Spatial proteomics reveal that the protein phosphatase PTP1B interacts with and may modify tyrosine phosphorylation of the rhomboid protease RHBDL4

Abstract

Rhomboid-like proteins are evolutionarily conserved, ubiquitous polytopic membrane proteins, including the canonical rhomboid intramembrane serine proteases and also others that have lost protease activity during evolution. We still have much to learn about their cellular roles, and evidence suggests that some may have more than one function. For example, RHBDL4 (rhomboid-like protein 4) is an endoplasmic reticulum (ER)–resident protease that forms a ternary complex with ubiquitinated substrates and p97/VCP (valosin-containing protein), a major driver of ER-associated degradation (ERAD). RHBDL4 is required for ERAD of some substrates, such as the pre–T-cell receptor α chain (pTα) and has also been shown to cleave amyloid precursor protein to trigger its secretion. In another case, RHBDL4 enables the release of full-length transforming growth factor α in exosomes. Using the proximity proteomic method BioID, here we screened for proteins that interact with or are in close proximity to RHBDL4. Bioinformatics analyses revealed that BioID hits of RHBDL4 overlap with factors related to protein stress at the ER, including proteins that interact with p97/VCP. PTP1B (protein-tyrosine phosphatase nonreceptor type 1, also called PTPN1) was also identified as a potential proximity factor and interactor of RHBDL4. Analysis of RHBDL4 peptides highlighted the presence of tyrosine phosphorylation at the cytoplasmic RHBDL4 C terminus. Site-directed mutagenesis targeting these tyrosine residues revealed that their phosphorylation modifies binding of RHBDL4 to p97/VCP and Lys 63 -linked ubiquitinated proteins. Our work lays a critical foundation for future mechanistic studies of the roles of RHBDL4 in ERAD and other important cellular pathways.

RHBDL4 protects against endoplasmic reticulum stress by regulating the morphology and distribution of ER sheets

“…5d). Significantly, this interaction was also found in our previous large-scale interaction screen of the RHBDL4 interactome -CLIMP-63 aka CKAP4 was one of the reproducible hits in HeLa and HEK 293 cells 49 . We could not detect an interaction between RHBDL4 and RTN4 or ATL1, or between RHBDL4 and CNX, another ER sheet membrane protein (not shown).…”

Intramembrane protease RHBDL4 cleaves oligosaccharyltransferase subunits to target them for ER-associated degradation

“…Mapping of the cleavage sites revealed that the scissile peptide bonds in L1 and L9 are spaced apart from the TM helix charge, supporting a model that docking and formation of the scission complex are distinct steps. Overall, the emerging picture is that recognition of RHBDL4 substrates is influenced by multiple factors, also including substrate ubiquitination by ERAD E3 ligases (Fleig et al, 2012), integration of signaling from G-protein-coupled receptors (Wunderle et al, 2016), tyrosine phosphorylation of the RHBDL4 cytoplasmic domain (Ikeda and Freeman, 2019) and sensing of the membrane lipid composition (Paschkowsky et al, 2018).…”

Intramembrane protease RHBDL4 cleaves oligosaccharyltransferase subunits to target them for ER-associated degradation

“…Mapping of the cleavage sites revealed that the scissile peptide bonds in L1 and L9 are spaced apart from the TM helix charge, supporting a model that docking and formation of the scission complex are distinct steps. Overall, the emerging picture is that recognition of RHBDL4 substrates is influenced by multiple factors, also including substrate ubiquitination by ERAD E3 ligases (Fleig et al, 2012), integration of signaling from G-protein coupled receptors (Wunderle et al, 2016), tyrosine phosphorylation of the RHBDL4 cytoplasmic domain (Ikeda and Freeman, 2019) and sensing of the membrane lipid composition .…”