E3 ubiquitin ligases in ErbB receptor quantity control

Seminars in Cell & Developmental Biology 21 (2010) 936–943

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Review

E3 ubiquitin ligases in ErbB receptor quantity control夽 Kermit L. Carraway III ∗ UC Davis Cancer Center, Research Building III, Room 1400, 4645 2nd Avenue, Sacramento, CA 95817, United States

a r t i c l e

i n f o

Article history: Available online 22 September 2010 Keywords: ErbB receptor E3 ubiqutin ligase Ubiquitination Degradation Cancer

a b s t r a c t Signaling through ErbB family growth factor receptor tyrosine kinases is necessary for the development and homeostasis of a wide variety of tissue types. However, the intensity of receptor-mediated cellular signaling must fall within a precise range; insufficient signaling can lead to developmental abnormalities or tissue atrophy, while over-signaling can lead to hyperplastic and ultimately neoplastic events. While a plethora of mechanisms have been described that regulate downstream signaling events, it appears that cells also utilize various mechanisms to regulate their ErbB receptor levels. Such mechanisms are collectively termed “ErbB receptor quantity control.” Notably, studies over the past few years have highlighted roles for post-transcriptional processes, particularly protein degradation, in ErbB quantity control. Here the involvement of ErbB-directed E3 ubiquitin ligases is discussed, including Nrdp1-mediated ErbB3 degradation, ErbB4 degradation mediated by Nedd4 family E3 ligases, and CHIP-mediated ErbB2 degradation. The hypothesis is forwarded that protein degradation-based ErbB quantity control mechanisms play central roles in suppressing receptor overexpression in normal cells, and that the loss of such mechanisms could facilitate the onset or progression of ErbB-dependent tumors. © 2010 Elsevier Ltd. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. ErbB receptor overexpression in cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. E3 ubiquitin ligase-mediated protein ubiquitination and degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Post-transcriptional regulation of ErbB receptor levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Mouse models of ErbB2 overexpression-induced mammary cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Ligand-induced suppression of ErbB receptor levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nrdp1-mediated ErbB3 degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Characterization of Nrdp1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Nrdp1 regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Nrdp1 involvement in ErbB3 overexpression in mammary tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ErbB4 degradation mediated by Nedd4 family proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Itch-mediated ErbB4 degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. WWP1-mediated ErbB4 degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Degradation of soluble ErbB4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CHIP-dependent ErbB2 degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary and concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction 1.1. ErbB receptor overexpression in cancer The four members of the ErbB family of receptor tyrosine kinases are necessary for the development and maintenance of a variety of

夽 The author is supported by NIH grants GM068994 and CA123541. ∗ Tel.: +1 916 734 3114; fax: +1 916 734 0190. E-mail address: [email protected]. 1084-9521/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcdb.2010.09.006

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tissue types, including heart, lung, skeletal muscle, and the central and peripheral nervous systems [1–4]. Overexpression and concomitant aberrant activation of ErbB receptors have been implicated in the development and progression of a variety of solid tumor types [5,6]. For example, overexpression of epidermal growth factor receptor [ErbB1 or EGFR] or its oncogenic splice variants is found in over 50% of malignant gliomas, 30–90% of advanced colorectal tumors, and is a common feature of lung, breast, head and neck, and other epithelial cancers [7–9]. A particularly well-studied example, erbB2 gene amplification is observed in 25–30% of breast cancer

