Summary
α4 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 α4 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 α4 integrin cytoplasmic domain with 14-3-3ζ. This interaction depends on serine phosphorylation of α4 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 α4 integrin and 14-3-3ζ is enhanced by the direct association between 14-3-3ζ and paxillin, resulting in the formation of a ternary complex that stabilises the recruitment of each component. Although pair-wise interaction between α4 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 α4 integrin-dependent migration.
Footnotes
Supplementary material available online at http://jcs.biologists.org/cgi/content/full/122/10/1654/DC1
This work was supported by grants 045225 and 074941 from the Wellcome Trust (to M.J.H.). The Bioimaging Facility microscopes and the Biomolecular Analysis Facility Q-Trap mass spectrometer used in this study were purchased with grants from BBSRC, Wellcome Trust and the University of Manchester Strategic Fund. We would like to thank Blanche Schwappach (University of Manchester, Manchester, UK) for discussion of 14-3-3 ligand engagement. Deposited in PMC for release after 6 months.
↵* These authors contributed equally to this work
↵‡ Present address: Department of Cell and Developmental Biology, SUNY Upstate Medical University, Syracuse, NY, USA
↵§ Present address: Department of Biochemistry, Shiraz University of Medical Sciences, Shiraz, Iran
- Accepted February 5, 2009.
- © The Company of Biologists Limited 2009
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