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Address correspondence to S.J. Shattil, Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Rd., VB-5, La Jolla, CA 92037. Tel.: (858) 784-7148. Fax: (858) 784-7422. email: shattil{at}scripps.edu
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Abstract |
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Key Words: signaling; Src; Syk; FAK; bioluminescence
Abbreviations used in this paper: BiFC, bimolecular fluorescence complementation; BRET, bioluminescence resonance energy transfer; BSA, bovine serum albumin; Rluc, Renilla luciferase.
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Introduction |
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The molecular basis for IIbß3 activation of tyrosine kinases is incompletely understood. c-Src and several other Src family kinases coimmunoprecipitate with
IIbß3 from resting platelets. In contrast, Syk coimmunoprecipitates with
IIbß3 only after platelet adhesion to fibrinogen (Obergfell et al., 2002). Work in CHO cells indicates that c-Src and Syk activation requires
IIbß3 clustering and an intact ß3 cytoplasmic tail (Hato et al., 1998; Arias-Salgado et al., 2003). Because c-Src, Syk, and FAK can bind directly to ß3 tail peptides or model ß3 tail proteins in vitro (Schaller et al., 1995; Woodside et al., 2002; Arias-Salgado et al., 2003), direct interactions between ß3 and tyrosine kinases may promote outside-in signaling.
To better define IIbß3tyrosine kinase interactions in living cells, we have used two protein interaction reporter assays that can detect proximal (nm) interactions, bioluminescence resonance energy transfer (BRET), and bimolecular fluorescence complementation (BiFC). BRET between a Renilla luciferase (Rluc) chimera and another chimera with a luminescence acceptor, such as GFP, has been used to study homooligomerization of receptors, including integrins, by luminometry (Angers et al., 2000; Boute et al., 2002; Ramsay et al., 2002; Buensuceso et al., 2003). In BiFC, two potential interacting proteins are fused to NH2- and COOH-terminal half-molecules of YFP, respectively. If the transfected proteins interact, YFP may be reconstituted (Hu et al., 2002; Hu and Kerppola, 2003). Thus, BiFC may complement information derived from BRET by enabling visualization and subcellular localization of
IIbß3tyrosine kinase complexes by fluorescence microscopy. Both techniques require expression of chimeric proteins, but they have the potential to supplement biochemical and genetic analyses of integrins. Using BRET, BiFC, and proteins fused to suitable reporters, we establish here that proximal
IIbß3tyrosine kinase interactions occur in a coordinated and sequential manner within membrane ruffles and specific adhesion structures, providing a spatial context for outside-in
IIbß3 signaling.
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Results and discussion |
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BiFC was performed by fusing IIb to the COOH-terminal half-molecule of YFP (
IIbYC) and c-Src to the NH2-terminal half of YFP (SrcYN; Fig. 2 A). BiFC between
IIbYCß3 and SrcYN was observed around the periphery of nonadherent cells and at the ruffling edges of lamellipodia of fibrinogen-adherent cells, where it colocalized with antibody-labeled
IIbß3 and c-Src (Fig. 2 B, i). In contrast, no BiFC was observed in cells expressing ß3
724 (Fig. 2 B, ii). BiFC between
IIbYCß3 and SrcYN also colocalized with cortactin within a distance of 34 µm from the leading edge (Fig. 2 C, i), and with vinculin and F-actin in small focal complexes and larger focal adhesions (Fig. 2 C, ii). Similarly, when unfixed cells were analyzed by real-time fluorescence microscopy, BiFC between
IIbYCß3 and SrcYN was detected within membrane ruffles, focal complexes, and focal adhesions (Fig. 3 A and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200402064/DC1). Over a 6-min observation period,
IIbYCß3SrcYN complexes appeared to move from membrane ruffles to the focal adhesion structures, a conclusion supported by the predominant colocalization of the BiFC signal with known markers of these structures in fixed and stained cells (Fig. 2 C). Thus, the proximity of
IIbß3 to c-Src and the dynamic nature of
IIbß3c-Src complexes upon integrin ligation provide a spatial context for initiation of outside-in
IIbß3 signaling. Similarly, interaction between c-Src and the related integrin,
Vß3, may occur within podosomes of osteoclasts (Linder and Aepfelbacher, 2003), whereas the absence of such an interaction may help to explain the overlapping bone resorbtion defects in mice deficient in either
Vß3 or c-Src (Soriano et al., 1991; McHugh et al., 2000).
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These results raise additional questions. First, previous work with fibrinogen-adherent platelets and IIbß3-CHO cells has shown that Src activity promotes Syk activation (Gao et al., 1997; Obergfell et al., 2002). To determine if Src activity is required to recruit Syk to
IIbß3, BiFC experiments were performed in the presence of a Src kinase inhibitor, either PP2 or SU6656 (Hanke et al., 1996; Blake et al., 2000). When 5 µM PP2 or 2 µM SU6656 was added to cells before plating on fibrinogen, interactions between
IIbYCß3 and SykYN were partially inhibited, whereas the inactive congener PP3 had no effect (Fig. 5 A). Similarly, PP2 or SU6656 inhibited interactions between SykYC and SrcYN. When PP2 was added to cells already adherent to fibrinogen, it caused a rapid loss of BiFC signals between
IIbYCß3 and SykYN or between SykYC and c-SrcYN (unpublished data). Thus, Src activity is required for the recruitment of Syk to
IIbß3 and possibly for the maintenance of a critical density of integrinSyk complexes in adherent cells.