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patients and overexpression of the ErbB2 protein correlates with poor prognosis [10–12]. erbB2 amplification and overexpression are also common to other carcinomas as well, including tumors of the ovary, bladder and gastrointestinal tract [13]. Countless studies with cultured cells suggest that ErbB2 overexpression is sufficient to activate its protein tyrosine kinase activity and that ErbB2 signaling mediates cellular transformation. ErbB2 overexpression in the mouse mammary gland is sufficient to induce metastatic tumors [14], underscoring its oncogenic potential. Collectively, expression and functional studies point to a model where EGFR and ErbB2 overexpression enhances the signaling capacity of these receptors, in turn facilitating the initiation and progression of solid tumors. Although it has been clear for over 15 years that the properties of ErbB3 place it at the center of oncogenic signaling, an appreciation for the role of ErbB3 in tumor biology is just now beginning to develop [15–17]. ErbB3 harbors an intrinsically impaired kinase activity [18], and thus has little oncogenic activity on its own [19]. However its strong propensity to heterodimerize with and stimulate ErbB2 [16,20–22], as well as its unique ability to very efficiently engage the PI3-kinase oncogenic pathway [23,24], raise the possibility that ErbB3 could play a necessary role in oncogenesis induced by other ErbB receptors. Indeed, it is has been suggested that signaling by the ErbB2–ErbB3 heterodimer is the most potent of the ErbB signaling species [22], and the ErbB2–ErbB3 complex is a proposed oncogenic unit [25]. Consistent with this, preclinical studies support a key function for ErbB3 in promoting the growth properties of ErbB2-positive breast cancer cells [26]. Importantly, several reports also point to roles for ErbB3 in conferring resistance to cancer therapeutics [16,17,27–29]. ErbB3 overexpression has also been observed in solid tumors. For example, its overexpression relative to normal tissue has been reported in up to 63% of primary breast tumors [30] and has been linked to lymph node metastases, recurrence and poor prognosis [31,32]. Several studies have established a strong link between the coordinate overexpression and activation of ErbB2 and ErbB3 in breast tumor cell lines and patient samples [32,33]. Finally, the role of ErbB4 in cancer biology remains a question, with studies pointing to both oncogenic and tumor suppressive roles for the receptor [34,35]. As discussed further below, some confusion may arise from the existence of erbB4 splice variations that give rise to receptor proteins with markedly differing signaling properties. These splice variants are not easily distinguished in patient samples using standard staining methods and reagents, complicating the interpretation of outcomes studies [35]. Collectively, these observations confirm that ErbB overexpression is a lesion common to solid tumors, underscore the active contributions of overexpressed proteins to tumor cell growth properties, and point to the potential of ErbB-directed therapies. However, while EGFR and ErbB2-directed antibody and small molecule inhibitors have exhibited some clinical efficacy [6,36], and ErbB3-directed inhibitors are under development [16,17], inherent and acquired resistance to these therapeutics remains a vexing clinical problem [36]. A more thorough understanding of ErbB receptor overexpression mechanisms may offer new avenues of therapeutic intervention [32,37].

energy of ATP to activate ubiquitin, covalently attaching its terminal carboxylate group to a cysteine residue within the enzyme. The thioester-linked ubiquitin is then transferred to a specific cysteine residue of E2 ubiquitin conjugating enzymes in preparation for target protein ubiquitination. Finally, E3 ubiquitin ligases mediate the transfer of ubiquitin from an E2 to a target protein, either by physically bringing ubiquitin-charged E2 proteins together with targets to mediate transfer or by serving as thioester intermediates themselves. The ubiquitin moiety is then often recognized by specific intracellular proteins that influence the activity, localization or stability of the target protein. Most ubiquitination events that have been characterized thus far involve the transfer of ubiquitin moieties to the ␧-amino group of a lysine residue within the target protein to form an isopeptide bond. However, ubiquitination of the amino terminus has been demonstrated to mediate the degradation of some proteins [42,43], and the ubiquitination of cysteine, serine and threonine residues within proteins has been reported [44,45]. Importantly, ubiquitin itself has seven lysine residues, each of which may be ubiquitinated in an iterative manner to give rise to polyubiquitinated target proteins. Canonically, proteins polyubiquitinated through ubiquitin lysine 48 (K48) are recognized for degradation by proteins of the 26S proteasome, while monoubiquitinated membrane proteins are often trafficked to the lysosome for degradation [38,46,47]. As the factors responsible for coupling intracellular proteins to the degradation machinery, E3 ligases play central roles in governing the stability of proteins essential to cellular function. These proteins play key roles in essentially all major cellular processes, including cell cycle, viability, differentiation, gene transcription, DNA repair, protein trafficking, and ER quality control, to name a few. E3 ubiquitin ligases are responsible for the specificity of ubiquitin-mediated degradation, determining which target proteins are degraded and how efficiently that process is carried out. E3 ligases are multi-domain proteins containing one or more domains that couple to E2 enzymes and at least one other domain that recognizes target proteins to be degraded. The three major subgroups of E3 ligases contain a RING (Really Interesting New Gene) domain, a HECT (Homologous to E6-AP Type) domain or a U-box, each of which is responsible for binding ubiquitin conjugating enzymes [41]. E3 ligases, and thus the efficiency of degradation of their target proteins, may be regulated at a variety of levels. First, there are numerous examples of E3 ligases that are regulated by abundance, and in some instances autoubiquitination and degradation have been implicated in the regulation of these proteins [48–50]. In addition, post-translational modifications such as phosphorylation, ubiquitination and modification by ubiquitin-like proteins such as Nedd8 have been demonstrated to play central roles in the regulation of E3 ligases [51–56]. Not surprisingly, the dysregulation of E3 ligase function has been implicated in various disease states such as neurodegenerative disorders and cancer [57–61]. This review will focus on the role of E3 ubiquitin ligases in governing the steady-state levels of ErbB receptor tyrosine kinases, emphasizing the notion that the dysregulation of these factors in cells may have profound effects on receptor overexpression and hyper-activity.

