Ubiquitination and proteasomal activity is required for transport of the EGF receptor to inner membranes of multivesicular bodies

EGF, but not TGFα, efficiently induces degradation of the EGF receptor (EGFR). We show that EGFR was initially polyubiquitinated to the same extent upon incubation with EGF and TGFα, whereas the ubiquitination was more sustained by incubation with EGF than with TGFα. Consistently, the ubiquitin ligase c-Cbl was recruited to the plasma membrane upon activation of the EGFR with EGF and TGFα, but localized to endosomes only upon activation with EGF. EGF remains bound to the EGFR upon endocytosis, whereas TGFα dissociates from the EGFR. Therefore, the sustained polyubiquitination is explained by EGF securing the kinase activity of endocytosed EGFR. Overexpression of the dominant negative N-Cbl inhibited ubiquitination of the EGFR and degradation of EGF and EGFR. This demonstrates that EGF-induced ubiquitination of the EGFR as such is important for lysosomal sorting. Both lysosomal and proteasomal inhibitors blocked degradation of EGF and EGFR, and proteasomal inhibitors inhibited translocation of activated EGFR from the outer limiting membrane to inner membranes of multivesicular bodies (MVBs). Therefore, lysosomal sorting of kinase active EGFR is regulated by proteasomal activity. Immuno-EM showed the localization of intact EGFR on internal membranes of MVBs. This demonstrates that the EGFR as such is not the proteasomal target

Association of physical activity with overall mortality among long-term testicular cancer survivors: A longitudinal study

Physical activity (PA) has been associated with reduced mortality among cancer survivors, but no study has focused on testicular cancer survivors (TCSs). We aimed to investigate the association of PA measured twice during survivorship with overall mortality in TCSs. TCSs treated during 1980 to 1994 participated in a nationwide longitudinal survey between 1998 to 2002 (S1: n = 1392) and 2007 to 2009 (S2: n = 1011). PA was self-reported by asking for the average hours per week of leisure-time PA in the past year. Responses were converted into metabolic equivalent task hours/week (MET-h/wk) and participants were categorized into: Inactives (0 MET-h/wk), Low-Actives (2-6 MET-h/wk), Actives (10-18 MET-h/wk) and High-Actives (20-48 MET-h/wk). Mortality from S1 and S2, respectively, was analyzed using the Kaplan-Meier estimator and Cox proportional hazards models until the End of Study (December 31, 2020). Mean age at S1 was 45 years (SD 10.2). Nineteen percent (n = 268) of TCSs died between S1 and EoS, with 138 dying after S2. Compared to Inactives at S1, the mortality risk among Actives was 51% lower (HR 0.49, 95% CI: 0.29-0.84) with no further mortality reduction among High-Actives. At S2, the mortality risk was at least 60% lower among the Actives, High-Actives and even the Low-Actives compared to the Inactives. Persistent Actives (≥10 MET-h/wk at S1 and S2) had a 51% lower mortality risk compared to Persistent Inactives (<10 MET-h/wk at S1 and S2; HR 0.49, 95% CI: 0.30-0.82). During long-term survivorship after TC treatment, regular and maintained PA were associated with an overall mortality risk reduction of at least 50%

Activation of the Epidermal Growth Factor (EGF) Receptor Induces Formation of EGF Receptor- and Grb2-Containing Clathrin-Coated Pits

In HeLa cells depleted of adaptor protein 2 complex (AP2) by small interfering RNA (siRNA) to the μ2 or α subunit or by transient overexpression of an AP2 sequestering mutant of Eps15, endocytosis of the transferrin receptor (TfR) was strongly inhibited. However, epidermal growth factor (EGF)-induced endocytosis of the EGF receptor (EGFR) was inhibited only in cells where the α subunit had been knocked down. By immunoelectron microscopy, we found that in AP2-depleted cells, the number of clathrin-coated pits was strongly reduced. When such cells were incubated with EGF, new coated pits were formed. These contained EGF, EGFR, clathrin, and Grb2 but not the TfR. The induced coated pits contained the α subunit, but labeling density was reduced compared to control cells. Induction of clathrin-coated pits required EGFR kinase activity. Overexpression of Grb2 with inactivating point mutations in N- or C-terminal SH3 domains or in both SH3 domains inhibited EGF-induced formation of coated pits efficiently, even though Grb2 SH3 mutations did not block activation of mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K). Our data demonstrate that EGFR-induced signaling and Grb2 are essential for formation of clathrin-coated pits accommodating the EGFR, while activation of MAPK and PI3K is not required