|Home | About | Journals | Submit | Contact Us | Français|
Ubiquitination is an important posttranslational modification that plays a pivotal role in numerous biological functions, such as cell growth, proliferation, apoptosis, DNA damage response, innate immune response, and neuronal degeneration. Although ubiquitination is thought to achieve these functions by targeting proteins for proteasome-dependent degradation, recent studies suggest that ubiquitination also has nonproteolytic functions, such as protein trafficking, kinase and phosphatase activation, which are involved in cell survival and cancer development. These progresses have advanced our current understanding of the novel functions of ubiquitination in signal transduction pathways and may provide novel paradigms for the treatment of human cancers.
Protein kinases are important signaling mediators that can convey extracellular clues, such as growth factors, from outside of the cells to the nucleus by eliciting protein phosphorylation (Hynes & MacDonald, 2009; Klein & Levitzki, 2009). Phosphorylation is an important posttranslational modification known to regulate the activity and expression of protein kinases. Like phosphorylation, ubiquitination represents another important posttranslational modification that plays a pivotal role in a myriad of biological functions including cell growth, apoptosis, DNA damage repair, innate immune response, and neuron degeneration (Bhoj & Chen, 2009; Giasson & Lee, 2003; Hoeller & Dikic, 2009). Although the ubiquitination is traditionally viewed as an important signature to target proteins for proteasome-dependent degradation (Pickart, 2001), recent studies reveal that ubiquitination has other nonproteolytic functions involved in the regulation of protein localization and activity (Bhoj & Chen, 2009; Chen & Sun, 2009; Giasson & Lee, 2003; Hoeller & Dikic, 2009; Raiborg & Stenmark, 2009). These novel and interesting findings have opened up a new avenue for this posttranslational modification in regulating signal transduction pathways important for numerous biological functions.
Ubiquitin is a highly evolutionarily conserved protein consisting of 76-amino acid polypeptides. Ubiquitination is elicited by an enzymatic cascade involving three distinct classes of enzymes named E1 (ubiquitin activating enzyme), E2 (ubiquitin conjugating enzyme), and E3 (ubiquitin ligase) (Fig. 1). Ubiquitination is a covalent reaction that attaches the ubiquitin(s) to one or more lysine residues of proteins. Seven lysine (K) residues (K6, K11, K27, K29, K33, K48, and K63) are found in the ubiquitin, and polyubiquitin chains involving those sites have been identified. K48-linked ubiquitination is recognized by the 26S proteasome and triggers protein degradation (Fig. 1). K63-linked ubiquitination does not trigger protein degradation in vivo, although it may facilitate in vitro proteasome-dependent protein degradation like K48-linked ubiquitination (Hofmann & Pickart, 2001). Instead it plays non-proteolytic functions (Fig. 1) (Bhoj & Chen, 2009; Chen & Sun, 2009; Giasson & Lee, 2003; Hoeller & Dikic, 2009; Raiborg & Stenmark, 2009), Although other types of ubiquitin modifications are also observed, the exact role that they play is less understood. Recent studies suggest that they can be involved in either protein degradation or non-proteolytic functions (Fig. 1) (Adhikari & Chen, 2009; Heemers & Tindall, 2009; Xu et al., 2009).
In this review, we will summarize recent advances in the novel role of ubiquitination in regulating the localization and activity of protein kinases and phosphatases with an emphasis on the Akt ubiquitination and potential therapeutic strategies for cancers.
In general, the E2 determines which type of ubiquitin modification has occurred, whereas the E3 determines the substrate specificity. There are 2 E1s, roughly 50 E2s, and 600 E3s identified in human genome (Bhoj & Chen, 2009). Except for UBC13, most E2s trigger the K48-linked ubiquitination, in turn leading to proteasome-dependent degradation. UBC13, with the assistance of its cofactor UEV1A, is a major E2 known to induce K63-linked ubiquitination. The K63-linked ubiquitination provides a molecular platform for protein-protein interaction important for kinase signaling activation, receptor endocytosis, protein trafficking, and DNA damage repair.