1.2. E3 ubiquitin ligase-mediated protein ubiquitination and degradation

2. Post-transcriptional regulation of ErbB receptor levels

Cells make extensive use of the ubiquitin system to control the abundance of key signaling and regulatory proteins, and accumulating evidence suggests that ErbB trafficking and degradation is mediated by receptor ubiquitination [38–40]. In general, the small protein ubiquitin is covalently attached to proteins destined for degradation as a result of the coordinated actions of three classes of enzymes [41]. E1 ubiquitin activating enzymes use the

While much effort over the past 15–20 years has gone into understanding the mechanisms by which ErbB overexpression and aberrant activation contribute to the cellular properties associated with malignancy, relatively little effort has been put into understanding how these proteins become overexpressed by tumors. Some evidence has accumulated for aberrant transcriptional regulation in promoting receptor overexpression. For example, FOXP3

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is an X-linked breast tumor suppressor transcriptional regulator that represses erbB2 gene expression, and deletion, somatic mutations, and downregulation of the foxp3 gene correlate with ErbB2 overexpression in patient breast cancer samples [62]. On the other hand, ZNF217 is a transcriptional regulator amplified at the 20q13 locus in approximately 20% of breast tumors whose product expression in breast cancer correlates with ErbB3 protein levels. Znf217 expression and knockdown elevate and suppress, respectively, erbB3 gene expression in cultured breast cancer cells [63]. Thus, erbB transcriptional regulation likely contributes to receptor overexpression in a population of breast tumors. However, by far the most widely acknowledged mechanism implicated in ErbB receptor overexpression involves gene amplification, where gene doses up to 20-fold over normal are thought to give rise to protein overexpression. Most notably, numerous publications have found a strong correlation between erbB2 gene amplification and ErbB2 protein overexpression, and it is generally accepted that elevated gene dosage in tumors is sufficient to drive ErbB2 overexpression. However, ErbB2 protein overexpression is observed in the absence of gene amplification [11,12], and amplification of the erbB3 gene in tumors is rare despite the relative frequency of protein overexpression [64,65]. These observations indicate that other mechanisms may also contribute to aberrantly high ErbB protein accumulation in cells. 2.1. Mouse models of ErbB2 overexpression-induced mammary cancer Transgenic mouse models of ErbB2 overexpression-induced mammary tumorigenesis point to the intriguing notion that posttranscriptional events play key roles in allowing ErbB receptors to become overexpressed during tumor development. As noted above, transgenic overexpression of ErbB2 protein using the murine mammary tumor virus (MMTV) promoter/enhancer gives rise to metastatic mammary tumors [14], underscoring the malignant potential of this receptor. However, tumors in this model develop with a significantly longer latency and lower frequency than in many other oncogene models, suggesting that additional processes might be necessary to drive tumorigenesis. Indeed, analysis of the erbB2 transgene itself in this model revealed small deletions within the extracellular juxtamembrane region of the receptor that facilitate receptor homodimerization and constitutive signaling [66,67]. However, transgenic expression of these deleted forms in the mammary gland still yield low tumor rates and latencies [68], suggesting further rate-limiting events may be required prior to tumor onset. Curiously, our lab has observed that ErbB2 protein levels in uninvolved (non-tumor) mammary tissue from ErbB2 transgenic animals are similar to levels in wild-type animals [32,69]. Hence, the elevated ErbB2 transcript derived from the transgene is not sufficient to produce elevated ErbB2 protein in the mammary gland, suggesting that normal mammary epithelial cells harbor mechanisms that suppress ErbB2 protein overexpression. Moreover, as transgenic overexpression in the mouse is a model for gene amplification in patients, these observations raise the possibility that erbB2 gene amplification alone may not be sufficient to elicit ErbB2 protein overexpression in breast tumors. In contrast, tumors from transgenic mice overexpress ErbB2 protein by 10–100-fold compared with uninvolved tissue when normalized for epithelial content [30,69; see Fig. 1A]. These observations suggest that tumor cells dismantle mechanisms that keep ErbB2 protein in check in normal tissue, permitting transgene-driven receptor overexpression. Interestingly, ErbB2 overexpression-induced mouse mammary tumors also markedly overexpress endogenous ErbB3 protein relative to uninvolved tissue [30,68,69; Fig. 1A], underscoring synergistic roles of the two receptors in promoting tumor onset and

Fig. 1. Post-transcriptional upregulation of ErbB receptor proteins in mouse mammary tumors (Refs. [30,69,92]). (A) Normal (N) and tumor (T) mammary tissue from two FvB strain MMTV-ErbB2 transgenic mice were analyzed by immunoblotting for ErbB2, ErbB3, Nrdp1 and cytokeratin-18. (B) Real-time RT-PCR analysis was carried out with normal and tumor tissue from four mice.

