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J Biol Chem. 2014 Oct 31;289(44):30443-58. doi: 10.1074/jbc.M114.608992. Epub 2014 Sep 15.

A kinase inhibitor screen reveals protein kinase C-dependent endocytic recycling of ErbB2 in breast cancer cells.

Author information

1
From the Eppley Institute for Research in Cancer and Allied Diseases.
2
From the Eppley Institute for Research in Cancer and Allied Diseases, Departments of Genetics, Cell Biology, and Anatomy, and.
3
From the Eppley Institute for Research in Cancer and Allied Diseases, Biochemistry & Molecular Biology, College of Medicine, and.
4
From the Eppley Institute for Research in Cancer and Allied Diseases, Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, Nebraska 68198-5950.
5
From the Eppley Institute for Research in Cancer and Allied Diseases, Departments of Genetics, Cell Biology, and Anatomy, and Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, Nebraska 68198-5950.
6
From the Eppley Institute for Research in Cancer and Allied Diseases, Departments of Genetics, Cell Biology, and Anatomy, and Biochemistry & Molecular Biology, College of Medicine, and Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, 985950 Nebraska Medical Center, Omaha, Nebraska 68198-5950 hband@unmc.edu.

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.

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

PMID:
25225290
PMCID:
PMC4215227
DOI:
10.1074/jbc.M114.608992
[Indexed for MEDLINE]
Free PMC Article

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