The E3s are categorized into two major types, one consisting of a homologous to the HECT (E6-AP carboxyl terminus) domain and the other containing RING (a really interesting new gene) or RING-like domain (e.g. U-Box and PHD domain) (Bhoj & Chen, 2009). Most of E3s recognize target proteins for K48-linked ubiquitination. However, there are a few E3s, such as HectH9, Mdm2, TRAF6 (tumor necrosis factor receptor-associated factor 6), c-IAP1/2 (cellular inhibitor of apoptosis protein 1/2), and RNF8 (ring finger protein 8) targeting proteins for K63-linked ubiquitination (Adhikary et al., 2005; Bertrand et al., 2008; Bhoj & Chen, 2009; Huen et al., 2007; Mailand et al., 2007). HectH9, Mdm2, and c-IAP1/2 trigger not only K63-linked ubiquitination, but also K48-linked ubiquitination. For instance, HectH9 can not only elicit K63-linked Myc ubiquitination, which is an important step for Myc transcriptional activation, but can also trigger ubiquitination-dependent degradation of MCL-1 and p19Arf involved in apoptosis (Adhikary et al., 2005; Chen et al., 2005; Zhong et al., 2005). So far, TRAF6 and RNF8 are known E3s that selectively regulate K63-linked ubiquitination important for the innate immune response and DNA damage response, respectively (Bhoj & Chen, 2009; Huen et al., 2007; Mailand et al., 2007).
Akt is a serine/threonine protein kinase that plays a key role in mediating growth factor and cytokine signaling (Brazil et al., 2002; Cantley, 2002; Datta et al., 1999; Gonzalez & McGraw, 2009; Yang et al., 2010). It regulates numerous biological functions such as cell survival and metabolism by phosphorylating its downstream effectors. Akt is a relatively stable protein, and its protein levels are regulated by various stimuli that induce its ubiquitination. The stability of Akt is maintained by its binding to HSP90 (heat-shock protein 90), as inhibiting HSP90 activity by 17-AAG (17-allylamino-17-demethoxygeldanamycin) induces Akt ubiquitination and proteasome-dependent degradation (Basso et al., 2002; Solit et al., 2003). Subsequent experiments reveal that CHIP (carboxyl terminus of Hsc-70 interacting protein) induces Akt ubiquitination and its silencing prevents 17-AAG-mediated Akt degradation (Dickey et al., 2008), suggesting that CHIP is required for Akt degradation upon the inhibition of HSP90 activity (Fig. 2). Since HSP90 positively regulates Akt activity (Sato et al., 2000), it is possible that CHIP may preferentially target the inactive Akt. Additionally, Akt stability is also controlled by Akt T450 phosphorylation triggered by the mTORC2 containing mTOR, Sin1, Rictor, and Lst1, as Sin1 deficiency or mTOR knockdown impaired Akt T450 phosphorylation, accompanied by enhancing Akt ubiquitination and degradation (Facchinetti et al., 2008).
A recent study suggests that TTC3 (tetratricopeptide repeat domain 3) is an E3 ligase responsible for Akt ubiquitination and degradation (Suizu et al., 2009; Toker, 2009). TTC3 interacts with active Akt, but not inactive Akt, in the nucleus (Suizu et al., 2009; Toker, 2009), suggesting that TTC3-mediated Akt ubiquitination may be an important step to terminate nuclear Akt signaling. Interestingly, TTC3 is phosphorylated at S378 by Akt and this phosphorylation is required for TTC3 E3 ligase activity (Suizu et al., 2009; Toker, 2009), providing a negative feedback loop to attenuate Akt signaling. BRCA1 tumor suppressor is also shown to interact with active Akt and to induce its ubiquitination and degradation (Xiang et al., 2008). Thus, it appears that the stability of inactive Akt is regulated by CHIP, whereas that of active Akt is regulated by either TTC3 or BRCA1. Although it is unclear which type of Akt ubiquitination is triggered by these E3 ligases, it is highly possible that K48-linked ubiquitination may be involved (Fig. 2).
On the other hand, we found that Akt ubiquitination is also triggered by TRAF6 E3 ligase. TRAF6 interacts with and induces Akt ubiquitination in vitro and in vivo dependently of its E3 ligase activity (Yang et al., 2009; Yang et al., 2010), suggesting that TRAF6 is a direct E3 ligase for Akt (Fig. 2). Interestingly, this Akt ubiquitination occurs through the K63-linked modification and does not trigger Akt degradation. However, it facilitates Akt membrane recruitment and subsequent Akt phosphorylation and activation. The ubiquitination of Akt occurs in the K8 and K14 residues within the PH (pleckstrin homology) domain of Akt, and both Akt-K8R and Akt-K14R mutants display defects in Akt ubiquitination, membrane recruitment, and phosphorylation (Yang et al., 2009; Yang et al., 2010), highlighting the critical role of Akt ubiquitination and TRAF6 in Akt signaling activation.