growth. Remarkably, despite enormous differences in the levels of ErbB2 and ErbB3 protein produced by uninvolved and tumor tissue in nulliparous animals, real-time PCR indicates that transcript levels for the two genes are virtually identical when normalized for epithelial content [30,32,69; Fig. 1B]. These observations indicate that ErbB negative regulatory pathways that influence protein translation or stability are responsible for the gatekeeper mechanisms that keep ErbB protein levels in check in normal tissue. Overall, mouse modeling reveals that normal mammary epithelial cells harbor powerful post-transcriptional mechanisms that suppress ErbB protein overexpression. Such ErbB quantity control mechanisms undoubtedly serve as a barrier to oversignaling, preventing ErbB signaling-induced hyperplastic events at times of tissue quiescence. Since ErbB protein overexpression coincides with tumor onset it is possible that the loss of these ErbB negative regulatory mechanisms is requisite for ErbB2-induced tumor formation. It is also possible that these mechanisms are relieved during periods of growth. For example, as ErbB receptors have been demonstrated to contribute to epithelial outgrowth during puberty and to proliferation and differentiation during pregnancy [70–72], it is possible that the partial suppression of ErbB negative regulatory processes couples with hormonal regulatory events [73] to allow sufficient receptor accumulation and signaling to support these processes. During the transition to tumor, preneoplastic cells could mimic these events to facilitate ErbB receptor overexpression and promote tumor initiation and progression. 2.2. Ligand-induced suppression of ErbB receptor levels Lysosome-mediated protein degradation is a mechanism widely employed by cells to suppress receptor tyrosine kinase function following ligand activation as a means of preventing oversignaling. Growth factor stimulation causes many receptor tyrosine kinases to localize to clathrin coated pits, internalize, and traffic to endosomes. Receptors are sorted in endosomes according to whether they are to be recycled to the cell surface or degraded in lysosomes based on their ubiquitination state. EGFR is a very well-studied model of ligand-induced down-regulation and degradation of cell surface receptors [40]. Ligand binding stimulates the multiple monoubiquitination and K63 polyubiquitination of EGFR [46,74,75], and it has been demonstrated that monoubiquitination is sufficient to drive EGFR internalization and degradation [46,74]. EGF receptor ubiquitination is mediated at least in part by the RING finger E3 ubiquitin ligase cbl [76], which is recruited to ligand-stimulated EGFR by binding to phosphorylated receptor tyrosine 1045 [77–79]. cbl-mediated ubiquitination of specific lysine residues of the kinase domain [75] facilitates receptor sorting into endosomal multivesicular bodies and ultimately trafficking to lysosomes for degradation. While it is clear that cbl plays a key role in mediating the degradation of activated EGF receptor, cbl interaction with ErbB2 occurs

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under artificial circumstances [80–83] and its interaction with ErbB3 or ErbB4 has not been observed [80]. Curiously, both ErbB2 and ErbB4 contain putative phosphorylation sites with surrounding sequences that are highly homologous to EGFR Y1045. Substitution of these sequences into EGFR preserves EGF-stimulated cbl interaction [84], suggesting that cbl-mediated degradation of activated ErbB2 and ErbB4 may play physiological roles that remain to be delineated. It has also been suggested that ErbB2, ErbB3 and ErbB4 are impaired relative to EGFR in terns of their ligand-induced internalization, down-regulation and degradation [85]. However, we have observed that the growth factor NRG1␤ induces ErbB2 and ErbB3 degradation in a subset of cell lines [86; unpublished observations], raising the possibility that ErbB degradation mechanisms may be context-dependent. Cbl-mediated EGFR down-regulation establishes a paradigm for the suppression of ErbB receptor levels, underscoring the notion that E3 ubiquitin ligases play central roles in ErbB receptor degradation. However, the example of ligand-stimulated ligase recruitment cannot be extended to account for ErbB quantity control because these mechanisms are operative even in the absence of growth factor. Thus it is likely that other E3 ligases additionally act on ErbB receptors independent of ligand stimulation, and indeed three examples of this phenomenon have emerged over the last 10 years. 3. Nrdp1-mediated ErbB3 degradation Work from our lab has established that a particular RING finger domain-containing E3 ubiquitin ligase, which we named Nrdp1 (Neuregulin receptor degradation protein-1), controls steadystate levels of the ErbB3 and ErbB4 receptors. We identified Nrdp1 in a yeast two-hybrid screen for proteins that interact with the intracellular domain of ErbB3 [87]. As ErbB3 lacks significant autophosphorylation activity [18], the screen was specifically geared to identify proteins that interact in an activation-independent manner; such proteins would likely be involved in constitutive receptor trafficking or localization of receptors to membrane subdomains. 3.1. Characterization of Nrdp1 Nrdp1 is a member of a large family of proteins termed RBCC (Ring finger, B-box, coiled-coil) or TRIM (tripartite motif) based on their domain organizations. The amino terminal regions of RBCC proteins are characterized by a RING finger domain, thought to couple to E2 ubiquitin conjugating enzymes, followed by a B-box zinc binding domain and a coiled-coil motif. Their carboxy terminal substrate binding regions diverge markedly, possibly accounting for the pleiotropic activities of RBCC family members. RBCC proteins are involved in numerous cellular processes including apoptosis, cell cycle control and viral response [88]. The fragment of Nrdp1 identified by the yeast screen, which we call clone 32, encompasses its coiled-coil and carboxy terminal regions and corresponds to a naturally occurring splice variant that lacks the amino terminal RING finger and B-box domains. The crystal structure of the Nrdp1 carboxy terminal domain has been solved and represents a novel fold [89], and amino acids critical for ErbB3 binding have been identified [90]. We found that Nrdp1 co-immunoprecipitates with ErbB3 and ErbB4, mediates receptor ubiquitination, and suppresses the levels of these receptors but not EGF receptor or ErbB2 when expressed in cells [30,87,91]. These activities occur in the absence of added growth factor, indicating that Nrdp1 acts to regulate steady-state levels of receptor. Nrdp1 knockdown or expression of clone 32 markedly stabilizes endogenous ErbB3 [30], often by 10-fold or more, suggesting that the vast majority of the ErbB3 protein syn-

