a quantitative framework based on systems biology and mathematical modelling is needed to efficiently understand their roles in cell signalling.
KeywordsUbiquitination Ubiquitination-phosphorylation crosstalk Quantitative modelling Phosphorylation-induced ubiquitination MAPK signalling
Cell signalling crucially depends on a repertoire of posttranslational modification (PTM) mechanisms for its regulation. Protein ubiquitination, the covalent attachment of the short protein modifier ubiquitin to target proteins, has emerged as a prevalent modification utilised by signalling processes to regulate a range of functional behaviours. First recognised as a targeting signal to send proteins to the proteosomal degradation pathway, ubiquitination has since been implicated in the non-degradative regulation of a plethora of cellular processes, including signal transduction, enzymatic activation[2, 3], endocytosis and trafficking, chromatin rearrangement and DNA repair.
Over the past few years, the interplay between ubiquitination and phosphorylation has emerged as a prominent posttranslational crosstalk and a key principle in eukaryotic cell signalling. Phosphorylation often serves as a marker that triggers subsequent ubiquitination, in particular where ubiquitination leads to degradation[9–11]. In many cases, phosphorylation of substrate E3 ligases acts as a signal that can dramatically influence their activity. In other cases, ubiquitination provide a switching mechanism that can turn on/off the kinase activity of certain proteins. Understanding of how these two major PTMs interact to regulate signal transduction is an important topic in cell signalling. In this review, we discuss the involvement of ubiquitination in the regulation of the epidermal growth factor (EGF)-mediated extracellular signal-regulated kinase (ERK) signalling pathway via the EGF receptor (EGFR), and highlight the interplay between ubiquitination and phosphorylation in this system, which beyond its many physiological functions is also a major player in human cancer. The review contains two parts. In the first part we survey recent biological findings related to ubiquitination and crosstalk with phosphorylation as means for the functional control of the components of the EGFR-mediated ERK pathway, and highlight some remaining open questions. In the second part, we demonstrate the rich and versatile dynamics of crosstalk between ubiquitination and phosphorylation by using quantitative modelling and analysis of various network motifs where such crosstalk is often observed. Multiple lines of evidence from both theoretical and experimental studies have shown that intricate dynamics including bistable switches, mutistability and sustained oscillation can be brought about as a result of the interplay between feedback regulations and nonlinear post-translational modification cascades, such as phosphorylation[13–16], ubiquitination and GTPase cascades. Oscillations in GTPase cascades drive periodic protrusion and retraction of lamellipodia during cell migration[18, 19]. In addition, short-period (20 min) and long-period (4–5 hrs) ERK oscillations have been experimentally reported[15, 16]. It is likely that these complex dynamics may also emerge from crosstalk between phosphorylation and ubiquitination. Our aim here is to illuminate non-trivial dynamics arising from these generic crosstalk mechanisms that would apply not only to the EGFR pathway but to many other pathways. We argue that given the overwhelming complexity originating from interconnected PTMs, a quantitative framework based on systems biology and mathematical modelling is needed to efficiently understand their regulatory roles in cell signalling.
Involvement of ubiquitination in EGFR-mediated MAPK signalling pathway
Ubiquitin-mediated regulation of EGFR, adaptor proteins and roles in endocytosis
The function of ubiquitination as a regulatory mechanism in Receptor Tyrosine Kinases (RTKs) endocytosis was one of the early findings of the non-proteolytic roles of this PTM in cell signalling[21, 22]. Ubiquitination of the receptor and endocytic adaptor proteins was found critically important in mediating EGFR internalisation and downstream signal transduction. The proteins of the Cbl family, consisting of three mammalian homologs c-Cbl, Cbl-b and Cbl-3, are the best characterized E3 ligases that regulate the EGFR endocytosis pathway. Located next to the RING finger domain, which is responsible for transferring ubiquitin to substrates, the Cbl N-terminal region is composed of three conserved domains: a 4 helix bundle domain (4H), an EF hand-like domain, and a SH2-like domain (Figure 1c). Together, these conserved regions form the TKB (tyrosine kinase binding) domain that enables Cbl to recognise phosphotyrosine residues and interact with phosphotyrosine-containing proteins. Following ligand binding and activation of EGFR by autophosphorylation, Clb directly binds to activated EGFR via the TKB domain[23–25]. Cbl can also be recruited to activated RTKs through its constitutive binding partner Grb2 which directly binds to RTK phosphotyrosines via its SH2 domain[26–28]. Recent structural studies suggested that once bound, Cbl becomes phosphorylated on a critical tyrosine (371 in c-Cbl and 363 in Cbl-b) due to the opening-up of the compact structure within Cbl that previously hides the E2 binding site[29, 30]. This phosphorylation enables full rotation of the Cbl linker region which exposes the RING domain enabling binding of the ubiquitin-loaded E2 complex. This then triggers allosteric activation of the E2 and stimulates Cbl E3 ligase activity resulting in the subsequent multi-monoubiquitination and polyubiquitination of the EGFR[29, 30].