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The current results serve to further emphasize the dynamic and heterogeneous nature of integrin-based signaling complexes (Miyamoto et al., 1995; Zamir and Geiger, 2001), and they reveal a new level of complexity in IIbß3 signaling. On a broader level, BRET and BiFC should provide a facile means to monitor binary interactions between integrins and many other proteins. Along with FRET and other recent elegant innovations in imaging (Kraynov et al., 2000; Webb et al., 2003), these two approaches should complement biochemical and genetic investigations into the molecular basis of integrin signaling.
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Materials and methods |
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Construction of recombinant proteins
For BRET assays, plasmid templates encoding human IIb, Syk, or mouse c-Src were amplified by PCR to place appropriate restriction sites, and PCR products were subcloned into pGFPN2 and pRlucN2 mammalian expression vectors (Buensuceso et al., 2003) to produce the BRET chimeras in Fig. 1 A. For BiFC assays, plasmid templates encoding human
IIb, Syk, mouse c-Src, or the NH2-terminal (YN) or COOH-terminal (YC) halves of YFP (a gift from T. Kerppola and C.-D. Hu, University of Michigan, Ann Arbor, MI; Hu et al., 2002) were amplified to place appropriate restriction sites, and PCR products were subcloned into pCDNA3 to produce the BiFC chimeras in Fig. 2 A. All coding sequences were verified by DNA sequencing, and expression plasmids were purified with the Qiafilter plasmid maxi kit (QIAGEN).
Cell culture and transfections
CHO cells were transiently transfected (Obergfell et al., 2001) with ß3 and the appropriate BRET or BiFC chimeras. In experiments where interactions between two tyrosine kinases were being studied, the tyrosine kinase chimeras were transfected into A5 CHO cells stably expressing IIbß3 or
IIbß3
724 (Obergfell et al., 2001).
BRET assays
48 h after transfection of BRET chimeras, CHO cells were resuspended to 3 x 106/ml in BRET buffer (Buensuceso et al., 2003), and 30 µl aliquots were added to microtiter wells precoated with 250 µg/µl fibrinogen or 5 mg/ml BSA . After 45 min at RT, 50 µl of luciferase substrate (coelenterazine, 10 µM final concentration) were added and BRET was determined by luminometry using a 410 nm/80 nm bandpass filter for Rluc and a 515 nm/30 nm bandpass filter for GFP. Results are expressed as the BRET ratio calculated as follows: (Emission at 515 nm BG515)/(Emission at 410 nm BG410), where BG515 is the emission at 515 nm and BG410 is the emission at 410 nm of a 5-µM solution of coelenterazine prepared in BRET buffer (Xu et al., 1999; Buensuceso et al., 2003).
BiFC assays
48 h after transfection of BiFC chimeras, CHO cells were resuspended in Tyrode's buffer supplemented with 2 mM MgCl2 and CaCl2 and plated for 2 h at 30°C on coverslips precoated with 100 µg/ml fibrinogen to allow cell spreading and maturation of the YFP fluorophore (Hu et al., 2002). In parallel, cells were plated on 5 mg/ml BSA and maintained in suspension. Cells were fixed in 3% formaldehyde, permeabilized with 0.1% Triton X-100, and stained with primary and secondary antibodies. Fluorescence images were acquired with a laser scanning confocal microscope (model MRC 1024; Bio-Rad Laboratories) and processed with imaging software (ISee Imaging Systems). Colocalization of BiFC fluorescence with other fluorescence images was quantified at the single pixel level, and data were accumulated for at least 20 cells over three experiments. In accordance with current definitions, a focal complex was defined here as a round, integrin-based structure smaller than 1 µm2, located 03 µm from the leading edge of a migrating cell, whereas a focal adhesion was defined as an elongated, integrin-based structure of 23 µm2 linked to actin stress fibers that could be located throughout the basal surface of the cell (Burridge et al., 1988; Jockusch et al., 1995). To monitor BiFC in real time (Kiosses et al., 1999), cells were plated on fibrinogen for 90 min at 30°C. Images were captured using an inverted fluorescence microscope (model TE300; Nikon) equipped with a CCD camera (Model Micromax 1024B ; Roper Scientific) and analyzed with ISEE imaging software.
Online supplemental material
Video 1 shows interactions between IIbß3 and c-Src. This Quicktime movie shows a BiFC signal in real time between
IIbYCß3 and c-SrcYN in a CHO cell starting 90 min after plating on fibrinogen. Fluorescence images were acquired at 1-min intervals (1 frame/min) for 34 min as described in Materials and methods. The movie is repeated a second time zooming in on a region of growing lamellipodium and shows the BIFC signal at the cell periphery moving inward to focal complexes and adhesions.
Video 2 shows interactions between c-Src and Syk. This Quicktime movie shows a BiFC signal in real time between c-SrcYN and SykYC in a CHO cell starting 90 min after plating on fibrinogen. Fluorescence images were acquired at 1-min intervals (1 frame/min) for 54 min. The movie is repeated three times over a 9-min period zooming in on a region of growing lamellipodium that shows the BiFC signal at the cell periphery moving inward and reappearing always first at the cell edge. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200402064/DC1.
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Acknowledgments |
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This work was supported by grants from the National Institutes of Health.
Submitted: 11 February 2004
Accepted: 22 March 2004
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