TRAF6 is previously known to be involved in innate immune response and apoptosis by regulating Toll-like receptor and TGF-β signaling involved in NF-κB activation and p38 activation, respectively (Chen, 2005; Heldin et al., 2009; Sorrentino et al., 2008; Yamashita et al., 2008). Our study expands its known roles not only in the immune response, but also in growth factor receptor-mediated Akt signaling activation and cell survival.
The PH domain of Akt is known to be responsible for phosphoinositol (3,4,5) triphosphate (PIP3) binding critical for Akt membrane localization (Brazil et al., 2002; Varnai et al., 2005). The K14 residue is within the PIP3 binding pocket, while the K8 residue is not (Bellacosa et al., 1998; Carpten et al., 2007; Rong et al., 2001). As expected, the Akt-K14R mutant fails to bind to PIP3, whereas the Akt-K8R mutant retains its ability to interact with PIP3 (Yang et al., 2009). This result suggests that the regulation of Akt membrane recruitment by Akt ubiquitination does not result from its impact on the PIP3 binding.
Ubiquitination was previously shown to regulate the membrane localization and endocytosis of receptor proteins, such as epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF-1R), and prolactin receptor (Fallon et al., 2006; Monami et al., 2008; Varghese et al., 2008). As the K63-linked ubiquitination plays an important role in protein-protein interaction, the K63-linked ubiquitination of Akt may serve as a scaffold to recruit the adaptors for Akt, in turn facilitating Akt membrane recruitment and activation. We think that such Akt adaptors are likely to be ubiquitin-binding proteins (UBPs), which contain ubiquitin-binding domains (UBDs) capable of binding to monoubiquitin or polyubiquitin chains (Hicke et al., 2005). Several UBDs, such as UIM (ubiquitin-interacting motif), UBA (ubiquitin-associated) domain, CUE (coupling of ubiquitin conjugation to endoplasmic reticulum degradation) domain, GAT (Gga and TOM1), PAZ (polyubiquitin-associated zinc finger) domain, VHS (Vps27, HRS, STAM), NZF (Npl4 zinc finger) motif, GLUE (GRAM-like ubiquitin-binding in Eap45), and UEV (ubiquitin-conjugating enzyme variant) motif, are discovered (Hicke et al., 2005). Similar to ubiquitination, UBPs are also found to be involved in protein degradation, kinase activation, receptor endocytosis, and DNA damage response (Hicke et al., 2005).
Is TRAF6-mediated Akt ubiquitination relevant and important for human cancers? Akt-E17K mutant is found in a subset of human cancers, such as breast and colon cancer, and exhibits higher PIP3 binding, membrane localization, and activation (Brazil et al., 2002; Varnai et al., 2005). Interestingly, we found that this Akt mutant also displays a hyperubiquitination of Akt, and disruption of this hyperubiquitination of Akt profoundly impairs Akt membrane localization, phosphorylation, and activation (Carpten et al., 2007; Yang et al., 2009). Accordingly, these results suggest that the Akt-E17K mutant gains two important properties—hyperubiquitination and hyper-PIP3 binding. Both events appear to contribute to its constitutive membrane localization and activation.
Another Akt mutation E49K was recently identified in bladder cancers. Similar to the Akt-E17K mutant, the Akt-E49K mutant also displays Akt hyperphosphorylation and activation (Askham et al., 2010). Although this Akt mutation is also located at the PH domain of Akt, it is not in the PIP3 binding pocket, suggesting that the mutation on this site may not affect Akt binding to the PIP3. Since this Akt mutant gains an additional lysine residue, it is very likely that the Akt-E49K mutant may also display hyperubiquitination, in turn contributing to its hyperactivation and hyper-oncogenic potential.
