The epidermal growth factor receptor (EGFR) is a critical determinator of cell fate. Signalling from this receptor tyrosine kinase is spatially regulated by progression through the endocytic pathway, governing receptor half-life and accessibility to signalling proteins and phosphatases. Endocytosis of EGFR is required for interaction with the protein tyrosine phosphatase PTP1B (ref. 1), which localizes to the cytoplasmic face of the endoplasmic reticulum (ER), raising the question of how PTP1B comes into contact with endosomal EGFR. We show that EGFR-PTP1B interaction occurs by means of direct membrane contacts between the perimeter membrane of multivesicular bodies (MVBs) and the ER. The population of EGFR interacting with PTP1B is the same population that undergo ESCRT-mediated (endosomal sorting complex required for transport) sorting within MVBs, and PTP1B activity promotes the sequestration of EGFR on to MVB internal vesicles. Membrane contacts between endosomes and the ER form in both the presence and absence of stimulation by EGF. Thus membrane contacts between endosomes and the ER may represent a global mechanism for direct interaction between proteins on these two organelles.

The tight junction protein ZO-1 inhibits G1/S-phase transition by cytoplasmic sequestration of a complex formed by CDK4 and the transcription factor ZONAB. Canine ZONAB is the homologue of human DbpA, an E2F target gene that is overexpressed in different carcinomas. Since the ZONAB target genes that are involved in G1/S-phase transition are unknown, we employed the mammary epithelial cell line MCF-10A and cDNA arrays to screen for such genes. We identified genes encoding cell cycle and replication proteins whose expression was altered due to increased ZONAB expression. For proliferative cell nuclear antigen and cyclin D1 genes, we show that increased mRNA levels resulted in increased protein levels and we identified ZONAB-responsive elements in their promoters by using different approaches, including chromatin immunoprecipitation assays. RNA interference and overexpression of ZONAB affected the proliferation of both MCF-10A and MDCK cells as well as the differentiation of MDCK cells into polarized cysts in three-dimensional cultures. These results indicate that ZONAB regulates the transcription of genes that are important for G1/S-phase progression and links tight junctions to the transcriptional control of key cell cycle regulators and epithelial cell differentiation.

SummaryMembrane contact sites between the ER and multivesicular endosomes/bodies (MVBs) play important roles in endosome positioning and fission and in neurite outgrowth. ER-MVB contacts additionally function in epidermal growth factor receptor (EGFR) tyrosine kinase downregulation by providing sites where the ER-localized phosphatase, PTP1B, interacts with endocytosed EGFR before the receptor is sorted onto intraluminal vesicles (ILVs). Here we show that these contacts are tethered by annexin A1 and its Ca2+-dependent ligand, S100A11, and form a subpopulation of differentially regulated contact sites between the ER and endocytic organelles. Annexin A1-regulated contacts function in the transfer of ER-derived cholesterol to the MVB when low-density lipoprotein-cholesterol in endosomes is low. This sterol traffic depends on interaction between ER-localized VAP and endosomal oxysterol-binding protein ORP1L, and is required for the formation of ILVs within the MVB and thus for the spatial regulation of EGFR signaling.

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TGFβ induces various responses, including Rho signaling. How TGFβ stimulates Rho is poorly understood. Our data indicate that GEF-H1 is a target and effector of TGFβ to regulate Rho signaling, gene expression, and cell migration, suggesting that it represents a new marker and possible therapeutic target for degenerative and fibrotic diseases.