Ubiquitination-related mechanisms regulating the adaptor proteins also play crucial roles in the functioning of the endocytotic pathway, including cargo recognition and delivering. These adaptors include proteins at the plasma membrane including the clathrin coat, the EGFR substrate 15 (EPS15), a member of the EPS15-interacting protein family (EPSIN1–EPSIN3), and hepatocyte growth factor-regulated Tyr kinase substrate (HRS) at the endosomes. Adaptor proteins, which contain ubiquitin binding domains (UBD) such as the ubiquitin-interacting motif (UIM) can recognise the ubiquitin molecules on the ubiquitinated EGFR. This leads to the assembly of active receptors in clathrin-coated pits of the plasma membrane, endosomes and the multivesicular bodies (MVBs). Adaptor proteins also undergo ubiquitination upon ligand stimulation through a process known as coupled monoubiquitination, which requires the presence of an intact UBD. For instance, upon EGF stimulation EPS15 interacts directly with NEDD4 via its UBD and is ubiquitinated by NEDD4, a homologous to the E6AP carboxyl terminus (HECT) E3 ligase. NEDD4 then transfers the thiolester-conjugated ubiquitin from its catalytic cysteine residue to the adaptor protein, inducing monoubiquitination. This directs progression of the ubiquitinated receptors toward lysosomal degradation through the ESCRT complexes[31, 33].
Ubiquitin-mediated EGFR endocytosis affects the signalling dynamics of the downstream pathways, thereby modulating the cellular decisions. Cells have evolved ways to reverse ubiquitination events through de-ubiquitinating enzymes. The STAM-binding protein (STAMBP, also known as AMSH) is a DUB specifically cleaving the lysine 63 and 48-linked ubiquitin chains anchored at the endosome via interaction with the clathrin coat. Thus, STAMBP counteracts the ubiquitin-dependent sorting of receptors to lysosomes. Another DUB which can abrogate the endocytosis of EGFR receptors is USP8. Before being incorporated into internal vesicles of MVBs, the ubiquitinated EGFR can undergo USP8-induced deubiquitination which moves the EGFR into the recycling pathway back to the plasma membrane. Interestingly, USP8 can be tyrosine and serine phosphorylated in an EGFR- and Src-kinase dependent manner. Since decreased USP8 tyrosine phosphorylation is associated with enhanced endosomal recycling of EGFR when cells are stimulated by TGFα, it is likely that USP8 phosphorylation may regulate its DUB activity. Further research is required to shed more light on this issue.