K63-linked ubiquitination also regulates the localization and activity of other kinases. The IGF-1R is internalized into the early endosome and lipid raft compartments in response to various concentrations of IGF-1 (Sehat et al., 2008). The low dose of IGF-1 triggers K63-linked ubiquitination of IGF-1R (IGF-1 receptor), while high dose of IGF-1 induces K48-linked IGF-1R ubiquitination (Sehat et al., 2008). Mdm2 is shown to trigger K63-linked ubiquitination of IGF-1R and promotes the internalization of IGF-1R to the early endosome (Table 1). In contrast, c-Cbl induces K48-linked IGF-1R ubiquitination and promotes the internalization of IGF-1R to the lipid raft. In addition, NEDD4-1 also triggers the ubiquitination of IGF-1R and regulates its internalization to the early endosome and lipid raft (Monami et al., 2008). These results suggest that diverse types of ubiquitination may regulate the trafficking of IGFR to distinct compartments. However, it is unclear whether the ubiquitination of IGF-1R driven by these E3 ligases directly regulates the trafficking and signaling transduction of IGF-1R.
Although the activation of protein kinases is normally regulated by their phosphorylation, K63 ubiquitination also plays important roles in the activation of kinases involved in the Toll-like receptor and TGF-β receptor signaling. TAK1 (transforming growth factor-β activating kinase 1) is an important player that mediates NF-κB, p38, and JNK activation in response to the activation of Toll-like receptor and TGF-β receptor. Upon stimulation with TGF-β, TAK1 undergoes K63-linked ubiquitination, which is elicited by TRAF6 (Table 1) (Sorrentino et al., 2008; Yamashita et al., 2008). Ubiquitination of TAK1 on K34 is critical for TAK1 autophosphorylation and subsequent activation (Sorrentino et al., 2008), although the underlying mechanism by which it regulates TAK1 activation remains unclear. It is possible that K63-linked ubiquitination of TAK1 may induce TAK1 conformational change, which then induces its autophosphorylation and activation. Alternatively, it may serve as a scaffold to recruit the TAB2/TAB3 complex, which then triggers TAK1 autophosphorylation and activation. Once activated, TAK1 then triggers IKK activation, in turn activating NF-κB.
Other study further shows that the ubiquitination of TAK1 serves as a scaffold to recruit mitogen-activated MEKK3 (protein kinase kinase kinase 3) to activate the MAP kinase pathway (Yamazaki et al., 2009). Another example in which ubiquitination regulates kinase activation is MLK3 (mixed linage kinase 3) (Table 1). MLK3 is a family of mitogen-activated protein kinase kinase kinase (MAP3K) responsible for the activation of multiple MAPK pathways in response to various stimuli, such as tumor necrosis factor (TNF). The K63-linked ubiquitination of MLK3 triggered by TRAF6 is also critical for the activation of MLK3 (Korchnak et al., 2009).
Recent studies reveal that K63-linked ubiquitination of IKKγ (also known as NEMO) is also critical for IκB kinase (IKK) activation (Table 1). IKK, consisting of IKKα, IKKβ, and IKKγ regulatory subunit, is an upstream kinase responsible for IκB phosphorylation and degradation (Bhoj & Chen, 2009; Chen, 2005). Degradation of IκB releases NF-κB from heterdimerizing with IκB and facilitates NF-κB nuclear translocation and activation. Although IKKγ by itself does not contain kinase activity, its interaction with IKKα and IKKβ is required for IKK activation. Abin-1 is a UBP that promotes IKKγ K27-linked ubiquitination and degradation, thus inhibiting IKK activity and NF-κB activation (Ashida et al., 2010). However, IKKγ undergoes TRAF6-mediated K63-linked ubiquitination upon the engagement of T-cell receptor (TCR), which appears to be critical for IKK and NF-κB activation (Lynch & Gadina, 2004; Shambharkar et al., 2007; Sun et al., 2004).
RICK (also known as RIP2) is a caspase-recruitment domain-containing kinase critical for NF-κB activation upon Nod1 and Nod2 stimulation (Inohara et al., 2000; Park et al., 2007). RICK is also known to undergo K63-linked ubiquitination upon Nod1 and Nod2 stimulation (Table 1). The RICK ubiquitination on K209 located within its kinase domain triggers the oligomerization of RICK and the recruitment of TAK1, in turn facilitating IKK activation (Hasegawa et al., 2008).
In addition, K63-linked ubiquitination of IRAK-1 (IL-1 receptor associated kinase-1) involved in Toll-like receptor and IL-1R-mediated NF-κB activation is also critical for IRAK-1 activation (Table 1). Upon IL-1 treatment, IRAK1 undergoes K63-linked ubiquitination presumably by E3 ligase TRAF6, which then recruits and activate IKKγ and NF-κB (Conze et al., 2008). Accordingly, these results suggest that K63-linked ubiquitination appears to be an important step for kinase activation.
