Abstract
The overactivation of epidermal growth factor (EGF) receptor (EGFR) is implicated in various cancers. Endocytosis plays an important role in EGFR-mediated cell signaling. We previously found that EGFR endocytosis during mitosis is mediated differently from interphase. While the regulation of EGFR endocytosis in interphase is well understood, little is known regarding the regulation of EGFR endocytosis during mitosis. Here, we found that contrary to interphase cells, mitotic EGFR endocytosis is more reliant on the activation of the E3 ligase CBL. By transfecting HeLa, MCF-7, and 293T cells with CBL siRNA or dominant-negative 70z-CBL, we found that at high EGF doses, CBL is required for EGFR endocytosis in mitotic cells, but not in interphase cells. In addition, the endocytosis of mutant EGFR Y1045F-YFP (mutation at the direct CBL binding site) is strongly delayed. The endocytosis of truncated EGFR Δ1044-YFP that does not bind to CBL is completely inhibited in mitosis. Moreover, EGF induces stronger ubiquitination of mitotic EGFR than interphase EGFR, and mitotic EGFR is trafficked to lysosomes for degradation. Furthermore, we showed that, different from interphase, low doses of EGF still stimulate EGFR endocytosis by non-clathrin mediated endocytosis (NCE) in mitosis. Contrary to interphase, CBL and the CBL-binding regions of EGFR are required for mitotic EGFR endocytosis at low doses. This is due to the mitotic ubiquitination of the EGFR even at low EGF doses. We conclude that mitotic EGFR endocytosis exclusively proceeds through CBL-mediated NCE.
Highlights
The epidermal growth factor (EGF) receptor (EGFR), like other receptor tyrosine kinases (RTKs), regulates key events in cell growth, differentiation, survival and migration [1,2,3]
EGFR expression at the plasma membrane does not change from interphase to mitosis [17,18,51]
Whereas interphase EGFR levels remain constant throughout 45 min of low-dose EGF treatment, we found that mitotic EGFR levels drop drastically with time (Figure 10C)
Summary
The epidermal growth factor (EGF) receptor (EGFR), like other receptor tyrosine kinases (RTKs), regulates key events in cell growth, differentiation, survival and migration [1,2,3]. Aberrant signaling from EGFR has been implicated in many diseases [2,4]. It was the first of this large family of transmembrane receptors to be cloned, and the first for which a clear connection between aberrant receptor function and cancer could be drawn [2]. EGFR activation stimulates various signaling pathways that regulate multiple cell functions [1,2,3]. Binding of EGF stimulates the rapid internalization of EGFR [13]. EGFR endocytosis and EGFR-mediated cell signaling are mutually regulated [14,15]
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