Activated ErbB3 Translocates to the Nucleus via Clathrin-independent Endocytosis, Which Is Associated with Proliferating Cells

Abstract

Members of the receptor tyrosine kinase family (RTK) have been shown to be present in the nucleus of cells; however, the mechanisms underlying their trafficking to the nucleus, and their relevance once there are poorly understood. In the present study, we focus on the RTK ErbB3 and elucidate the mechanisms regulating its trafficking. We show that heregulin-stimulation induces trafficking of phosphorylated ErbB3 from the plasma membrane to the nucleus via a clathrin-independent mechanism. Nuclear import of ErbB3 occurs via importin ␤1, which drives the receptor through the nuclear pore complex. In the nucleus, ErbB3 interacts with transcription complexes, and thereby has a role in transcriptional regulation. Our results also demonstrate that ErbB3 nuclear localization is transient as it is exported out of the nucleus by the nuclear receptor protein crm-1. Analysis of normal, regenerating tissues, and tumors showed that ErbB3 nuclear translocation is a common event in proliferating tissues.The epidermal growth factor receptor family (EGFR,3 ErbB2, ErbB3, and ErbB4) comprises some of the best characterized members of the receptor tyrosine kinases (RTKs). Their activation influences cellular signaling via the mitogen-activated protein kinase (MAPK)/ERK and the phosphatidylinositol 3-kinase (PI3K)-AKT pathways, both of which regulate essential cellular mechanisms including cell proliferation, survival, and differentiation. Dysregulation of these pathways is regularly found in carcinogenesis, tumor progression, and metastasis (1, 2). Furthermore, overexpression of EGFR, ErbB2, and ErbB3 in tumor tissue is often associated with poor patient outcome (3). ErbB3 is the least studied member of the EGFR family, However, recent evidence supports a key role for ErbB3 in cell transformation and malignancy of tumors (4). In addition, therapeutic interventions directed toward EGFR family members lead to the activation of ErbB3, which in turn is associated with the development of chemoresistance (5-8). However, the consequences of signaling activation and trafficking of ErbB3, as well as its functional relevance are poorly understood.Previous reports have shown that EGFR family members are present in the nucleus, either as full-length molecules, as in the case of EGFR (9) and ErbB2 (10), or truncated as for ␥-secretase-cleaved ErbB4 (11). In the present study, we demonstrate that nuclear translocation of ErbB3 is not limited to cancer, but appears to be a general phenomenon in proliferating tissues. To clarify ErbB3 trafficking from the cell surface to the nucleus, and its function once there, we applied a comprehensive approach using subcellular fractionation, cell surface protein labeling, immunostaining, and live cell imaging to investigate the transfer of ErbB3 to the nucleus. In addition, we studied the activation state, the translocation mechanisms, and the nuclear entry of ErbB3. To understand its function in the nucleus, we investigated the prevalence of nuclear ErbB3 in both controlled and diseased tissues. Our findings ...

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