Ubiquitin-mediated regulation of Ras as a major EGFR effector
In an effort to identify the molecular mechanism by which Ras ubiquitination is regulated, Xu et al. found that Rabex-5 (Rab5 GDP/GTP exchange factor), known previously as a GEF for Rab5[46, 47], is also an E3 ligase for H- and N-Ras. This discovery was supported by the earlier knowledge that Rabex-5 possesses a zinc finger (ZnF) domain similar to that of A20 with E3 ligase activity[49–51], and that Rabex-5 interacts with Ras[52, 53]. Using in vivo and in vitro ubiquitination assays along with RNAi technology, the authors showed that Rabex-5 is necessary and sufficient to catalyse H/N-Ras ubiquitination, promoting their endosomal localisation and resulting in suppressed ERK activation (Figure 2). Overexpression of Rabex-5 did not induce K-Ras ubiquitination, suggesting Rabex-5 is specific to H/N-Ras. Importantly, a mutation in the ZnF domain but not the GEF domain blocked Rabex-5’s ability to ubiquitinate Ras, indicating that Rabex-5 GEF activity is not required for ubiquitination. Interestingly, this is not the case for Rin1, which is a Rab5-directed GEF, where the GEF function is required for enhancing Rabex-5-dependent Ras ubiquitination (Figure 2). Because Rin1 is a Ras effector, this constitutes a negative feedback which serves to attenuate Ras-mediated ERK signalling. This mechanism is consistent with earlier observations that Rin1 competes with Raf-1 for binding to Ras[54, 55]. What remains unclear is how these distinct mechanisms of diminishing ERK signalling interplay at specific cell locations. Adding to the already complex picture, Rabex-5 was known to undergo coupled monoubiquitination, determined by its ability to bind ubiquitin through two independent ubiquitin binding domains (UBDs)[49, 51]. However, what is the function of this autoubiquitination and how it is involved in Ras ubiquitination are open questions.
Although the studies by the Bar-Sagi group[48, 57, 58] did not find ubiquitination of K-Ras, it has been reported that K-Ras could be monoubiquitinated in HEK293T cells, preferably at lysine 147. These discrepancies are most likely due to the usage of different cell types, which may differ in the expression of E3 ligases or the DUBs which determine the detectable levels of K-Ras ubiquitination. Interestingly, the ubiquitination of K-Ras strongly enhances ERK signalling as opposed to H-Ras ubiquitination, indicating dramatic isoform-specific functional difference. Monoubiquitination of K-Ras results in its enhanced GTP loading, whereas for the oncogenic G12V-K-Ras mutant, monoubiquitination increases Ras binding to its main downstream effectors including Raf-1 and PI3K. In identifying the molecular mechanism responsible for the monoubiquitination-mediated activation of K-Ras, Baker et al. recently showed that monoubiquitination at lysine 147 does not alter K-Ras’s intrinsic biochemical properties, but strongly inhibits GAPs-mediated hydrolysis resulting in increased GTP-bound population of monoubiquitinated Ras in vivo. Combined, these findings illuminate a novel role for ubiquitin in controlling Ras activity, in addition to regulating its spatial location. It however remains to be discovered whether a similar regulatory mechanism exists for other Ras isoforms under other cellular contexts. It is also noteworthy that all Ras isoforms are subject to polyubiquitination mediated by the F-box protein b-TrCP (b-transducin repeat–containing protein), leading to proteasome-dependent degradation of Ras. In conclusion, the above studies suggest that ubiquitination is an essential mechanism controlling Ras compartmentalisation and its signalling output.
Ubiquitin-mediated regulation of components of the Raf/MEK/ERK MAPK cascade
The transduction of a cellular signal as it propagates through the MAPK cascades, exemplified by the Raf/MEK/ERK module, is predominantly controlled by phosphorylation events where typically, each kinase in the cascade is activated by an upstream kinase and inactivated by relevant phosphatases. However, accumulating evidence has revealed that components of this cascade also can undergo ubiquitination, which not only leads to the degradation of the substrate proteins but also appears to regulate their activity and/or localisation.
