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- Materials and Methods
- Dsiclosure Statement
- Supporting Information
Overexpression of the ErbB2/HER2 receptor tyrosine kinase contributes to tumorigenesis. However, mechanisms regulating ErbB2 protein levels remain largely unclear. Here, we identified novel mechanisms of ErbB2 downregulation. ErbB2 constitutively binds to an adaptor protein FRS2β. We found that FRS2β bound to CD2AP and CIN85, which induces endosomal trafficking that targets lysosomes. FRS2β colocalized with CIN85 in the cytoplasm. Expression of wild type FRS2β but not its CIN85 non-binding mutant, downregulated the ErbB2 protein and inhibited anchorage-independent cell growth. Moreover, the E3 ubiquitin-protein ligase Cbl was contained within a complex of FRS2β and CIN85. Knockdown of both CIN85 and CD2AP or of Cbl, or treatment with lysosomal degradation inhibitors diminished FRS2β downregulation of ErbB2. In addition, knockdown of endogenous FRS2β caused upregulation of ErbB2 in primary neural cells. Finally, immunohistochemical analysis showed that human breast cancer tissues that overexpress ErbB2 expressed low levels of FRS2β. Thus, an FRS2β-CIN85/CD2AP-Cbl axis for downregulation of ErbB2 may regulate ErbB2 protein levels in physiological and pathological settings. Molecular targeting drugs that can increase or stabilize the ErbB2-FRS2β-CIN85/CD2AP-Cbl axis may have promise for the control of ErbB2-overexpressing tumors.
The epidermal growth factor receptor (EGFR) and its family member ErbB2 are typical receptor tyrosine kinases (RTK) and are involved in many aspects of biology, including physiological aspects such as development, and pathological aspects such as cancer. Epidermal growth factor (EGF) binds to the EGFR, leading to EGFR homo-dimerization or hetero-dimerization with its family members, which activates its RTK activity, resulting in the initiation of a variety of cellular signaling pathways that ultimately lead to many cellular processes including cell proliferation, differentiation, migration, survival and adhesion. There are also many negative feedback mechanisms to fine-tune this signaling in a physiological setting.[2, 3] In fact, overexpression of the EGFR or ErbB2 contributes to tumorigenesis in many types of cancer, which may be partly due to the fact that negative feedback regulation is insufficient in cells that express too many of these receptors. For example, the ErbB2 gene is amplified in approximately 20%–25% of human breast cancers It is therefore important to clarify the mechanisms of negative regulation of these receptors in a physiological setting in order to find a way to control pathological processes. Receptor downregulation is a major mechanism for negative regulation of the EGFR. This mechanism involves EGF-induced receptor endocytosis, which targets the receptor to the lysosome for degradation. Although there appears to be no ligand for ErbB2, the extracellular domain of ErbB2 has an extended conformation that mimics the ligand bounded-extracellular domain of the EGFR, which endows ErbB2 with the capacity to easily homo- and hetero-dimerize and thereby activate cellular signaling pathways. However, it is also known that ErbB2 is not degraded as efficiently as the EGFR, and the mechanisms by which ErbB2 is downregulated are still unclear.
FRS2β belongs to the FRS2 family of docking/scaffolding adaptor proteins.[7-9] This family comprises two members: FRS2α/FRS2 and FRS2β/FRS3. Both members contain N-terminal myristoylation sites, resulting in their membrane distribution, and a phosphotyrosine-binding (PTB) domain, which facilitates binding to a limited number of receptor tyrosine kinases (RTKs). FRS2α constitutively binds to the fibroblast growth factor (FGF) receptor via its PTB domain regardless of FGF stimulation and acts as a central mediator in FGF signaling. FRS2α becomes phosphorylated on tyrosine residues in response to FGF, leading to the activation of major signaling pathways including the Ras/ERK pathway.[10-13] FRS2β also constitutively binds to the FGF receptor, becomes tyrosine phosphorylated upon FGF stimulation and may function redundantly with FRS2α. However, a growing body of evidence suggests that the main functions of FRS2β are different from those of FRS2α. FRS2β constitutively binds to the EGFR and ErbB2 via its PTB domain regardless of receptor activation, but acts as a feedback inhibitor for EGF signaling.[3, 15, 16] FRS2β does not become phosphorylated on tyrosine residues in response to EGF, but does bind to activated ERK thereby inhibiting translocation of ERK to the nucleus, and also inhibits dimerization of the EGFR or ErbB2. In contrast to the ubiquitous expression of FRS2α from embryos to adults, FRS2β expression is restricted to only a few tissues: for example, neural tissues and epithelial cells in lung and breast tissues.[14, 17] The FRS2β protein colocalizes with intracellular vesicles including lysosomes in neural cells. This localization gave rise to the hypothesis that FRS2β functions in intracellular degradation systems.
The CBL-interacting protein 85 kDa (CIN85) and CD2-associated protein (CD2AP) belong to the same family of adaptor proteins.[18-22] CIN85 and CD2AP each have three SH3 domains that mediate protein–protein interactions by binding to proline (P)-rich motifs of a variety of proteins and are involved in many cellular processes including the degradation of EGFRs that are transported in endosomes. Cbl, a E3 ubiquitin ligase, is one such interacting protein and is responsible for directing the lysosomal degradation of these RTKs by their ubiquitination.
