title: A kinase inhibitor screen reveals protein kinase C-dependent endocytic recycling of ErbB2 in breast cancer cells.
abstract: ErbB2 overexpression drives oncogenesis in 20-30% cases of breast cancer. Oncogenic potential of ErbB2 is linked to inefficient endocytic traffic into lysosomes and preferential recycling. However, regulation of ErbB2 recycling is incompletely understood. We used a high-content immunofluorescence imaging-based kinase inhibitor screen on SKBR-3 breast cancer cells to identify kinases whose inhibition alters the clearance of cell surface ErbB2 induced by Hsp90 inhibitor 17-AAG. Less ErbB2 clearance was observed with broad-spectrum PKC inhibitor Ro 31-8220. A similar effect was observed with Go 6976, a selective inhibitor of classical Ca(2+)-dependent PKCs (α, β1, βII, and γ). PKC activation by PMA promoted surface ErbB2 clearance but without degradation, and ErbB2 was observed to move into a juxtanuclear compartment where it colocalized with PKC-α and PKC-δ together with the endocytic recycling regulator Arf6. PKC-α knockdown impaired the juxtanuclear localization of ErbB2. ErbB2 transit to the recycling compartment was also impaired upon PKC-δ knockdown. PMA-induced Erk phosphorylation was reduced by ErbB2 inhibitor lapatinib, as well as by knockdown of PKC-δ but not that of PKC-α. Our results suggest that activation of PKC-α and -δ mediates a novel positive feedback loop by promoting ErbB2 entry into the endocytic recycling compartment, consistent with reported positive roles for these PKCs in ErbB2-mediated tumorigenesis. As the endocytic recycling compartment/pericentrion has emerged as a PKC-dependent signaling hub for G-protein-coupled receptors, our findings raise the possibility that oncogenesis by ErbB2 involves previously unexplored PKC-dependent endosomal signaling.
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, Nebraska 68198-5950.
authors.forename: Tameka A
authors.initials: TA
authors.lastname: Bailey
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, Nebraska 68198-5950; Departments of Genetics, Cell Biology, and Anatomy, and University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, Nebraska 68198-5950.
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, Nebraska 68198-5950; Departments of Biochemistry & Molecular Biology, College of Medicine, and University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, Nebraska 68198-5950.
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, Nebraska 68198-5950.
authors.forename: Timothy Alan
authors.initials: TA
authors.lastname: Bielecki
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, Nebraska 68198-5950; Departments of Biochemistry & Molecular Biology, College of Medicine, and University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, Nebraska 68198-5950.
authors.forename: Bhopal
authors.initials: B
authors.lastname: Mohapatra
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, Nebraska 68198-5950.
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, Nebraska 68198-5950.
authors.forename: Manju
authors.initials: M
authors.lastname: George
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, Nebraska 68198-5950; Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, Nebraska 68198-5950.
authors.forename: David L
authors.initials: DL
authors.lastname: Kelly
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, Nebraska 68198-5950; Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, Nebraska 68198-5950.
authors.forename: Amarnath
authors.initials: A
authors.lastname: Natarajan
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, Nebraska 68198-5950; Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, Nebraska 68198-5950.
authors.forename: Srikumar M
authors.initials: SM
authors.lastname: Raja
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, Nebraska 68198-5950; Departments of Genetics, Cell Biology, and Anatomy, and University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, Nebraska 68198-5950; Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, Nebraska 68198-5950.
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, Nebraska 68198-5950; Departments of Genetics, Cell Biology, and Anatomy, and University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, Nebraska 68198-5950; Departments of Biochemistry & Molecular Biology, College of Medicine, and University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, Nebraska 68198-5950; Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, Nebraska 68198-5950. Electronic address: hband@unmc.edu.
authors.forename: Hamid
authors.initials: H
authors.lastname: Band
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D016227:Benzoquinones / Q000494:pharmacology; D001943:Breast Neoplasms; D002227:Carbazoles / Q000494:pharmacology; D063646:Carcinogenesis / Q000378:metabolism; D045744:Cell Line, Tumor; D004354:Drug Screening Assays, Antitumor; D004705:Endocytosis / Q000187:drug effects; D011992:Endosomes / Q000378:metabolism; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D025461:Feedback, Physiological; D005260:Female; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D020935:MAP Kinase Signaling System; D010766:Phosphorylation; D051571:Protein Kinase C-alpha / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051745:Protein Kinase C-delta / Q000037:antagonists & inhibitors / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D016227:Benzoquinones; D002227:Carbazoles; D048049:Extracellular Signal-Regulated MAP Kinases; D007211:Indoles; D047029:Lactams, Macrocyclic; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D047428:Protein Kinase Inhibitors; D018719:Erb-b2 Receptor Tyrosine Kinases; C081021:Go 6976; C112765:tanespimycin; C508053:ERBB2 protein, human; C496628:PRKCA protein, human; C497258:PRKCD protein, human; C064758:Ro 31-8220
keywords: Breast Cancer; Confocal Microscopy; Endocytic Traffic; Endocytosis; ErbB2; High-Content Fluorescence Microscopy; Kinase Inhibitors; Protein Kinase C (PKC); Receptor Tyrosine Kinase; Small Molecule Screening