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First published online 28 April 2009
doi: 10.1242/jcs.049130

Journal of Cell Science 122, 1654-1664 (2009)
Published by The Company of Biologists 2009
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An integrin-{alpha}4–14-3-3{zeta}–paxillin ternary complex mediates localised Cdc42 activity and accelerates cell migration

Nicholas O. Deakin1,*,{ddagger}, Mark D. Bass1,*, Stacey Warwood1, Julia Schoelermann2,3, Zohreh Mostafavi-Pour1,§, David Knight1, Christoph Ballestrem1 and Martin J. Humphries1,¶

1 Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK
2 Department of New Materials and Biosystems, Max-Planck-Institute for Metals Research, Heisenbergstrasse 3, D-70569, Germany
3 Department of Biophysical Chemistry, University of Heidelberg, INF 235, 69120 Heidelberg, Germany


Figure 1
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  		Fig. 1. The cytoplasmic motifs of fibronectin-binding integrin {alpha}-subunits. (A-D) CHO-B2 cells expressing {alpha}5 and {alpha}4 integrin subunits or a chimeric integrin comprising the {alpha}5 extracellular domain and the {alpha}4 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 {alpha}4 and {alpha}5 integrin subunits indicating the {alpha}4 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).

 

Figure 2
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  		Fig. 2. Phosphorylation of the {alpha}4 cytoplasmic domain. (A,B) The {alpha}5/{alpha}4 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 {alpha}5 integrin extracellular domain using H-104, and phosphorylation of the {alpha}4 cytoplasmic domain was measured by autoradiography. (C) 2 fmol of synthetic peptide corresponding to the {alpha}4 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 {alpha}4 integrin, immunoprecipitated from A375 melanoma cell, was analysed using the protocol optimised with the synthetic peptide.

 

Figure 3
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  		Fig. 3. 14-3-3{zeta} interacts with the cytoplasmic domain of {alpha}4 integrin in adhesion contacts. CHO-B2 cells transiently transfected with (A) {alpha}5/{alpha}4-GFP, (B) {alpha}5/{alpha}4-S978A-GFP and (C) {alpha}5/{alpha}4-S978D-GFP, were co-transfected with mRFP–14-3-3{zeta} 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.

 

Figure 4
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  		Fig. 4. 14-3-3{zeta} interacts with full-length {alpha}4 integrin in adhesion contacts. CHO-B2 cells transiently transfected with (A) {alpha}4-GFP, (B) {alpha}4-S978A-GFP and (C) {alpha}4-S978D-GFP, were co-transfected with mRFP–14-3-3{zeta} 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.

 

Figure 5
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  		Fig. 5. Paxillin associates with {alpha}4 integrin in adhesion contacts. CHO-B2 cells transiently transfected with (A) {alpha}5/{alpha}4-GFP and (B) {alpha}5/{alpha}4-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.

 

Figure 6
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  		Fig. 6. Recombinant 14-3-3{zeta} interacts with both paxillin and {alpha}4 integrin. (A) CHO-B2 cell lysate was incubated with protein G-Sepharose beads conjugated to GST or GST–14-3-3{zeta} 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, 14-3-3{zeta} and {alpha}-actinin. (C) Lysates of mutant {alpha}5/{alpha}4 CHO-B2 lines were incubated with protein G-Sepharose beads conjugated to GST or GST–14-3-3{zeta} 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.

 

Figure 7
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  		Fig. 7. Formation of a ternary complex stabilises recruitment of 14-3-3{zeta} and paxillin to {alpha}4 integrin. CHO-B2 cells transiently transfected with (A,B) {alpha}5/{alpha}4-Y991A-GFP and (C,D) {alpha}4-Y991A-GFP, were co-transfected with mRFP–14-3-3{zeta} and subjected to acceptor photobleaching FRET. Representative images of FRET efficiency and overlay of {alpha}5/{alpha}4-Y991A-GFP fluorescence before and after mRFP photobleaching are shown. Line profiles indicate fluorescence intensity emitted by {alpha}5/{alpha}4Y991A-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 {alpha}5/{alpha}4, {alpha}5/{alpha}4-Y991A and {alpha}5/{alpha}4-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.

 

Figure 8
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  		Fig. 8. The interactions of {alpha}4 integrin with 14-3-3{zeta} and paxillin regulate localised GTPase activation. Rac1 activity (A) and Cdc42 activity (B) of {alpha}5/{alpha}4-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) {alpha}5/{alpha}4, (D) {alpha}5/{alpha}4-S978A, (E) {alpha}5/{alpha}4-Y991A and (F) {alpha}5/{alpha}4-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 {alpha}5/{alpha}4 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 {alpha}5 subunit. Data are the average of four individual experiments, performed in quadruplicate, error bars represent s.e.m., *P<0.05.

 

Figure 9
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  		Fig. 9. Schematic model of the effects of 14-3-3{zeta} or paxillin binding to {alpha}4 integrin in different areas of the membrane.

 

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© The Company of Biologists Ltd 2009