Raf proteins are the main effectors of Ras[63, 64] and direct activators of MEK[65, 66], serving as essential connectors linking Ras to the MEK-ERK pathway. Extensive work focusing on Raf regulation have revealed a complex, yet still incomplete, picture of the Raf activation/inactivation cycle where phosphorylation events play major regulatory roles (reviewed in). In contrast, the involvement of ubiquitination in the modulation of Raf has received far less attention and remains largely elusive. Raf-1 exists in a complex with the heat shock protein HSP90 and this association is essential for Raf-1 stability. Using NIH3T3 cells treated with GA (the benzoquinone ansamycin Geldanamycin) to disrupt the Raf-1-HSP90 complex which induces rapid Raf-1 degradation, Schulte et al. then used different inhibitors for various proteolytic systems to investigate the mechanisms responsible for the degradation of Raf-1. Inhibition of the proteosome, rather than of the lysosome or other proteases, prevented the observed enhanced Raf-1 degradation. Moreover, the Raf-1 fraction protected from GA-induced degradation showed a smearing pattern typical of polyubiquitinated proteins. These data indicate that Raf degradation involves ubiquitination and the proteosome-mediated pathway. The next important question emerges as to how Raf’s proteosomal degradation is regulated. Investigating if the kinase activity of Raf-1 is regulating its degradation, Noble et al. argued that that Raf-1 kinase activity is required to induce an (in cis) autophosphorylation of the site S621 which helps stabilise Raf-1. Interestingly, autophosphorylation does not appear to regulate B-Raf stability, since the equivalent S729 site is not autophosphorylated in B-Raf, and B-Raf activity has no effect on its expression level. Clearly, additional work must be done to further elucidate the Raf ubiquitination-related regulation.
Although evidence pointing to an ubiquitination-related mechanism involving MEK in mammalian cells is sparse, the yeast MEK protein Ste7 has been shown by multiple studies to undergo ubiquitination and regulate MAPK specificity[71–73]. The terminal kinases of the cascade, ERK1 and ERK2 have been shown to be ubiquitinated by MEKK1, a MAP kinase kinase of the STE11 family. MEKK1 phosphorylates several MEKs, and its major targets are MKK3 and MKK4, which in turn activates JNK[75, 76]. In addition to activating JNK, MEKK1 is also known to regulate ERK signalling. Lu et al. showed that MEKK1 has a dual role as a kinase that also has E3 ligase activity due to a separate kinase domain and a RING-finger like structure containing the PHD domain. Under stress stimulation induced by sorbitol, MEKK1 directly interacts with and poly-ubiquitinates ERK1/2, sending it for degradation which subsequently leads to down-regulation of ERK activity. This however is not the case for serum or EGF stimulation. The dual role of MEKK1 appears to provide opposing controls over ERK, with activating function and also inhibiting function as a direct de-stabiliser. It is important though to note that the existence of multiple regulatory mechanisms does not necessarily imply that they are simultaneously active, but one may be favoured over another under certain physiological conditions. Interestingly, the MEKK1 kinase activity was found to be involved in ERK1/2 ubiquitination. Furthermore, MEKK1 undergoes non-proteolytic self-ubiquitination which inhibits its catalytic activity as a kinase, attenuating MEKK1-mediated phosphorylation of MKK3/4 and resulting in inhibition of ERK1/2 signalling. This represents a rather interesting case where ubiquitination modifies the kinase activity rather than ligase activity of the modified protein. A recent study further reported that under hyperosmotic stress, another MAPK kinase kinase, MEKK2, mediates the transient activation of ERK. However, unlike MEKK1, MEKK2 is instead controlled by an external E3 ligase, the carboxyl terminus of Hsc70-interacting protein (CHIP). CHIP depletion attenuates the degradation of MEKK2 and prolongs ERK activity.
Roles of ubiquitination in crosstalked pathways
Functional roles of Itch in the EGFR/ERK signalling pathway
ITCH is the HECT E3 ubiquitin ligase belonging to the NEDD4 protein family. It is characterised by the N-terminal C2 domain responsible for membrane localisation, 2 to 4 WW domains involved in substrate recognition, and the C-terminal catalytic HECT ligase domain. Although ITCH is better known for its role in the immune system development[80, 81] where its deficiency causes syndromic multisystem autoimmune disease, increasing evidence implicates ITCH involvement in EGF signalling and EGF-mediated anti-apoptosis.