In contrast to what protein kinase do, protein phosphatase removes the phosphate group from target proteins or lipids, therefore regulating their activity and functions (Shi, 2009). Although the activity of phosphatases is regulated by their phosphorylation, recent studies suggest that K63-linked ubiquitination may also be an important posttranslational modification to regulate the localization and activity of phosphatases. One phosphatase whose function is regulated by ubiquitination is PTEN (Phosphatase and tensin homolog) tumor suppressor (Table 2). PTEN, whose function is frequently lost in various human cancers, dephosphorylates PIP3 and negatively regulates Akt signaling to suppress tumor development (Di Cristofano & Pandolfi, 2000; Salmena et al., 2008). The ubiquitination of PTEN is driven by NEDD4-1 and is shown to be critical for nuclear localization and stability of PTEN (Trotman et al., 2007; Wang et al., 2007). It is proposed that PTEN nuclear localization appears to be important for its tumor suppressive functions such as cell cycle arrest and apoptosis (Chang et al., 2008; Trotman et al., 2007), although the exact function of PTEN in the nucleus remains to be determined. Interestingly, the PTEN mutant identified from the Cowden patients displays a profound reduction in PTEN ubiquitination, accompanied by the impairment of PTEN nuclear import (Trotman et al., 2007). This result suggests that the defect in PTEN ubiquitination may contribute to the development of Cowden disease.
Although NEDD4-1 triggers PTEN ubiquitination, which is critical for PTEN nuclear translocation in the in vitro knockdown system, the NEDD4-1 deficient MEFs display neither the impairment of PTEN ubiquitination nor the defect of PTEN nuclear localization (Fouladkou et al., 2008), suggesting that other E3 ligases are likely involved. XIAP (X-linked inhibitor of apoptosis protein) may be another E3 ligase for PTEN ubiquitination, as XIAP is shown to interact with PTEN and promotes PTEN ubiquitination (Van Themsche et al., 2009). XIAP−/− MEFs display the impairment in ubiquitination and nuclear localization of PTEN (Van Themsche et al., 2009). These results underscore the important role of PTEN ubiquitination in PTEN nuclear translocation, although it remains to be determined whether XIAP regulates the tumor suppressive function of PTEN.
SHIP2 (SH2-containing 5′-inositol phosphatase), which dephosphorylate PIP3 and negatively regulates PI3K (phosphoinositol 3-kinase)/Akt signaling(Lazar & Saltiel, 2006), also undergoes ubiquitination (Table 2). Ubiquitination of SHIP2 induced by growth factor EGF (epidermal growth factor) prevents SHIP2 binding to c-Cbl (De Schutter et al., 2009). Although c-Cbl interacts with SHIP2, it does not induce the ubiquitination of SHIP2. Future experiment aiming at defining the E3 ligase and the functional relevance of SHIP2 ubiquitination will be warranted.
Another phosphatase whose activity is regulated by ubiquitination is PP2A (protein phosphatase 2A). PP2A is a serine/threonine phosphatase that terminates Akt signaling by dephosphorylating Akt at T308 (Tremblay & Giguere, 2008; Trotman et al., 2006). PP2A consists of scaffold A subunit, regulatory B subunit, and catalytic c subunit (PP2Ac). Ubiquitination of PP2Ac perhaps by E3 ligase MID1 leads to PP2Ac proteasome-dependent degradation (Table 2) (Trockenbacher et al., 2001). Alpha 4 is a PP2Ac interacting protein whose function is to bridge the interaction between MID1 and PP2A (McConnell et al.). Alpha 4 null cells display enhanced PP2Ac ubiquitination and degradation, accompanied by a reduction in PP2A phosphatase activity (Kong et al., 2007). Since the ubiquitination of PP2A promotes PP2Ac degradation, it is very likely that this ubiquitination may act through K48-linked modification.
The ubiquitination is a reversible process in which the ubiquitin on proteins can be removed by the deubiquitinating enzymes (DUBs). 90 DUBs are present in human genome that can be divided into five subfamilies: ubiquitin-specific protease (USP), ubiquitin carboxyl-terminal hydrolase (UCH), ovarian tumor-like protease (OTU), JAMM/MPN metalloprotease, and Machado–Jakob-disease protease (MJD) (Bhoj & Chen, 2009; Hoeller & Dikic, 2009; Sun, 2009). Among them, the USP family proteins (USPs) are relatively well studied with known substrates (see below).
