The regulation of endocytic sorting and cholesterol transport by PTP1B-mediated ESCRT dephosphorylation at ER-endosome membrane contact sites

The transport of proteins and lipids to the right cellular location is critical for the function of all cells. Endocytosis is a mechanism of uptake of extracellular molecules and cell surface membrane proteins into intracellular membrane compartments, called endosomes. Within the endosome, proteins and lipids are sorted. Unwanted cell membrane proteins and lipids can then be removed by delivering them to lysosomes where they are degraded. This degradative pathway is balanced by new synthesis of membrane proteins and lipids in the endoplasmic reticulum (ER). Overproduction or impaired degradation of membrane proteins or lipids can lead to multiple diseases, including cancer and lipid storage disorders.

The degradative pathway is important for regulating signaling from growth factor receptors, such as the EGF receptor (EGFR), a cell surface signaling protein that regulates cell division. Dysregulated signaling from the EGFR occurs in about 50% of human cancers and is a target for cancer therapies. It activates signaling pathways by chemically modifying both itself and other signaling proteins. This modification, called phosphorylation, is reversible by dephosphorylation. The EGFR can be dephosphorylated by a phosphatase on the ER, PTP1B. Proteins and lipids can shuttle from cell membrane to membrane via small transport vesicles. Different cellular membranes can also form membrane contacts where they come very close together to form sites where proteins on the apposing membranes can interact and lipids can be flipped directly from one membrane to the other. We recently found that the ER and the endocytic pathway form membrane contacts and have identified molecules that tether the apposing membranes at the contact. We showed that ER:endosome membrane contacts are necessary for PTP1B to dephosphorylate endocytosed EGFR, and that PTP1B activity promotes delivery of EGFR to the lysosome for degradation. Some of the molecules that regulate lysosomal delivery of EGFR are themselves regulated by PTP1B-mediated dephosphorylation and one of those molecules, Hrs, has also been implicated in cholesterol exchange between endosomes and the ER.

Cholesterol is a critical component of cell membranes obtained by endocytosis of dietary LDL, or by new synthesis in the ER. Excess endocytosed cholesterol is transferred to the ER and packaged into lipid droplets for storage. However, the endocytic pathway needs a certain amount of cholesterol to function. So when LDL-derived cholesterol is low, newly synthesized cholesterol is transferred from the ER to endosomes. We showed that ER:endosome membrane contacts regulate exchange of cholesterol between the ER and endosomes. Regulation of endosomal cholesterol is important for multiple processes. Endosomal cholesterol is required for normal function of the degradative pathway, but impaired egress of endosomal LDL-cholesterol is associated with lipid storage diseases, such as the childhood neurological disorder, Neimann Pick Type C disease.

We hypothesise that PTP1B-mediated dephosphorylation of Hrs at membrane contact sites co-ordinates lysosomal targeting of EGFR and cholesterol exchange between endosomes and the ER. We will test this hypothesis using tools we've developed to manipulate membrane contacts, together with high-resolution electron microscopy (the only way to unequivocally identify membrane contacts) and newly developed super resolution microscopy techniques. Together these studies will provide a better understanding of how PTP1B activity at membrane contact sites regulates EGFR signaling and cholesterol exchange. This will determine how the relationship between PTP1B and EGFR can be exploited for the design of better combination cancer therapies and may yield novel therapeutic targets for the treatment of lipid storage diseases.

The EGF receptor (EGFR) activates signaling pathways through reversible phosphorylation of itself and downstream targets, has a well-established role in cancer and is a target for cancer therapies. Endocytic transport regulates EGFR signaling, as ubiquitinated receptor is sorted onto intraluminal vesicles (ILVs) of multivesicular bodies (MVBs), removing its catalytic domain from cytoplasmic targets before lysosomal delivery for degradation. We showed that endocytosed EGFR is dephosphorylated by the ER-localised phosphatase, PTP1B, at membrane contact sites (MCSs) tethered by annexinA1/S100A11 complexes, where the membranes of the ER and MVBs come into close apposition. These MCSs regulate ILV formation, most likely by PTP1B-mediated dephosphorylation of the ESCRT proteins, Hrs and STAM, that sort ubiquitinated cargo onto ILVs, and also by transport of ER-derived cholesterol to MVBs to support ILV formation when endosomal cholesterol is low. Intriguingly, Hrs is also required for cholesterol transport from endosomes to the ER, a process proposed to occur at MCSs. We hypothesise that modulation of Hrs/STAM phosphorylation at MCSs by PTP1B co-ordinates MVB sorting and cholesterol transport functions of Hrs. We will use our established tools to modulate PTP1B activity (PTP1B overexpression/depletion/inhibition), MCS formation (AnnexinA1/S100A11 depletion/overexpression) and cholesterol exchange (culture in delipidated serum/statins) and generate novel gene-edited HeLa cells expressing Hrs/STAM phosphorylation mutants. A combination of electron and super-resolution microscopy and biochemical analysis will be used to determine the role of Hrs/STAM phosphorylation in regulating sorting of EGFR onto ILVs and cholesterol transport at MCSs. These data will help to elucidate the role of PTP1B in EGFR-dependent tumorigenesis, will aid in the design of improved combination cancer therapies and will increase our understanding of the role of MCSs in lysosomal storage diseases.

The results of this research will be of benefit to academic researchers and healthcare professionals working in the fields of cancer, diabetes, obesity and neurodegenerative disorders characterized by lipid storage in the endocytic pathway. PTP1B regulates insulin signaling and is a validated therapeutic target for diabetes, obesity and breast cancer. Dysregulated signaling from the EGF receptor (EGFR) has a well-established role in human cancer and EGFR is a target for cancer therapies. In this proposal we will investigate the role of PTP1B in regulating EGFR trafficking, and signaling. An improved understanding of the interplay between PTP1B and EGFR has clear importance for the safety and efficacy of exploiting PTP1B-directed therapeutics for diabetes and cancer, the management of which currently places a huge financial burden on the healthcare system. We have shown that PTP1B and EGFR interact at membrane contact sites between endosomes and the ER, and have identified specific regulators of these contacts. This could provide an interesting means of targeting PTP1B at specific sites of interaction by targeting regulators of sub-populations of membrane contact sites. This project will also address the relatively poorly understood role of membrane contact sites in cholesterol transport, focusing on the role of Hrs. Loss of Hrs results in accumulation of cholesterol in the endocytic pathway, which is a characteristic of lysosomal storage diseases, such as Neiman-Pick type C, and is associated with a number of neurodegenerative disorders. This project will therefore contribute to research into neurodegenerative disease biology, with the potential to identify novel therapeutic targets that specifically facilitate cholesterol egress from the endocytic pathway.
The existence of membrane contact sites (MCSs) that provide a novel means of communication between organelles has only recently been recognized and due to an explosion of interest in the field, membrane contact site biology is a rapidly advancing field. Here, in combination with other established methods, we take a novel approach to studying endocytic traffic, taking advantage of our recent findings on MCS regulation to manipulate a specific subset of ER-endosome membrane contact sites. This approach could have wide-ranging applications to other research areas. We will additionally continue to optimise super-resolution methods for MCS visualisation. Since membrane contact sites provide potential sites of interorganellar lipid exchange and regulators of cytoplasmic calcium levels, our research would additionally benefit researchers working in the fields of calcium dynamics and lipid traffic.