title: EHD2 mediates trafficking from the plasma membrane by modulating Rac1 activity.
abstract: EHDs [EH (Eps15 homology)-domain-containing proteins] participate in different stages of endocytosis. EHD2 is a plasma-membrane-associated EHD which regulates trafficking from the plasma membrane and recycling. EHD2 has a role in nucleotide-dependent membrane remodelling and its ATP-binding domain is involved in dimerization, which creates a membrane-binding region. Nucleotide binding is important for association of EHD2 with the plasma membrane, since a nucleotide-free mutant (EHD2 T72A) failed to associate. To elucidate the possible function of EHD2 during endocytic trafficking, we attempted to unravel proteins that interact with EHD2, using the yeast two-hybrid system. A novel interaction was found between EHD2 and Nek3 [NIMA (never in mitosis in Aspergillus nidulans)-related kinase 3], a serine/threonine kinase. EHD2 was also found in association with Vav1, a Nek3-regulated GEF (guanine-nucleotide-exchange factor) for Rho GTPases. Since Vav1 regulates Rac1 activity and promotes actin polymerization, the impact of overexpression of EHD2 on Rac1 activity was tested. The results indicated that wt (wild-type) EHD2, but not its P-loop mutants, reduced Rac1 activity. The inhibitory effect of EHD2 overexpression was partially rescued by co-expression of Rac1 as measured using a cholera toxin trafficking assay. The results of the present study strongly indicate that EHD2 regulates trafficking from the plasma membrane by controlling Rac1 activity.
authors.affiliation: Department of Cell Research and Immunology, Tel Aviv University, Ramat Aviv, Israel.
authors.forename: Sigi
authors.initials: S
authors.lastname: Benjamin
authors.forename: Hilla
authors.initials: H
authors.lastname: Weidberg
authors.forename: Debora
authors.initials: D
authors.lastname: Rapaport
authors.forename: Olga
authors.initials: O
authors.lastname: Pekar
authors.forename: Marina
authors.initials: M
authors.lastname: Nudelman
authors.forename: Daniel
authors.initials: D
authors.lastname: Segal
authors.forename: Koret
authors.initials: K
authors.lastname: Hirschberg
authors.forename: Shulamit
authors.initials: S
authors.lastname: Katzav
authors.forename: Marcelo
authors.initials: M
authors.lastname: Ehrlich
authors.forename: Mia
authors.initials: M
authors.lastname: Horowitz
mesh_terms: D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D000072379:NIMA-Related Kinases; D011485:Protein Binding / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D051567:Proto-Oncogene Proteins c-vav / Q000235:genetics / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; C406446:EHD2 protein, human; D051567:Proto-Oncogene Proteins c-vav; C107953:RAC1 protein, human; C496618:VAV1 protein, human; C486224:NEK3 protein, human; D000072379:NIMA-Related Kinases; D017346:Protein Serine-Threonine Kinases; D020830:rac1 GTP-Binding Protein