title: SPIN90 knockdown attenuates the formation and movement of endosomal vesicles in the early stages of epidermal growth factor receptor endocytosis.
abstract: The finding that SPIN90 colocalizes with epidermal growth factor (EGF) in EEA1-positive endosomes prompted us to investigate the role of SPIN90 in endocytosis of the EGF receptor (EGFR). In the present study, we demonstrated that SPIN90 participates in the early stages of endocytosis, including vesicle formation and trafficking. Stable HeLa cells with knockdown of SPIN90 displayed significantly higher levels of surface EGFR than control cells. Analysis of the abundance and cellular distribution of EGFR via electron microscopy revealed that SPIN90 knockdown cells contain residual EGFR at cell membranes and fewer EGFR-containing endosomes, both features that reflect reduced endosome formation. The delayed early endosomal targeting capacity of SPIN90 knockdown cells led to increased EGFR stability, consistent with the observed accumulation of EGFR at the membrane. Small endosome sizes and reduced endosome formation in SPIN90 knockdown cells, observed using fluorescent confocal microscopy, strongly supported the involvement of SPIN90 in endocytosis of EGFR. Overexpression of SPIN90 variants, particularly the SH3, PRD, and CC (positions 643 - 722) domains, resulted in aberrant morphology of Rab5-positive endosomes (detected as small spots located near the cell membrane) and defects in endosomal movement. These findings clearly suggest that SPIN90 participates in the formation and movement of endosomes. Consistent with this, SPIN90 knockdown enhanced cell proliferation. The delay in EGFR endocytosis effectively increased the levels of endosomal EGFR, which triggered activation of ERK1/2 and cell proliferation via upregulation of cyclin D1. Collectively, our findings suggest that SPIN90 contributes to the formation and movement of endosomal vesicles, and modulates the stability of EGFR protein, which affects cell cycle progression via regulation of the activities of downstream proteins, such as ERK1/2, after EGF stimulation.
authors: Bio Imaging and Cell Dynamics Research Center, School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Cheomdan Gwagi-ro 261, Gwangju Metrocity, Korea.
authors: Hyejin
authors: H
authors: Oh
authors: Hwan
authors: H
authors: Kim
authors: Kyung-Hwun
authors: KH
authors: Chung
authors: Nan Hyung
authors: NH
authors: Hong
authors: Baehyun
authors: B
authors: Shin
authors: Woo Jin
authors: WJ
authors: Park
authors: Youngsoo
authors: Y
authors: Jun
authors: Sangmyung
authors: S
authors: Rhee
authors: Woo Keun
authors: WK
authors: Song
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D001693:Biological Transport, Active / Q000502:physiology; D002453:Cell Cycle / Q000502:physiology*; D019938:Cyclin D1 / Q000096:biosynthesis / Q000235:genetics; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000235:genetics / Q000378:metabolism*; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation / Q000502:physiology
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C530104:CCND1 protein, human; D009124:Muscle Proteins; C486062:NCKIPSD protein, human; D019938:Cyclin D1; C512478:EGFR protein, human; D066246:ErbB Receptors; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3