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thesized by some cell lines is degraded by the Nrdp1 pathway. Importantly, knockdown or clone 32 expression enhances cellular response to NRG1, indicating that Nrdp1 mediates the degradation of signaling-competent receptors. From these studies we conclude that Nrdp1 represents a key component of a novel pathway that contributes to the maintenance of steady-state ErbB3 and ErbB4 protein levels by mediating the efficient degradation of these proteins. 3.2. Nrdp1 regulation In working with Nrdp1, we noticed that the protein is extremely unstable when transfected or transduced in a variety of cell lines. Often Nrdp1 protein cannot be detected by immunoblotting unless cells are treated with a proteasome inhibitor such as MG132 [50]. Point mutations in the RING finger domain that disrupt zinc ion coordination and thus coupling to E2 ubiquitin conjugating enzymes markedly stabilize Nrdp1, raising the possibility that autoubiquitination makes a major contribution to Nrdp1 lability. Given that Nrdp1 overexpression and disruption respectively suppress and augment ErbB3 protein levels, we considered the possibility that the activity of Nrdp1 may be regulated at the level its stability. To begin to unravel mechanisms of Nrdp1 regulation, we carried out an affinity chromatography/tandem mass spectrometry screen for interacting proteins. Among the proteins identified by this screen was USP8 [50], a deubiquitinating enzyme of unknown function at the time. We found that the carboxy terminal domain of Nrdp1 binds to the rhodanese domain of USP8, and that USP8 very efficiently deubiquitinates and stabilizes Nrdp1 [50]. Moreover, we observed that treatment of cells with the NRG1␤ growth factor stabilizes Nrdp1 through USP8 [86]. NRG1␤ stimulation of the ErbB2/ErbB3 heterodimer engages PI3-kinase, in turn activating Akt, which phosphorylates murine USP8 on threonine 907. USP8 phosphorylation leads to its activation and stabilization, which in turn stabilizes Nrdp1. The resulting feedback negative regulatory loop provides a mechanism for ligand-induced down-regulation of ErbB3, and we have demonstrated that Nrdp1 or USP8 knockdown in MCF7 breast tumor cells suppresses NRG1␤-stimulated ErbB3 ubiquitination and degradation [86]. 3.3. Nrdp1 involvement in ErbB3 overexpression in mammary tumors The mechanisms underlying ErbB3 overexpression by tumors are not understood. Amplification of the erbB3 gene has been reported, but PCR and FISH results suggest that this is not as prevalent as erbB2 amplification [64,65] and does not account for the frequency of elevated ErbB3 protein observed in tumors. Given that ErbB3 overexpression is a common occurrence in breast tumors, and that ErbB3 levels may be regulated post-transcriptionally in mouse mammary tumors [30,67,69], a key question concerns whether the Nrdp1 pathway is dysregulated in tumors, and if so whether dysregulation contributes to tumor initiation or progression. As shown in Fig. 1, Nrdp1 protein is lost in ErbB2-driven mammary tumors from transgenic mice and this loss cannot be attributed to a reduction in message levels. Moreover, we have observed that Nrdp1 protein is suppressed in 57% of tumors when comparing patient-matched normal and tumor breast tissues, and that there is a strong inverse correlation between Nrdp1 and ErbB3 protein levels in patient tumors [30]. These observations point to the possibility that Nrdp1 loss contributes to the ErbB3 protein overexpression in both human and mouse tissue samples. To determine whether Nrdp1 restoration to mouse mammary tumors suppresses tumor onset, we created transgenic mice expressing human Nrdp1 cDNA under the MMTV promoter. MMTV-

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Nrdp1 female mice exhibited 10-fold elevated Nrdp1 transcript in the mammary gland, exhibited elevated Nrdp1 protein and suppressed ErbB3 levels, but exhibited only a minor delay in development of the mammary gland during puberty [92]. This is a milder phenotype than that observed when mammary buds from ErbB3−/− embryos were transplanted into the cleared mammary glands of immunocompromised mice [93], possibly the result of either incomplete ErbB3 removal by Nrdp1 or mouse strain differences between the two studies. Surprisingly, crossing MMTV-Nrdp1 mice into the ErbB2 overexpression model had little effect on tumor latency or burden, or on ErbB3 protein overexpression [92], even though Nrdp1 transcript levels were 10-fold higher in bigenic animals than in MMTV-ErbB2 mice. While initially puzzling, immunoblotting studies revealed that the ErbB2-induced mouse mammary tumors lacked both endogenous and transgene-derived Nrdp1 protein. Consistent with this, cultured ErbB2-positive breast tumor cells are resistant to the expression of exogenous Nrdp1, while non-transformed cells efficiently support Nrdp1 expression [92]. While the question of whether Nrdp1 loss plays a causal role in ErbB2-driven tumorigenesis remains open, these observations indicate that like ErbB2 and ErbB3, Nrdp1 levels are dysregulated in tumor cells by posttranscriptional mechanisms. Overall, Nrdp1 regulation of ErbB3 via degradation offers novel insight into the mechanisms by which ErbB receptor levels are maintained in normal tissue, and the strategies that tumors might employ to inactivate these mechanisms.

4. ErbB4 degradation mediated by Nedd4 family proteins ErbB4 is somewhat unique among the ErbB family members in that two splicing variations give rise to four different protein products with significantly different biochemical and signaling properties. One splice variation yields alternative structures in the extracellular juxtamembrane region of the receptor. The resulting protein products, termed JM1-a and JM-b, exhibit differential sensitivity to the ADAM17 metalloprotease. JM-a cleavage by ADAM17 releases a 120 kDa ErbB4 extracellular domain fragment, leaving behind an 80 kDa fragment (m80) that encompasses a few amino acids of the extracellular region along with the transmembrane and intracellular domains [94]. Removal of the bulk of the extracellular domain then confers ErbB4 sensitivity to the ␥-secretase protease, which cleaves m80 within its transmembrane domain to release the entire intracellular (s80) region of the receptor into the cytosol. s80 then translocates to the nucleus where it regulates transcription patterns to slow cellular growth and stimulate the differentiation of tissues such as breast [72,95,96]. The other splice variation in ErbB4 results in the inclusion (Cyt-1) or deletion (Cyt-2) of a 16 amino acid segment in the intracellular carboxy terminal tail region of the receptor. Thus, Cyt-1 forms contain a sequence (PPAYTPM) that is predicted to couple to both PI3-kinase through a phosphorylated YXXM motif and to proteins containing WW domains through a PY motif. Preferential coupling of Cyt-1 isoforms to PI3-kinase has been reported to mediate ligand-induced cellular proliferation, survival and motility [97]. In addition to Nrdp1, ErbB4 levels are also regulated by members of the Nedd4 subfamily of E3 ubiquitin ligases. The Nedd4 subfamily, including Nedd4, Nedd4L, Itch and WWP1, comprise nine of the 28 HECT domain-containing E3 ligases encoded by the human genome [39,98]. Nedd4 family members contain an amino terminal C2 domain, responsible for phospholipid binding and membrane localization, two to four WW domains and a carboxy terminal HECT domain. Evidence accumulated over the past few years indicates that Nedd4 family ligases interact with ErbB4 through association of their WW domains with the PY motif of Cyt-1 receptor forms, resulting in the accelerated degradation of full-length receptors as

