An integrin-α4-14-3-3ζ-paxillin ternary complex mediates localised Cdc42 activity and accelerates cell migration

alpha 4 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 alpha 4 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 alpha 4 integrin cytoplasmic domain with 14-3-3 zeta. This interaction depends on serine phosphorylation of alpha 4 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 alpha 4 integrin and 14-3-3. is enhanced by the direct association between 14-3-3. and paxillin, resulting in the formation of a ternary complex that stabilises the recruitment of each component. Although pair-wise interaction between alpha 4 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 alpha 4 integrin-dependent migration.