Novel Protein Interactions with Endoglin and Activin Receptor-like Kinase 1: Potential Role in Vascular Networks

Abstract

Endoglin and activin receptor-like kinase 1 are specialized transforming growth factor-beta (TGF-␤) superfamily receptors, primarily expressed in endothelial cells. Mutations in the corresponding ENG or ACVRL1 genes lead to hereditary hemorrhagic telangiectasia (HHT1 and HHT2 respectively). To discover proteins interacting with endoglin, ACVRL1 and TGF-␤ receptor type 2 and involved in TGF-␤ signaling, we applied LUMIER, a high-throughput mammalian interactome mapping technology. Using stringent criteria, we identified 181 novel unique and shared interactions with ACVRL1, TGF-␤ receptor type 2, and endoglin, defining potential novel important vascular networks. In particular, the regulatory subunit B-beta of the protein phosphatase PP2A (PPP2R2B) interacted with all three receptors. Interestingly, the PPP2R2B gene lies in an interval in linkage disequilibrium with HHT3, for which the gene remains unidentified. We show that PPP2R2B protein interacts with the ACVRL1/TGFBR2/endoglin complex and recruits PP2A to nitric oxide synthase 3 (NOS3). Endoglin overexpression in endothelial cells inhibits the association of PPP2R2B with NOS3, whereas endoglin-deficient cells show enhanced PP2A-NOS3 interaction and lower levels of endogenous NOS3 Serine 1177 phosphorylation. Our data suggest that endoglin regulates NOS3 activation status by regulating PPP2R2B access to NOS3, and that PPP2R2B might be the HHT3 gene. Furthermore, endoglin and ACVRL1 contribute to several novel networks, including TGF-␤ dependent and independent ones, critical for vascular function and potentially defective in HHT. Molecular & Cellular Proteomics 13: 10.1074/mcp.M113.033464, 489-502, 2014. Transforming growth factor-␤ (TGF-␤)1 superfamily ligands, including TGF-␤s, activins and bone morphogenic proteins (BMPs), regulate several pathways essential for vascular development and function (1). Responses to these ligands are controlled by type I and II serine kinase receptors, coreceptors and signaling SMAD intermediates. Endothelial cells express the coreceptor, endoglin, and the specialized type I receptor, ACVRL1 (activin receptor-like kinase 1 or ALK1); both molecules are critical for regulation of angiogenesis and vasomotor function by TGF-␤ superfamily ligands (2, 3).Mutations in ENG and ACVRL1 genes lead to hereditary hemorrhagic telangiectasia (HHT), types 1 and 2, respectively (4). HHT affects 1 in 5000 -8000 people worldwide and is characterized by arteriovenous malformations (AVMs) in mul-

Increased endothelial cell permeability in endoglin-deficient cells

“…Our recent study (11) demonstrated protein interactions between many Rho GTPases and TGF‐β receptors. Pak‐1 interacted with ENG, TGFBR2, ALK1, and ALK5 (11, 16), whereas Rac‐2 and Rab38 interacted with the first 3. RhoH interacted with ALK1, RhoJ and Rasl with ALK1 and TGFBR2, and Arl8B with ENG and TGFBR2 (11).…”

“…Human embryonic kidney‐293T cells were seeded in 6‐well plates at 2.5 × 10 5 cells perwell and transfected with 1 μg total plasmid DNA (RhoA‐Flag, with or without ENG) using FuGENE 6 (Hoffmann‐La Roche Limited, Mississauga, ON, Canada). The preparation of cell lysates, immunoprecipitation (IP), and Western blot (WB) were carried out as described (11). The P4A4 mAb to ENG (1 μg) was used for IP and the mouse anti‐Flag (M2) antibody (Sigma‐Aldrich, Oakville, ON, Canada) for WB.…”

“…Furthermore, VE‐cadherin concentrated at cell‐to‐cell adherens junctions, interacts with many components of the TGF‐β receptor complex, including ENG, TGFBR2, and ALK1 (10). Our previous study, using a high‐throughput luminescence‐based mammalian interactome assay (LUMIER), documented novel ENG, TGFBR2, and ALK1 interactions with several proteins including p21‐activated kinase (PAK‐1), GTP‐binding proteins, and Ras‐related C3 botulinum toxin substrate 2 (Rac‐2) involved in endothelial barrier maintenance, cytoskeletal remodeling, and cell migration (11). Thus, ENG, directly, or as a component of the TGF‐β receptor complex may regulate EC integrity; its absence could result in vascular hyperpermeability and contribute to the fetal lethality associated with the homozygous null genotype (9, 12).…”

“…(continued on next page) several proteins including p21-activated kinase (PAK-1), GTP-binding proteins, and Ras-related C3 botulinum toxin substrate 2 (Rac-2) involved in endothelial barrier maintenance, cytoskeletal remodeling, and cell migration (11). Thus, ENG, directly, or as a component of the TGF-b receptor complex may regulate EC integrity; its absence could result in vascular hyperpermeability and contribute to the fetal lethality associated with the homozygous null genotype (9,12).…”

Systems medicine: evolution of systems biology from bench to bedside

“…High‐throughput interactome mapping recently identified 181 novel interactors between ACVRL1, the TGF‐β receptor‐2, and endoglin, including protein phosphatase subunit β (PPP2RB). In turn, PPP2RB was shown to disrupt endothelial nitric oxide synthase signaling in endoglin‐deficient cells in vitro , identifying a potential role for PPP2RB in the pathobiology of HHT …”