well as truncated cytosolic forms. The clinical importance of Nedd4 family E3 ligases is illustrated by Liddle Syndrome, a rare heritable form of hypertension arising from mutations in the PY motif of the renal epithelial sodium channel that uncouple its degradation from Nedd4-2 [99,100]. 4.1. Itch-mediated ErbB4 degradation In the first of a series of four studies concerning Nedd4 familymediated ErbB4 degradation, Omerovic et al. used a peptide containing the ErbB4 sequence PPAYTPM to identify the Nedd4 family member Itch as a Cyt-1 binding protein [101]. These authors demonstrated that wild-type Itch but not its ligase-inactive point mutant S830A mediates the ligand-independent ubiquitination and degradation of full-length and 80 kDa ErbB4, while Itch knockdown exhibited opposite effects on an endogenous ErbB4 population. Although their in vitro studies indicated that the WW domains of both Nedd4 and Itch are capable of interacting with Cyt-1, co-immunoprecipitation studies suggested only Itch efficiently interacts with ErbB4 in cells. Inhibitor studies demonstrated that Itch-mediated ErbB4 degradation is mediated by the lysosome. Effects of Itch are specific to ErbB4 relative to the other ErbB family members, and Cyt-1 appears to be the preferred substrate relative to Cyt-2. Sundvall et al. reached similar conclusions, although their studies distinctly focused on ErbB4 isoform internalization and degradation [35]. These investigators observed that the Cyt-1 splice variant is more highly localized to endocytic vesicles and has a faster rate of ligand-independent degradation than Cyt-2. Point mutations in the second proline (P1054A) or the tyrosine (Y1056F) residues of the PPAYTPM sequence of Cyt-1 disrupted these differences, pointing to a possible role for a WW domain-containing E3 ubiquitin ligase. These investigators observed that ErbB4 coimmunoprecipitates with Itch but not Nedd4, that P1054A and Y1056F mutants do not efficiently interact with Itch and that Itch augments ErbB4 monoubiquitination, internalization and degradation. Taken together, these two studies point to a model whereby Itch mediates the ubiquitination of membrane-bound Cyt-1-containing ErbB4 forms, facilitating their trafficking to the lysosome for degradation. 4.2. WWP1-mediated ErbB4 degradation Using microarray analysis, Feng et al. observed that expression of the soluble 80 kDa (s80) form of ErbB4 in mouse mammary cells elevates transcript levels of another Nedd4 family member, WWP1 [102], pointing to a potential the existence of a feedback negative regulatory loop. The investigators presented evidence that the WW domains of WWP1 mediate its association with PY motifs of Cyt1 but not other ErbB receptors, and that association contributes to ErbB4 ubiquitination and degradation. Moreover, they demonstrated that membrane-bound full-length and m80 ErbB4 forms are preferentially targeted by WWP1 and that the deletion of the WWP1 C2 domain facilitates its degradation of s80, suggesting a role for C2-mediated membrane targeting in WWP1 discrimination of ErbB4 forms. Li et al. similarly observed that WWP1 preferentially targets Cyt-1 relative to Cyt-2 for degradation, and further implicated the Nedd4 family member Nedl1 in suppressing steadystate ErbB4 levels [103]. 4.3. Degradation of soluble ErbB4 Overall, the four studies discussed above strongly suggest that Nedd4 family members play a central role in regulating steady-state ErbB4 levels by mediating the ligand-independent ubiquitination and lysosomal degradation of PY motif-containing

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membrane-associated ErbB4 forms. It should be noted that in building upon their observations that the Cyt-2 form of s80 preferentially translocates to the nucleus because the Cyt-1 form is more rapidly degraded, Zeng et al. found that Nedd4 plays a role in Cyt-1 s80 ubiquitination and degradation in MDCK epithelial cells [104]. While the discrepancy between this study and other studies concerning Nedd4 involvement in ErbB4 degradation is not clear, their observations point to the notion that Cyt-2 translocation results from the resistance of this isoform to Nedd4-mediated degradation. Strunk et al. demonstrated that S80 can also be degraded in a cell cycle-dependent manner via anaphase promoting complex (APC) recognition of a D-box motif in the receptor. These investigators demonstrated that s80 suppressed the ability of polyoma middle T antigen-expressing HC11 cells to form tumors when injected into mouse mammary glands, while disruption of the D-box abrogated tumor formation [105]. These observations strongly link the biological activity of ErbB4 to its stability, underscoring the critical role of protein degradation as a key regulator of ErbB receptor function. 5. CHIP-dependent ErbB2 degradation A subset of cellular proteins is acted upon by chaperones, or proteins that assist in the proper folding of client proteins. Together with a host of co-chaperones, the chaperone Hsp90 helps to maintain many signaling proteins in their fully folded state by using its ATPase activity to fuel the renaturation of partially unfolded proteins [106,107]. Cancer cells utilize the Hsp90 machinery to protect mutationally activated and overexpressed oncoproteins from misfolding and degradation, thereby contributing to oncogene addiction and tumor cell survival [108]. Hsp90 acts in conjunction with a second chaperone, Hsp70, to ensure that only fully folded client proteins accumulate within cells. Clients whose folding becomes trapped in an unproductive state exchange their association with Hsp90 for association with an Hsp70 complex that includes the E3 ubiquitin ligase CHIP. CHIP then mediates the ubiquitination and ultimate degradation of misfolded Hsp90 clients. Notably, Hsp90 may be inhibited by compounds of the benzoquinone ansamycin family such as geldanamycin (GA), and GA treatment of cells suppresses levels of Hsp90 client proteins by promoting the accumulation of denatured forms that are acted upon by Hsp70/CHIP. Over a dozen years ago it was first observed that Hsp90 inhibitors promote the degradation of EGFR and ErbB2 in cancer cell lines that overexpress receptors [109,110], although their dependence on Hsp90 appears to differ for the two. Hsp90 inhibition specifically induces the degradation of newly synthesized receptors in the endoplasmic reticulum [109], while the stability of both immature and mature ErbB2 forms appear to be dependent on Hsp90 [111]. Very recently it was reported that the degradation of nascent ErbB3 is also induced by Hsp90 inhibition [112]. In the case of ErbB2, CHIP has been demonstrated to associate with the receptor following Hsp90 inhibition to mediate its ubiquitination and degradation [111,113]. The ligases responsible for Hsp90 inhibition-mediated EGFR and ErbB3 remain to be delineated. 6. Summary and concluding remarks The picture that emerges from the studies described above is that E3 ubiquitin ligases can play key roles in suppressing cellular levels of ErbB receptor tyrosine kinases in cells. In the case of CHIP, this function can be exploited with Hsp90 inhibitors to suppress ErbB receptor levels in overexpressing cells. Clinical trials analyzing the impact of Hsp90 inhibitors toward cancer patients are ongoing [107], and it will be interesting to determine whether these inhibitors will be of therapeutic benefit to ErbB2-positive

