title: An integrin-alpha4-14-3-3zeta-paxillin ternary complex mediates localised Cdc42 activity and accelerates cell migration.
abstract: Alpha4 integrins are used by leukocytes and neural crest derivatives for adhesion and migration during embryogenesis, immune responses and tumour invasion. The pro-migratory activity of alpha4 integrin is mediated in part through the direct binding of the cytoplasmic domain to paxillin. Here, using intermolecular FRET and biochemical analyses, we report a novel interaction of the alpha4 integrin cytoplasmic domain with 14-3-3zeta. This interaction depends on serine phosphorylation of alpha4 integrin at a site (S978) distinct from that which regulates paxillin binding (S988). Using a combination of metabolic labelling and targeted mass spectrometry by multiple reaction monitoring we demonstrate the low stoichiometry phosphorylation of S978. The interaction between alpha4 integrin and 14-3-3zeta is enhanced by the direct association between 14-3-3zeta and paxillin, resulting in the formation of a ternary complex that stabilises the recruitment of each component. Although pair-wise interaction between alpha4 integrin and paxillin is sufficient for normal Rac1 regulation, the integrity of the ternary complex is essential for focused Cdc42 activity at the lamellipodial leading edge and directed cell movement. Taken together, these data identify a key signalling nexus mediating alpha4 integrin-dependent migration.
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK.
authors.forename: Nicholas O
authors.initials: NO
authors.lastname: Deakin
authors.forename: Mark D
authors.initials: MD
authors.lastname: Bass
authors.forename: Stacey
authors.initials: S
authors.lastname: Warwood
authors.forename: Julia
authors.initials: J
authors.lastname: Schoelermann
authors.forename: Zohreh
authors.initials: Z
authors.lastname: Mostafavi-Pour
authors.forename: David
authors.initials: D
authors.lastname: Knight
authors.forename: Christoph
authors.initials: C
authors.lastname: Ballestrem
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Humphries
mesh_terms: D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002465:Cell Movement; D006224:Cricetinae; D003412:Cricetulus; D005353:Fibronectins / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D039441:Integrin alpha4 / Q000235:genetics / Q000378:metabolism*; D039041:Integrin alpha4beta1 / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000378:metabolism; D009154:Mutation; D051419:Paxillin / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011554:Pseudopodia / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
chemical_list: D048948:14-3-3 Proteins; D005353:Fibronectins; D039041:Integrin alpha4beta1; D046912:Multiprotein Complexes; C496061:PXN protein, human; D051419:Paxillin; D011993:Recombinant Fusion Proteins; D039441:Integrin alpha4; D012694:Serine; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein