title: Cbl-family ubiquitin ligases and their recruitment of CIN85 are largely dispensable for epidermal growth factor receptor endocytosis.
abstract: Members of the casitas B-lineage lymphoma (Cbl) family (Cbl, Cbl-b and Cbl-c) of ubiquitin ligases serve as negative regulators of receptor tyrosine kinases (RTKs). An essential role of Cbl-family protein-dependent ubiquitination for efficient ligand-induced lysosomal targeting and degradation is now well-accepted. However, a more proximal role of Cbl and Cbl-b as adapters for CIN85-endophilin recruitment to mediate ligand-induced initial internalization of RTKs is supported by some studies but refuted by others. Overexpression and/or incomplete depletion of Cbl proteins in these studies is likely to have contributed to this dichotomy. To address the role of endogenous Cbl and Cbl-b in the internalization step of RTK endocytic traffic, we established Cbl/Cbl-b double-knockout (DKO) mouse embryonic fibroblasts (MEFs) and demonstrated that these cells lack the expression of both Cbl-family members as well as endophilin A, while they express CIN85. We show that ligand-induced ubiquitination of EGFR, as a prototype RTK, was abolished in DKO MEFs, and EGFR degradation was delayed. These traits were reversed by ectopic human Cbl expression. EGFR endocytosis, assessed using the internalization of (125)I-labeled or fluorescent EGF, or of EGFR itself, was largely retained in Cbl/Cbl-b DKO compared to wild type MEFs. EGFR internalization was also largely intact in Cbl/Cbl-b depleted MCF-10A human mammary epithelial cell line. Inducible shRNA-mediated knockdown of CIN85 in wild type or Cbl/Cbl-b DKO MEFs had no impact on EGFR internalization. Our findings, establish that, at physiological expression levels, Cbl, Cbl-b and CIN85 are largely dispensable for EGFR internalization. Our results support the model that Cbl-CIN85-endophilin complex is not required for efficient internalization of EGFR, a prototype RTK.
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, NE 68198-5950, USA.
authors.forename: Gulzar
authors.initials: G
authors.lastname: Ahmad
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, NE 68198-5950, USA; Department of Biochemistry & Molecular Biology, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, NE 68198-5950, USA.
authors.forename: Bhopal C
authors.initials: BC
authors.lastname: Mohapatra
authors.affiliation: Department of Pharmacology & Experimental Neuroscience, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, NE 68198-5950, USA.
authors.forename: Nancy A
authors.initials: NA
authors.lastname: Schulte
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, NE 68198-5950, USA; Department of Genetics, Cell Biology & Anatomy, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, NE 68198-5950, USA.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Nadeau
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, NE 68198-5950, USA; Department of Genetics, Cell Biology & Anatomy, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, NE 68198-5950, USA.
authors.forename: Haitao
authors.initials: H
authors.lastname: Luan
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, NE 68198-5950, USA; Department of Pathology & Microbiology, College of Medicine, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, NE 68198-5950, USA.
authors.forename: Neha
authors.initials: N
authors.lastname: Zutshi
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, NE 68198-5950, USA; Department of Biochemistry & Molecular Biology, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, NE 68198-5950, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Tom
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, NE 68198-5950, USA.
authors.forename: Cesar
authors.initials: C
authors.lastname: Ortega-Cava
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, NE 68198-5950, USA.
authors.forename: Chun
authors.initials: C
authors.lastname: Tu
authors.affiliation: Department of Pathology and Tumor Biology, Kyoto University, Yoshida-Konoe-Cho, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Sanada
authors.affiliation: Department of Pathology and Tumor Biology, Kyoto University, Yoshida-Konoe-Cho, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Seishi
authors.initials: S
authors.lastname: Ogawa
authors.affiliation: Department of Pharmacology & Experimental Neuroscience, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, NE 68198-5950, USA.
authors.forename: Myron L
authors.initials: ML
authors.lastname: Toews
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, NE 68198-5950, USA; Department of Genetics, Cell Biology & Anatomy, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, NE 68198-5950, USA; Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, NE 68198-5950, USA.
authors.forename: Vimla
authors.initials: V
authors.lastname: Band
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, NE 68198-5950, USA; Department of Biochemistry & Molecular Biology, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, NE 68198-5950, USA; Department of Genetics, Cell Biology & Anatomy, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, NE 68198-5950, USA; Department of Pathology & Microbiology, College of Medicine, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, NE 68198-5950, USA; Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, NE 68198-5950, USA. Electronic address: hband@unmc.edu.
authors.forename: Hamid
authors.initials: H
authors.lastname: Band
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D004705:Endocytosis; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009363:Neoplasm Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl / Q000378:metabolism*; D014162:Transfection
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; C433810:SH3KBP1 protein, human; C498782:Sh3kbp1 protein, mouse; D050721:Proto-Oncogene Proteins c-cbl; D066246:ErbB Receptors
keywords: CIN85; Cbl; Epidermal growth factor receptor; Internalization; Mouse embryonic fibroblast