Figure 1.
FAK activity and C-terminal PRR regions are required for cell motility.
(A) Schematic of FAK comprised of an N-terminal FERM domain, a central catalytic domain, three proline-rich regions (PRR) that are sites of SH3 domain binding, and a C-terminal FA-targeting domain (F.A.T) connecting FAK to integrins. Point-mutations are indicated that disrupt catalytic activity (lysine to arginine, R454), and SH3 domain binding to PRR2 (proline to alanine, A712/713) or PRR3 (proline to alanine, A876/877). (B) Lysates from the indicated GFP-FAK reconstituted FAK-/− MEFs showing GFP-FAK expression, FAK Y397 phosphorylation (pY397), and actin by immunoblotting. (C) Chemotaxis cell motility on FN-coated Millicell chambers over 4 h. Cell number is expressed as a percent of GFP-FAK WT (normalized to 100, ± SEM, ***p<0.001) from 3 independent experiments. (D) FA lifetime determination by counting sequential frames of mCherry paxillin fluorescence above background using time-lapse confocal microscopy. Images were acquired at 2 min intervals over 60 min in FAK−/− and FAK+/+ MEFs stimulated with growth media containing 50 ng/ml EGF. Data is the mean lifetime ± SEM of 125–150 FAs from at least 5 different cells (***p<0.001). (E) Co-localization of GFP-FAK and mCherry-paxillin at FAs in MEFs plating on FN-coated (10 µg/ml) coverslips for 60 min. Scale is 10 µm.
Figure 2.
FAK activity and C-terminal PRR regions are required for FA turnover.
(A) Scratch wound assay performed with FAK−/− fibroblasts expressing GFP or the indicated GFP-FAK constructs. Closure percentage was calculated by the change in area between 2 and 10 h. Experimental values are presented as ± SEM from 3 independent experiments (***p<0.001). (B) Representative image montage (10 to 30 min) from live-cell spinning disc confocal microscopy of FA-localized GFP-FAK after growth media supplemented with 50 ng/ml EGF stimulation. A merged image from the 10/20/30 min time points was pseudo-colored red (10 min), green (20 min) and blue (30 min) respectively to illustrate GFP-FAK localization over time. White regions indicate GFP-FAK localization overlap at 10 to 30 min. Scale is 10 µm. (C) Adhesion lifetime was determined by counting the number of sequential frames (2 min intervals within a 60 min time-lapse) of GFP-FAK FA-associated fluorescence above background. Data is the mean lifetime ± SEM of 150–200 FAs from at least 5 different cells from each of the indicated GFP-FAK reconstituted FAK−/− MEFs (**p<0.01, compared to FAK-WT).
Figure 3.
FAK PRR2 and PRR3 regions are direct binding sites for cortactin.
(A) Cortactin SH3 domain binds FAK as determined by GST or GST-cortactin SH3 domain pulldown assays using in vitro translated FAK protein. FAK immunoblotting shows binding and 5% of input. (B) Schematic of FAK containing an N-terminal FERM domain, three PRR (PRR1-PRR3) sites, a central kinase domain, and a C-terminal FA targeting region. Point-mutations are indicated that disrupt PRR2 (A712/713) and PRR3 (A876/877). The indicated FAK regions (below) were used as bait or prey in direct binding assays as either GFP, C-terminal TAP, or GST fusion proteins. (C) FAK C-terminal domain binds cortactin. GFP fusions of FAK 1-402, FAK 396–686, and FAK 411–686 with a C-terminal TAP tag or non-tagged FAK 686–1052 were in vitro translated in the presence of biotin-lysine and used in a direct binding assay with GST or GST-cortactin attached to beads. Streptavidin-HRP analyses show the amount of FAK bound or 5% of prey material used in the binding assay. (D) FAK PRR3 region binds cortactin. In vitro translated full-length cortactin was incubated with GST-FAK (853–946) or GST-FAK (947–1052) in a direct binding assay. Streptavidin-HRP analyses show the amount of cortactin bound or 5% of prey material used in the binding assay. (E) FAK PRR2 and PRR3 are individually required for cortactin binding. GFP-fusions of FAK WT, A713/713, or A876/877 were in vitro translated and incubated with GST-cortactin in a direct binding assay. Streptavidin-HRP analyses show the amount of GFP-FAK bound or 5% of prey material used in the binding assay.
Figure 4.
