A Kinase Inhibitor Screen Reveals Protein Kinase C-dependent Endocytic Recycling of ErbB2 in Breast Cancer Cells

• DOI: 10.1074/jbc.m114.608992
• Publication Date: 2014-09-16T05:17:36Z
• Authors: Tameka A. Bailey; Haitao Luan; Eric Tom; Timothy Alan Biel...; Bhopal Mohapatra; Gulzar Ahmad; Manju George; David L. Kelly; Amarnath Natarajan; Srikumar M. Raja; Vimla Band; Hamid Band
• Keywords: Feedback, Physiological; 0301 basic medicine; Indoles; Protein Kinase C-alpha; Carcinogenesis; MAP Kinase Signaling System; Lactams, Macrocyclic; Carbazoles; Antineoplastic Agents; Breast Neoplasms; Endosomes; Endocytosis; 3. Good health; 12. Responsible consumption; Enzyme Activation; Protein Kinase C-delta; 03 medical and health sciences; Cell Line, Tumor; Benzoquinones; Humans; Female; Drug Screening Assays, Antitumor; Phosphorylation; Extracellular Signal-Regulated MAP Kinases

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.