In order to obtain further insights into the specific functions of FRS2β, we screened for novel proteins that interact with FRS2β, but not with FRS2α using a high-throughput technique that combined affinity purification of interaction partners from cell lysates with subsequent liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS). We found that CIN85 and CD2AP were novel binding partners of FRS2β and that the complex between CIN85 or CD2AP with FRS2β contributed to downregulation of the ErbB2 protein.
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- Materials and Methods
- Dsiclosure Statement
- Supporting Information
In this study, using LC-MS/MS analysis we identified CIN85 and CD2AP as novel FRS2β binding proteins, and demonstrated that an FRS2β-CIN85/CD2AP complex downregulates the ErbB2 protein, probably by inducing its lysosomal degradation.
The mechanism of EGFR downregulation and that of ErbB2 downregulation that we describe here have many differences. EGF binding to the EGFR accelerates receptor internalization, which is followed by efficient lysosomal targeting of the internalized receptors and results in receptor downregulation. Upon EGF stimulation, the EGFR directly or indirectly binds to Cbl via tyrosine phosphorylated residues on the EGFR. Cbl binds to the SH3 domains of CIN85, and then CIN85 interacts with multiple proteins that are involved in endocytosis, in sorting into early and late endosomes or multi-vesicular bodies, and in targeting to lysosomes.[18, 19, 23] Cbl also ubiquitinates the EGFR and other proteins including CIN85 and it is thought that these ubiquitinated proteins are targeted to lysosomes and do not recycle back to the plasma membrane. We demonstrated that FRS2β can recruit the CIN85/CD2AP-Cbl degradation complex to ErbB2 regardless of ligand stimulation of ErbB family members. Since there is no ligand for ErbB2, this mechanism may play important roles in the negative regulation of ErbB2 signaling in a physiological setting.
Expression of FRS2β is limited to a few tissues. During development and in adults, FRS2β is expressed in the specific area of neural tissues in which differentiated neural cells are localized as well as in several epithelial cells in the lung, gastrointestinal tract and kidney.[14, 17] We detected reciprocal expression of FRS2β and ErbB2 in primary neural cells cultured under two different conditions. We also previously reported that FRS2β is expressed in Tuj1-positive differentiated neuronal cells. In contrast, it has been reported that ErbB2 is expressed in the ventricular zone in which NSPCs are localized. Our findings raise the intriguing possibility that ErbB2 signaling might be strictly controlled by FRS2β in vivo, by FRS2β-mediated downregulation of ErbB2 protein levels.
The EGFR protein does not appear to be downregulated by FRS2β in the absence of ligand stimulation by a mechanism similar to that of ErbB2 downregulation. In fact, we could not co-immunoprecipitate Cbl and the EGFR with FRS2β without ligand stimulation (data not shown). It is possible that the EGFR-FRS2β-CIN85 complex may not efficiently form a complex with Cbl due to a conformational problem, although it is difficult to prove such a hypothesis. However, since the EGFR protein is efficiently downregulated upon EGF stimulation of many common cell lines that do not express FRS2β, it appears that FRS2β does not play an important role in downregulation of the EGFR protein.
The CIN85 adaptor protein interacts with a number of proteins and is involved in a number of biological processes including receptor endocytosis. A recent screen, using an LC-MS/MS screening approach, identified approximately 100 proteins that can interact with CIN85 via its SH3 domains. However, this list did not include FRS2β, probably because the screen was performed using lysates of cells of the commonly used HeLa cell line, which do not express FRS2β. The site on FRS2β for CIN85/CD2AP binding that includes R251 is also used for FRS2β binding to activated ERK. It is possible that CIN85/CD2AP and ERK do not bind to the same FRS2β protein. We previously reported that ERK colocalizes with FRS2β in the plasma membrane. It is thus reasonable to assume that the FRS2β-CIN85/CD2AP complex and the FRS2β-ERK complex are separately localized in the endosome and plasma membrane, respectively.
Although FRS2β is weakly, or not at all, expressed in many cell lines, it was striking that FRS2β was expressed in many breast cancer tissues at various levels. These findings emphasize the notion that although expression of FRS2β may be disadvantageous to cell growth and/or survival in vitro, FRS2β does have in vivo roles. FRS2β expression levels tended to be low in breast cancer tissues with high ErbB2 expression levels. Thus, FRS2β may have a role in controlling ErbB2 protein levels even in pathological conditions including cancer.
Recently, it was reported that increased levels of CIN85 contributes to breast cancer malignancy. The authors examined functions of CIN85 in breast cancer cell lines including MCF-7 cells in which expression levels of FRS2β are very low. Since CIN85 is able to bind many other proteins including Arf GTPase and N-WASP, it appears to activate the pathways in which these proteins are involved. It is thus likely that functions of CIN85 are changed dependent on the main binding partners in cells.