UBTD1 regulates ceramide balance and endolysosomal positioning to coordinate EGFR signaling

Abstract

To adapt in an ever-changing environment, cells must integrate physical and chemical signals and translate them into biological meaningful information through complex signaling pathways. By combining lipidomic and proteomic approaches with functional analysis, we have shown that UBTD1 (Ubiquitin domain-containing protein 1) plays a crucial role in both the EGFR (Epidermal Growth Factor Receptor) self-phosphorylation and its lysosomal degradation. On the one hand, by modulating the cellular level of ceramides through ASAH1 (N-Acylsphingosine Amidohydrolase 1) ubiquitination, UBTD1 controls the ligand-independent phosphorylation of EGFR. On the other hand, UBTD1, via the ubiquitination of SQSTM1/p62 (Sequestosome 1) by RNF26 and endolysosome positioning, participates in the lysosomal degradation of EGFR. The coordination of these two ubiquitin-dependent processes contributes to the control of the duration of the EGFR signal. Moreover, we showed that UBTD1 depletion exacerbates EGFR signaling and induces cell proliferation emphasizing a hitherto unknown function of UBTD1 in EGFR-driven human cell proliferation.

Low Doses of PFOA Promote Prostate and Breast Cancer Cells Growth through Different Pathways

“…Human BCa cell line (MCF7) and human PCa cell line (DU145) were obtained from ATCC and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (respectively, 100 U/mL and 100 µg/mL, Gibco ® [Asnières sur Seine, France]) at 37 °C in a humidified atmosphere (5% CO 2 ). All cell lines used in this study were tested to confirm that they were free of mycoplasma [ 43 ].…”

“…For siRNA transfections, as previously described [ 43 ], siRNA control or siRNA pool directed against PlexinD1 (On Target Plus, Dharmacon ® [Lafayette, CL, USA]) were transfected with Lipofectamine RNAiMAX (InVitrogen ® [Asnières sur Seine, France]) according to the manufacturer’s instructions. Briefly, 120.000 cells/well in a 6-well plate were seeded overnight.…”

“…BrdU cell proliferation assay was performed using the Cell proliferation ELISA colorimetric kit (Sigma ® ) according to the manufacturer’s instructions. Briefly [ 43 ], cells were seeded overnight in 96-well plate at 4.000 cells/well. The day after, chemical treatments or siRNA transfections were performed.…”

“…Human Phospho-Kinase Antibody Array (RD Systems ® [Minneapolis, Minnesota]) was used to analyze the relative site-specific phosphorylation of 43 kinases and 2 related total proteins. Following the manufacturer’s instructions, we used 1.107 cells/mL for cell lysis corresponding to 400 µg of protein per array [ 43 ]. Phosphorylated proteins were detected using Syngene PXi by exposing membranes to luminescent signals generated after incubation of the membrane with ECL (WBKLS0500, Millipore ® [Darmstadt, Germany]).…”

“…All experiments were performed in triplicate with 3 wells per condition in each one. Significant differences between two conditions were assessed by unpaired or paired Student’s t-test and two-way ANOVA test followed by post hoc Fisher test when more than two conditions were analyzed [ 43 ]. A p -value < 0.05 was considered statistically significant.…”