title: Activation of EGF receptor endocytosis and ERK1/2 signaling by BPGAP1 requires direct interaction with EEN/endophilin II and a functional RhoGAP domain.
abstract: Rho GTPases are important regulators for cell dynamics. They are activated by guanine nucleotide exchange factors and inactivated by GTPase-activating proteins (GAPs). We recently identified a novel RhoGAP, BPGAP1, that uses the BNIP-2 and Cdc42GAP homology (BCH) domain, RhoGAP domain and proline-rich region to regulate cell morphology and migration. To further explore its roles in intracellular signaling, we employed protein precipitations and matrix-assisted laser desorption/ionization mass-spectrometry and identified EEN/endophilin II as a novel partner of BPGAP1. EEN is a member of the endocytic endophilin family but its function in regulating endocytosis remains unclear. Pull-down and co-immunoprecipitation studies with deletion mutants confirmed that EEN interacted directly with BPGAP1 via its Src homology 3 (SH3) domain binding to the proline-rich region 182-PPPRPPLP-189 of BPGAP1, with prolines 184 and 186 being indispensable for this interaction. Overexpression of EEN or BPGAP1 alone induced EGF-stimulated receptor endocytosis and ERK1/2 phosphorylation. These processes were further enhanced when EEN was present together with the wildtype but not with the non-interactive proline mutant of BPGAP1. However, EEN lacking the SH3 domain served as a dominant negative mutant that completely inhibited these effects. Furthermore, BPGAP1 with a catalytically inactive GAP domain also blocked the effect of EEN and/or BPGAP1 in EGF receptor endocytosis and concomitantly reduced their level of augmentation for ERK1/2 phosphorylation. Our findings reveal a concomitant activation of endocytosis and ERK signaling by BPGAP1 via the coupling of its proline-rich region, which targets EEN and its functional GAP domain. BPGAP1 could therefore provide an important link between cytoskeletal network, endocytic trafficking and Ras/MAPK signaling.
authors.affiliation: Cell Signaling and Developmental Biology Laboratory, Department of Biological Sciences, The National University of Singapore.
authors.forename: Bee Leng
authors.initials: BL
authors.lastname: Lua
authors.forename: Boon Chuan
authors.initials: BC
authors.lastname: Low
mesh_terms: D000595:Amino Acid Sequence; D004705:Endocytosis; D066246:ErbB Receptors / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000378:metabolism*; D020935:MAP Kinase Signaling System; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D018909:src Homology Domains
chemical_list: C479829:ARHGAP8 protein, human; D020690:GTPase-Activating Proteins; D047908:Intracellular Signaling Peptides and Proteins; C105159:SH3GL1 protein, human; C085379:rho GTPase-activating protein; D066246:ErbB Receptors; D048049:Extracellular Signal-Regulated MAP Kinases