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. 2009 May 15;122(Pt 10):1654-64.
doi: 10.1242/jcs.049130. Epub 2009 Apr 28.

An integrin-alpha4-14-3-3zeta-paxillin ternary complex mediates localised Cdc42 activity and accelerates cell migration

Nicholas O Deakin  1 , Mark D Bass, Stacey Warwood, Julia Schoelermann, Zohreh Mostafavi-Pour, David Knight, Christoph Ballestrem, Martin J Humphries
Affiliations

An integrin-alpha4-14-3-3zeta-paxillin ternary complex mediates localised Cdc42 activity and accelerates cell migration

Nicholas O Deakin et al. J Cell Sci. .

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.

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Figures

Fig. 1.
Fig. 1.
The cytoplasmic motifs of fibronectin-binding integrin α-subunits. (A-D) CHO-B2 cells expressing α5 and α4 integrin subunits or a chimeric integrin comprising the α5 extracellular domain and the α4 cytoplasmic domain were spread on recombinant fibronectin fragments, appropriate to the extracellular domain, and stained for vinculin (green) and actin (red). Scale bar: 10 μm. (E) Peptide sequence of the cytoplasmic domains of the α4 and α5 integrin subunits indicating the α4 paxillin-binding motif (green) and putative 14-3-3 mode II interaction motif (blue). Paxillin- (Y991A) and 14-3-3- (S978A) binding mutants were generated by site-directed mutagenesis (red).
Fig. 2.
Fig. 2.
Phosphorylation of the α4 cytoplasmic domain. (A,B) The α5/α4 chimera, including point substitutions S988A and S978A/S988A were immunoprecipitated from transgenic CHO-B2 cells, metabolically labelled with [32P]orthophosphate. Equivalent loading was tested by immunoblotting the α5 integrin extracellular domain using H-104, and phosphorylation of the α4 cytoplasmic domain was measured by autoradiography. (C) 2 fmol of synthetic peptide corresponding to the α4 integrin cytoplasmic domain, phosphorylated at S978 was digested with trypsin and added to a 6-protein mix. The mix was infused into the mass spectrometer and the retention spectrum was obtained for the peptide matching the specific neutral phosphate loss transition (534.7/485.7). (D) The light chain of endogenous α4 integrin, immunoprecipitated from A375 melanoma cell, was analysed using the protocol optimised with the synthetic peptide.
Fig. 3.
Fig. 3.
14-3-3ζ interacts with the cytoplasmic domain of α4 integrin in adhesion contacts. CHO-B2 cells transiently transfected with (A) α5/α4-GFP, (B) α5/α4-S978A-GFP and (C) α5/α4-S978D-GFP, were co-transfected with mRFP–14-3-3ζ and allowed to adhere to glass-bottomed Petri dishes coated with 10 μg/ml 50K. Spread cells were then subjected to acceptor photobleaching FRET. Line profiles indicate fluorescence intensity emitted by the GFP-conjugated integrin subunits in adhesion contacts before and after mRFP photobleaching. (D) The average peak FRET efficiency of all adhesion contacts per cell. Data are representative of 70-100 adhesion contacts, and the experiment analyzed 10 separate cells per line. Error bars represent s.e.m. *P<0.05. Scale bars: 5 μm.
Fig. 4.
Fig. 4.
14-3-3ζ interacts with full-length α4 integrin in adhesion contacts. CHO-B2 cells transiently transfected with (A) α4-GFP, (B) α4-S978A-GFP and (C) α4-S978D-GFP, were co-transfected with mRFP–14-3-3ζ and subjected to acceptor photobleaching FRET. (D) The average peak FRET efficiency of all adhesion contacts per cell. Data are representative of 70–100 adhesion contacts, and the experiment analyzed 10 separate cells per line. Error bars represent s.e.m. *P<0.05. Scale bar: 5 μm.
Fig. 5.
Fig. 5.
Paxillin associates with α4 integrin in adhesion contacts. CHO-B2 cells transiently transfected with (A) α5/α4-GFP and (B) α5/α4-Y991A-GFP, were co-transfected with mRFP-paxillin and subjected to acceptor photobleaching FRET. (C) The average peak FRET efficiency of all adhesion contacts per cell. Data are representative of 100-200 adhesion contacts, and the experiment analyzed five to ten separate cells per line. Error bars represent s.e.m. *P<0.005. Scale bars: 5 μm.
Fig. 6.
Fig. 6.
Recombinant 14-3-3ζ interacts with both paxillin and α4 integrin. (A) CHO-B2 cell lysate was incubated with protein G-Sepharose beads conjugated to GST or GST–14-3-3ζ and bound proteins analyzed by western blotting. Blots were probed for paxillin, vinculin and talin as candidate binding partners, and Raf1 and p130Cas as known 14-3-3 interacting proteins. (B) Protein complexes were immunoprecipitated from CHO-K1 cells using anti-paxillin or non-immune IgG. Precipitated proteins were blotted for paxillin, 14-3-3ζ and α-actinin. (C) Lysates of mutant α5/α4 CHO-B2 lines were incubated with protein G-Sepharose beads conjugated to GST or GST–14-3-3ζ and bound integrin was analyzed by western blotting. Blots of bound Raf1 and total integrin were used as controls and to standardise quantification. Error bars represent s.e.m., *P<0.05, n=4-8.
Fig. 7.
Fig. 7.
Formation of a ternary complex stabilises recruitment of 14-3-3ζ and paxillin to α4 integrin. CHO-B2 cells transiently transfected with (A,B) α5/α4-Y991A-GFP and (C,D) α4-Y991A-GFP, were co-transfected with mRFP–14-3-3ζ and subjected to acceptor photobleaching FRET. Representative images of FRET efficiency and overlay of α5/α4-Y991A-GFP fluorescence before and after mRFP photobleaching are shown. Line profiles indicate fluorescence intensity emitted by α5/α4Y991A-GFP in adhesion contacts before and after mRFP photobleaching. (B,D) The average peak FRET efficiency of all adhesion contacts per cell. Data are representative of 70-100 adhesion contacts, and each experiment analyzed seven separate cells. Error bars represent s.e.m.,*P≤0.05. Scale bars: 5 μm. (E) Images of lamellipodial leading edges of CHO-B2 cells expressing α5/α4, α5/α4-Y991A and α5/α4-S978D/Y991A integrin subunits. Cells were allowed to adhere to coverslips coated with 10 μg/ml 50K, fixed and stained for paxillin and actin. Paxillin images were converted to pixel intensity images. Arrows indicate focal adhesions (large adhesion plaques associated with the termini of actin stress fibres) and arrowheads the focal complexes (small adhesion plaques at the cell periphery). Scale bars: 3 μm. (F,G) Quantification of the pixel intensity at the leading edge of lamellipodia of cells stained for paxillin (F) and vinculin (G) Data are representative of three individual experiments and all protrusions in 15 cells/cell line were quantified. Error bars represent s.e.m. *P<0.05.
Fig. 8.
Fig. 8.
The interactions of α4 integrin with 14-3-3ζ and paxillin regulate localised GTPase activation. Rac1 activity (A) and Cdc42 activity (B) of α5/α4-expressing CHO-B2 lines spread on 50K, measured by affinity precipitation using GST-PAK. Error bars represent s.e.m., *P≤0.05, n=5 and 10 for Rac1 and Cdc42, respectively. (C-F) Raichu-Cdc42 and control Raichu-Cdc42Y40C FRET probes were transiently transfected into CHO-B2 cells expressing (C) α5/α4, (D) α5/α4-S978A, (E) α5/α4-Y991A and (F) α5/α4-S978D/Y991A adhered to a 50K substratum. Scale bar: 20 μm. (G) Quantification of cells for restriction of Cdc42 activity to the leading edge. Data are representative of 20 cells per experiment, performed on four separate occasions, error bars represent s.e.m., **P<0.005. (H) Quantification of the haptotactic migration of untransfected wild-type and α5/α4 variant-expressing CHO-B2 cells towards 10 μg/ml 50K. Haptotactic migration was calculated relative to the migration observed in CHO-B2 cells expressing the full-length α5 subunit. Data are the average of four individual experiments, performed in quadruplicate, error bars represent s.e.m., *P<0.05.
Fig. 9.
Fig. 9.
Schematic model of the effects of 14-3-3ζ or paxillin binding to α4 integrin in different areas of the membrane.
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