ITCH self-ubiquitination increases its activity
ITCH connects EGF signalling and apoptotic pathway
ITCH was demonstrated to interact with the truncated form of the proapototic protein Bid (tBid), ubiquitinate tBid and induce its proteosomal degradation (Figure 3). tBid is a truncated form of Bid arising through caspase mediated cleavage during apoptosis. tBid amplifies the mitochondrial apoptosis pathway by binding to and inactivating Bcl2 family proteins promoting mitochondrial permeability transition and apoptosis. In contrast, the full-length form of Bid does not interact with ITCH and is not subject to proteosomal degradation regardless of whether or not ITCH is present. Importantly, the ITCH-mediated down-regulation of tBid increased following EGF treatment. Furthermore, ITCH expression can significantly reduce cell apoptosis induced by tBid and influences the balance between cell survival and apoptosis in normal cell culture conditions. Taken together, these studies suggest a sequence of events involving ITCH that is initiated from the cell surface following EGF treatment: EGF triggers receptor activation which stimulates ITCH auto-ubiquitination partly due to EGF-mediated JNK phosphorylation. This leads to increased degradation of ITCH substrates, including truncated tBid, resulting in decreased apoptosis and thus promoting cell survival.
ITCH connects EGF signalling to apoptosis via the MST2 pathway
Another route through which ITCH links EGFR/Raf/ERK signalling to apoptosis is via the MST2/LATS1 pathway (Figure 3). Our group has shown that Raf-1 controls the proapoptotic kinase MST2 activity and restrains cell apoptosis via the Raf-1-MST2 complex formation, which occurs in two ways[89, 90]. First, Raf-1 binding interferes with MST2 dimerisation and subsequent activating autophosphorylation. Second, Raf-1 recruits a phosphatase that dephosphorylates the activating sites on MST2, thereby limit its activation. Furthermore, using a signalling pathway mapping strategy based on tracking dynamically changing protein interactions, we have mapped a multistep pathway from the cell membrane through MST2 activation to p73 dependent transcription in the nucleus, in which MST2 directly activates LATS1. Interestingly, ITCH has been recently reported as an E3 ligase for LATS1 as well as for p73, which targets these proteins for poly-ubiquitination and degradation[92, 93]. Thus, the involvement of ITCH as a degradation regulator of key components of the proapoptotic MST2/p73 pathway may link EGF signalling to apoptosis in a manner independent of the Raf-1-MST2 binding (Figure 3). It would be interesting in future studies to explore the role of ITCH in regulating apoptosis in this direction. Furthermore, since ITCH contains a consensus phosphorylation motif for LATS1 substrates, ITCH may be a substrate of LATS1. Understanding if LATS1 phosphorylates and alters ITCH activity therefore would be an interesting research avenue.
Fyn phosphorylation negatively regulates ITCH function
JNK is not the only kinase identified so far to target ITCH. Previous studies has indicated that Src-family tyrosine kinases are targeted for degradation by HECT-domain E3 ligase. Yang et al. therefore set out to investigate whether the Src-family kinase Fyn is ubiquitinated by ITCH in T cells, but instead discovered that ITCH is a substrate for Fyn. Fyn phosphorylates ITCH at Y371 located in the third WW domain. Importantly, an ITCH mutant where Y371 is replaced by phenylalanine causes a substantial increase in association of ITCH and one of its major substrate, JunB. Thus, the ubiquitin ligase activity of ITCH is regulated negatively by Fyn-mediated tyrosine phosphorylation and positively by JNK-mediated serine/threonine phosphorylation (Figure 3). Furthermore, Yang et al. found that ITCH Y371 to Phe mutation did not alter the self-ligase activity of ITCH in T cells, and hypothesised that Y371 phosphorylation results in a structural hindrance for JunB interaction. However, it remains unclear whether this tyrosine phosphorylation would affect the K63 self-ubiquitination of ITCH in other cell lines, such as HEK293, or whether it would affect Bid degradation and tBid-directed apoptosis. It is also open for investigation as to what are the inputs upstream of Fyn which triggers ITCH tyrosine phosphorylation. Nevertheless, it is intriguing to observe a signalling paradigm where two functionally opposing kinases act on a common E3 ligase to tune its activity. We anticipate this paradigm will become more commonly seen as more studies are carried out.