The DUBs are known to play important roles in controlling key signal transduction pathways involved in tumor suppressor and oncogenic pathways such as PTEN, p53, and NF-κB pathways (Bhoj & Chen, 2009; Salmena et al., 2008). One of the hallmarks for the DUBs is that UBDs are found in most DUBs, which allow them to interact with the ubiquitinated substrates and trigger protein deubiquitination. The USP7 (also known as HAUSP) binds to the ubiquitinated PTEN in the nucleus and induces the deubiquitination and nuclear export of PTEN (Table 3) (Song et al., 2008).
USP7 also interacts with the polyubiquitinated p53 and promotes p53 deubiquitination and stability (Li et al., 2002). However, USP7 deficiency paradoxically promotes p53 stabilization (Cummins & Vogelstein, 2004; Kon et al., 2009). This can be explained in part by the fact that USP7 also binds to polyubiquitinated Mdm2 and promotes Mdm2 stability (Brooks et al., 2007; Meulmeester et al., 2005a; Meulmeester et al., 2005b; Tang et al., 2006). Accordingly, these results suggest that USP7 may be an oncogenic event that can promote cancer development. Consistent with this notion, USP7 is amplified in human prostate cancer and its deficiency restricts cell proliferation (Song et al., 2008).
Apart from USP7, recent study suggests that USP10 is also a DUB for p53 (Table 3). Mdm2 is previously known to be an E3 ligase for p53 and promotes p53 nuclear export and degradation (Li et al., 2003). USP10 deubiquitinates p53 and reverses Mdm2-mediated p53 nuclear export and degradation (Yuan et al., 2010), suggesting that USP10 may act as a tumor suppressor by activating p53. Consistent with this notion, USP10 expression is frequently lost in renal cell carcinoma, and overexpression of USP10 suppresses cell transformation and tumorigenesis in a p53-dependent fashion (Yuan et al., 2010).
Accumulating evidence suggests that ubiquitination and degradation of oncogenic proteins are also regulated by USPs. For example, USP28 binds to Myc and causes the deubiquitination of Myc, resulting in protecting Myc from degradation (Table 3) (Amati & Sanchez-Arevalo Lobo, 2007; Popov et al., 2007). Importantly, USP28 is overexpressed in cancers, and its silencing reduces cell proliferation and transformation (Popov et al., 2007), suggesting that USP28 displays an oncogenic activity. Recent study reveals that USP2 is a DUB for Cyclin D1 and Mdm2. USP2 binds to Cyclin D1 and Mdm2 and induces their deubiquitination and stabilization, and its silencing causes downregulation of Cyclin D1 and Mdm2, leading to inhibiting cell proliferation (Shan et al., 2009; Stevenson et al., 2007).
A20 and CYLD (cylindromatosis tumor suppressor) are DUBs that negatively regulate NF-κB activation by inducing the deubiquitination of IKKγ and TRAF2, respectively (Table 3) (Bhoj & Chen, 2009; Brummelkamp et al., 2003; Kovalenko et al., 2003; Mauro et al., 2006; Sun, 2008; Sun, 2009). In line with the role of CYLD and A20 in NF-κB regulation, recent genetic evidence reveals that CYLD deficiency in mice or CYLD and A20 gene mutations in patients leads to NF-κB hyperactivation. This is accompanied by tumor susceptibility or tumor formation (Compagno et al., 2009; Kato et al., 2009; Novak et al., 2009; Sun, 2009).
As aforementioned, the ubiquitination and deubiquitination mediated through the E3 ligases and DUBs regulate the activity and functions of tumor suppressors and oncogenes important for cell proliferation, apoptosis, and tumorigenesis. Importantly, the deregulation of the E3 and DUBs is frequently found in human cancer, and mouse tumor models have revealed their important roles in cancer development, suggesting that targeting these molecules may have important therapeutic usage for the treatment of human cancer (Fig. 3).
