ReviewE3 ubiquitin ligases in ErbB receptor quantity control☆
Introduction
The four members of the ErbB family of receptor tyrosine kinases are necessary for the development and maintenance of a variety of tissue types, including heart, lung, skeletal muscle, and the central and peripheral nervous systems [1], [2], [3], [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], [8], [9]. A particularly well-studied example, erbB2 gene amplification is observed in 25–30% of breast cancer patients and overexpression of the ErbB2 protein correlates with poor prognosis [10], [11], [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], [16], [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], [21], [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], [28], [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].
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], [39], [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 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 ɛ-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], [49], [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], [52], [53], [54], [55], [56]. Not surprisingly, the dysregulation of E3 ligase function has been implicated in various disease states such as neurodegenerative disorders and cancer [57], [58], [59], [60], [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.
Section snippets
Post-transcriptional regulation of ErbB receptor levels
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 is an X-linked breast tumor suppressor transcriptional
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 steady-state 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
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
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
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
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The author is supported by NIH grants GM068994 and CA123541.