Quantitative modelling as a tool for analysis of ubiquitination-phosphorylation crosstalk networks
The last decade has witnessed an unprecedented explosion of biological knowledge and large data sets acquired for many signalling processes at the cellular level, largely due to the development of sophisticated and high-throughput biochemical techniques in proteomics and other omics. As part of this trend, the studies reviewed in the previous section, although still limited, have revealed a rather complex picture of how ubiquitination and phosphorylation interplay to regulate signal transduction pathways such as the EGFR. The huge complexity hampers our ability to interpret and predict the regulation of the network as a whole, which is essential to better understand EGFR signalling and its role in diseases. To unravel this complexity and obtain a systems-level understanding of network signalling, systems biology approaches employing quantitative frameworks in forms of mathematical and computational models are emerging as promising solutions. These mathematical models provide a platform for the description, prediction and understanding of the various regulatory mechanisms in a quantitative and integrative way[95–98]. In this section, we describe the rich and versatile dynamics of crosstalks between ubiquitination and phosphorylation by using mathematical modelling to analyse a number of network motifs largely motivated by the biological findings discussed in previous sections, and are commonly seen in other signalling processes besides the EGFR pathway.
Phosphorylation-mediated ubiquitination motif with feedback
Self-modification reactions can occur in either an intra-molecular or inter-molecular fashion, as depicted in Figure 7b. While our modelling analysis shows that the intra-molecular self-ubiquitination of the E3 ligase does not exhibit intricate dynamics, an inter-molecular form of self-ubiquitination, such as of ITCH discussed above, can bring about bistable behaviour to the system, even without the positive feedback loop[3, 13]. Figures 7c,d show bistability and hysteresis for the ubiquitinated forms of the ligase in response to the kinase abundance changes. Interestingly, E3-Ub and pE3-Ub have opposing off and on switches with the increasing kinase level. Similarly, the output also shows a bistable response, with the hysteresis curve being lower in the presence of high degradation rate (Figure 7e). Finally, we analyse motif 5 when the E3-to-Kinase positive feedback loop is also incorporated. Model analysis reveals that although self-ubiquitination or positive feedback alone is sufficient to give rise to bistability, adding the positive feedback appears to enhance self ubiquitination-induced bistability while adding self-ubiquitination does not necessarily enhance bistability established by the positive feedback (comparing blue to black curves, and blue to red in Figure 7f). Moreover, Figure 7f shows that the presence of both mechanisms brings the systems closer to irreversible hysteresis, indicated by the shift to the left of the corresponding hysteresis curve (blue line).
As discussed earlier, ubiquitination is a multi-step process which depends not only on the abundance and properties of the E3 ligase involved but also on other factors involving the preceding steps, including loading of ubiquitin onto conjugating enzymes E2s and ubiquitin transferring to the substrate. Consideration of these factors may be necessary for a detailed model of the control of the EGFR pathway by ubiquitination. Such work however would require comprehensive experimental effort to provide the missing kinetic data and other quantitative information to calibrate and validate the model.
Our findings of potentially bistable and oscillatory behaviour of the ubiquitination-phosphorylation motifs await experimental testing. In vitro experimental design based on the model analysis results could be the first step in confirming the predictions about the dynamics of the components of interest. An advantage of an in vitro system with purified forms of relevant E3 ligase, kinases and phosphatases is that it can be used to explore wide ranges of precisely set enzyme concentrations. To mimic the in vivo situation, some of these proteins may be embedded into a phospholipid membrane bilayer or liposomes if required, which can also facilitate the formation of protein complexes and increase reaction rates. For instance to detect oscillations in motif 4, the system can be started by addition of the relevant input signal, followed by addition of ubiquitin, the E1/E2 enzymes, E3 ligase, kinase and ATP to the reaction medium. At periodic selected time points, aliquots are taken, and the phosphorylated or ubiquitinated level of the substrate can be measured by immunoblotting using specific antibodies for phosphorylation or ubiquitination. It is however worth mentioning that assembling an oscillatory network in vitro is challenging due to a multitude of factors at play, including the adequate level of ubiquitin and the essential participation of the relevant E1/E2 enzymes. Therefore, direct in vivo approaches like imaging techniques using microscopy-based binding assay can be exploited for high temporal resolution measurements of components kinetics and may be a more favourable option. On the other hand, detection of switches such as in motif 5 can be done by similar measurement techniques in response to increasing titration of a dose component, in this case the involved kinase protein (Figure 7).