Akt signaling is an important oncogenic event, and aberrant Akt activation is associated with a variety of human cancers (Brazil et al., 2002; Cantley, 2002; Lin et al., 2010; Lin et al., 2009). Given the important role of the Akt signal in cancers, small molecule inhibitors targeting Akt have been developed and tested in the clinical trial (Hennessy et al., 2005; Liu et al., 2009; Wu et al., 2009). As we recently identified TRAF6 as a critical E3 ligase for Akt ubiquitination, membrane localization, and activation, small molecules targeting TRAF6 may be considered as single or adjuvant agents for the treatment of human cancer (Fig. 3a). Consistent with this notion, TRAF6 knockdown in prostate cancer cells reduces Akt activation and prostate cancer development (Yang et al., 2009). Furthermore, TRAF6 deficiency potentiates apoptosis induced by chemotherapy agents (Yang et al., 2009). Therefore, TRAF6 targeting can be also applied as an adjuvant approach to enhance the sensitivity of cancer cells to chemotherapy agents.
In addition to designing small molecule inhibitors for TRAF6, the manipulation of the microRNA (miR) pathways that negatively regulate TRAF6 expression can also be considered. Indeed, miR-145 and miR-146a were shown to repress TRAF6 protein translation and displayed a potent tumor suppressive effect on restricting the formation of primary and metastatic breast cancer (Hou et al., 2009; Hurst et al., 2009; Starczynowski et al., 2010; Yuan et al., 2010), suggesting that the therapeutic strategy by applying miR-145 and miR-146a for cancer treatment can also be considered.
Myc overexpression is associated with human cancers and targeting Myc was recently proven to be very effective in the treatment of human cancers (Adhikary & Eilers, 2005; Herold et al., 2009; Soucek et al., 2008). Cyclin D1 is also upregulated in human cancers, and its inactivation abolishes breast cancer development driven by oncogenic Ras or Neu (Lee et al., 2000; Yu et al., 2001). Although Cyclin D1 and Myc are important players for cancers, it may not be ideal to design small molecule inhibitors to target them, which lack enzymatic activities. Alternatively, therapeutic strategies for targeting USP28 and USP2 can be considered (Fig. 3b), as they are shown to play important roles in stabilizing oncogenic signals, such as Myc and Cyclin D1. Since USP2 and USP28 are DUBs, it is theoretically possible to design small molecule inhibitors to inhibit their deubiquitinating activity.
As USP7 silencing is shown to stabilize and enhance p53 activity, small molecule inhibitors targeting USP7 is expected to show an inhibitory effect on cancer cell growth. Indeed, a novel USP7 inhibitor was recently discovered and shown to induce p53-dependent apoptosis (Colland et al., 2009). Future experiments utilizing small molecule inhibitors targeting these Dubs in preclinical mouse tumor models may provide the proof of principle evidence that targeting the Dubs can be ideal therapeutic strategies for cancers (Fig. 3c).
Recent advances in the area of the ubiquitination study have unraveled several novel roles of ubiquitination in signaling transduction pathways involved in a wide range of biological functions. Depending on which type of the polyubiquitin chains formed within the proteins, ubiquitination can play a distinct role in the regulation of protein fate. K48-linked ubiquitination serves as a mark for protein degradation, whereas K63-linkd ubiquitination provides a scaffold for protein/protein interaction, in turn regulating the localization and activity of protein kinases and phosphates. Thus, this posttranslational modification has elicited an exciting avenue that has advanced our current understandings of how ubiquitination regulates protein degradation and activation.
Given the important role of ubiquitination in cancers, the effort towards designing small molecule inhibitors targeting this pathway has now been actively pursued. Some small molecule inhibitors targeting certain E3 ligases have developed and tested in clinical trials for cancers. The preliminary results have yielded some positive results, and we anticipate that more and more promising compounds targeting this pathway will be developed and eventually utilized for cancers.
Several outstanding questions remain mysterious and warrant for future investigations. Are there more E3s that can trigger K63-linked ubiquitination? How do the E3s and DUBs coordinate to regulate signaling activation for kinases and phosphatases? How does the ubiquitination regulate the localization and activation of kinases and phosphatases? Is ubiquitination a common mechanism to regulate the localization and activity of kinases and phosphatases? Addressing these important questions will lead to comprehensive understanding of the complicate mode of ubiquitination in signaling transduction pathways and shed new light on how growth factor receptor signaling is regulated.
We thank the members of the Lin’s lab for their comments and suggestions. Special thanks extend to Yuan Gao for her critical reading and editing on our manuscript. We apologize to many investigators whose important works were not cited here due to space limitations. This work is supported by M.D. Anderson Research Trust Scholar Fund, a RO1 grant from NCI, and New Investigator Award from Department of Defense to H.K. Lin.
Conflict of interest
The authors declare no conflict of interest.