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breast cancer patients. In the case of Nrdp1, the attenuation of ligase function by tumor cells likely contributes to ErbB3 overexpression. Indeed, it will be interesting to determine whether the dismantling of this pathway is requisite for ErbB3 protein overexpression and ultimately tumor onset and progression using mouse models. If true, the augmentation or restoration of these pathways may offer a novel approach to thwarting receptor activity in ErbB-dependent tumors. For example, our observations indicate that Nrdp1 loss during mammary normal-to-tumor transition occurs post-transcriptionally, most likely due to elevated autoubiquitination and degradation of the Nrdp1 protein [50,92]. Thus, the elucidation of pathways involved in maintaining Nrdp1 stability in normal tissue could uncover novel therapeutic targets whose inhibition would facilitate the restoration of ErbB3 to normal levels. Importantly, while Nrdp1 appears to target ErbB3 and ErbB4 receptors, it is likely that EGFR and ErbB2 are acted upon by similar E3 ligase-mediated degradation mechanisms to keep their levels in check. Thus, analysis of the Nrdp1-mediated ErbB3 degradation pathway and its dysregulation by tumors may offer novel insight into general mechanisms underlying ErbB receptor quantity control. Generalized key points are summarized:

1. Post-transcriptional mechanisms may play central roles in suppressing ErbB receptor levels in normal tissue, certainly in the mammary gland. This is based on mouse modeling, and a current question concerns the extent to which this principle extends to human biology. The relative advantages of post-transcriptional regulation as opposed to gene regulation are not clear; however, degradation mechanisms offer the advantage of very rapid responses to stressful conditions or fluctuations in hormones. 2. Growth factors can stimulate protein degradation pathways that govern steady-state ErbB receptor levels. In the case of Nrdp1, NRG1 stimulation of ErbB2/ErbB3 heterodimers in some cell lines stabilizes the ligase to promote ErbB3 ubiquitination and degradation [86]. Thus, under some circumstances growth factor stimulation can engage mechanisms involved in maintaining steady-state ErbB levels to elicit receptor down-regulation. However, cell types that inherently lack E3 ligases involved in ErbB receptor suppression, or cell lines derived from tumors that have suppressed these pathways, may not exhibit efficient ligand-induced ErbB down-regulation. 3. Tumors suppress post-transcriptional mechanisms to allow marked ErbB receptor overexpression, presumably as a mechanism to promote their own survival, growth and progression. A key question emerging from these observations is whether the loss of nodal ErbB regulatory proteins such as Nrdp1 is a rate-limiting step in ErbB-induced tumor formation. 4. ErbB negative regulators themselves can be posttranscriptionally suppressed in tumors. Nrdp1 loss in mammary tumors arises from a marked decrease in Nrdp1 protein in tumor cells relative to non-transformed cells, independent of changes in its message levels. These observations suggest that in some cases the dysregulation of ErbB negative regulatory mechanisms may be overlooked by gene expression profiling or other methods that are not based on protein quantification. 5. Different tumor types can utilize different mechanisms to suppress ErbB negative regulators. For example, although transcriptional suppression is not the cause of Nrdp1 loss in breast tumors, data mining suggests that Nrdp1 transcriptional suppression is a common feature of glioma (our unpublished observations). Overexpression of ErbB3 and ErbB4 is not a prominent feature of glioma, raising the possibility that Nrdp1 has functions beyond ErbB quantity control and that tumors benefit from the eradication of these functions.

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Curiously, Nrdp1-mediated ErbB3 degradation is inhibited by the proteasome inhibitor MG132 [91], suggesting that degradation of a cellular protein(s) is required for this process. Alternatively, Nrdp1mediated ErbB3 ubiquitination may mediate proteasomal rather than lysosomal receptor degradation, suggestive of a non-canonical cellular mechanism. Further investigation of the mechanisms underlying Nrdp1-mediated ErbB3 degradation could point to further general principles in ErbB receptor quantity control.

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