In summary, we have constructed mathematical models and carried out analysis for a number of commonly seen motifs of ubiquitination-phosphorylation crosstalk. The motifs, although simplified, show diverse dynamics including sustained oscillations and bistability. More importantly, the models have facilitated the identification of the conditions under which these dynamics may realise, which would have been infeasible if such models are not used. Modelling therefore provides a useful and necessary tool for efficient analysis of ubiquitination-phosphorylation crosstalk, thereby potentially improving our systems-level understanding of the integrated EGFR signalling.
Since the first discovery of protein ubiquitination more than three decades ago, extensive work has revolutionized our perception of its role in signalling networks. Not only protein ubiquitination serves as a main mechanism for protein degradation, emerging evidence has revealed that different types of ubiquitin chains can induce a variety of non-proteolytic functions and can dramatically alter the biological activities of a target protein. On top of that, ubiquitination is frequently observed to interplay with other PTMs such as phosphorylation or sumoylation to coordinate regulation of signalling processes in intricate manners. Such complexity arising from interconnected PTM networks poses enormous challenges for the systems level analysis of signalling processes. Mathematical modelling is emerging as a valuable tool to provide insight into their dynamic behaviour that would otherwise not be possible. Mathematical models help combine the mechanistic, molecular knowledge with rigorous analysis of the complex output dynamics of the PTM networks.
The expanding roles of ubiquitylation and phosphorylation in cell signalling, to large extent, have been uncovered thanks to recent advances in proteomics technologies which have enabled new ways for in-depth, unbiased and quantitative analysis of different PTMs on a global scale[106–110]. Techniques such as stable isotope labelling with amino acids in cell culture (SILAC) and label-free based mass spectrometry can quantify changes in expression of thousands of phosphoproteins and tens of thousands phosphorylation events in a single experiment and have become well established[106, 111]. Although proteome-wide analysis of endogenous ubiquitination has been more challenging, recent developments on antibodies-based enrichment methods demonstrate the feasibilities of similar large-scale, quantitative and site-specific investigations of this PTM. Moreover, novel methods that are aimed at identifying proteins comodified by both phosphorylation and ubiquitination have revealed exciting global details of the cross-regulation between these two PTMs. A major limitation with current mass spectrometry based methods however is the inability to distinguish among modifications by ubiquitination, NEDD8 or ISG15, due to an identical di-Gly remnant generated by trypsin proteolysis of the modified proteins. Nevertheless, it is likely that with the observed fast pace of technological advance, sophisticated methods capable of resolving at even higher quantitative resolution the extent of PTMs crosstalk and their distinct dynamics under different cellular perturbations are within close reach. These data will undoubtedly be valuable inputs to the construction of large-scale, next-level quantitative models. A global, data-driven modelling-based understanding of PTMs networks and the ability to simulate their behaviour and form testable predictions will open countless possibilities that can drive the frontiers of both biological and medical research.
Epidermal growth factor
Epidermal growth factor receptor
Extracellular signal-regulated kinase
Casitas b-lineage lymphoma
Receptor tyrosine kinase
Epidermal growth factor receptor substrate 15
Hepatocyte growth factor-regulated tyrosine kinase substrate
Homologous to the E6-AP carboxyl terminus
Signal transducing adaptor molecule
STAM binding protein
Ubiquitin specific peptidase 8
Ras-related protein Rab5
Guanine nucleotide exchange factor
Benzoquinone ansamycin Geldanamycin
c-Jun N-terminal kinase
MEK kinase 1
- ITCH Itchy:
E3 ubiquitin protein ligase
Neural precursor cell expressed developmentally down-regulated protein 4
Regulatory particle non-ATPase
Really interesting new gene 1 protein.
This work was supported by Science Foundation Ireland under Grant No. 06/CE/B1129 and the European Union Grant PRIMES No. FP7-HEALTH-2011-278568. We thank Alexander von Kriegsheim for critical reading of the manuscript.
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