Cortactin knockdown results in decreased FA turnover.
(A) GFP-FAK WT MEFs were transiently transfected with scrambled (Scr), cortactin, or p130Cas siRNA and protein levels determined after 48 h by immunoblotting. (B) Densitometry analyses of p130Cas and cortactin levels as a percent of Scr control. Values are means ±SD from two experiments. (C) Adhesion lifetime was determined by counting the number of sequential frames (2 min intervals from a 60 min time-lapse) for FA-associated GFP-FAK fluorescence above background. Data represent mean lifetime±SEM of 125–150 FAs from at least 5 different cells for Scr-, cortactin, or p130Cas siRNA-transfected GFP-FAK MEFs (***p<0.001). (D) Representative image montage (2 to 40 min) from live-cell spinning disc confocal microscopy of FA-localized GFP-FAK after growth media supplemented with 50 ng/ml EGF stimulation. As indicated, cells were transfected with control, anti-p130Cas, or anti-cortactin siRNA along with fluorescent marker (siGLO, not shown). A merged image from the 2/20/40 min time points was pseudo-colored red (2 min), green (20 min) and blue (40 min) respectively to illustrate GFP-FAK localization over time. White regions indicate GFP-FAK localization overlap at 2 and 40 min. Scale is 10 µm.
Figure 5.
Regulation of FAK and cortactin binding by FN adhesion.
(A) Lysates were made from MEFs held in suspension for 30 min or FN re-plated (30 and 60 min) and were analyzed by cortactin or FAK antibody immunoprecipitation (IP) followed by immunoblotting for cortactin and FAK. (B) Mutations disrupting FAK activity or within PRR2 either stabilize or prevent cortactin association with FAK by co-IP analyses, respectively. Lysates were made from the indicated GFP-FAK reconstituted MEFs held in suspension for 30 min or FN plated (30 and 60 min) and were analyzed by anti-GFP IPs followed by anti-cortactin, anti-FAK Y397 phosphorylation (pY397), and anti-GFP immunoblotting to determine the level of GFP-FAK.
Figure 6.
FAK PRR2 or FAK PRR3 regions are needed to promote transient cortactin co-localization with FAK at FAs.
FAK−/− or the indicated GFP-FAK reconstituted MEFs were serum starved, held in suspension, FN replated for 60 min, and then analyzed for endogenous paxillin (green) or cortactin (red) immuno-staining. GFP-FAK was visualized by intrinsic fluorescence. Shown is GFP-FAK or paxillin and cortactin distribution within cells and merged images were used to evaluate co-localization (yellow). Scale is 10 µM. Inset, 4X enlargement of the corresponding box.
Figure 7.
FAK tyrosine phosphorylation of cortactin during FN replating of MEFs.
(A) Lysates were made from the indicated GFP-FAK reconstituted MEFs held in suspension for 30 min or FN plated (30 and 60 min) and were analyzed by anti-cortactin IPs followed by anti-phosphotyrosine (pY) and anti-cortactin immunoblotting. (B) In vitro kinase assays using recombinant GST-Cortactin incubated in the presence (lanes 1, 2 and 4) or absence (lane 3) of recombinant FAK kinase, or presence (lanes 1,3 and 4) and absence (lane 2) of ATP. Proteins were analyzed by anti-phosphotyrosine (pY) immunoblotting and by Coomassie staining.
Figure 8.
Cortactin tyrosine to phenylalanine (3YF) mutation prevents FAK-mediated cell migration and FA turnover.
(A) Schematic of cortactin with an N-terminal acidic region (NTA), six cortactin tandem repeats, a helical region, a proline-rich region containing Y421, Y466, and Y482 phosphorylation sites (human numbering), and a carboxyl-terminal SH3 domain. (B) Reconstituted FAK−/− MEFs expressing GFP-FAK were transfected with RFP-cortactin WT, or RFP-cortactin with Y421, Y466, and Y482 mutated to phenylalanine (3YF) or mutated to glutamic acid (3YE). Representative images are shown as a kymograph of merged images extracted from live-cell spinning disc confocal microscopy after growth media supplemented with 50 ng/ml EGF stimulation (30 of 60 min time-lapse, 2 minute intervals). Images were pseudo-colored in red (RFP-cortactin) and in green (GFP-FAK) to illustrate GFP-FAK adhesion lifetime. White arrows indicate appearance of GFP-FAK in a given FA and when this particular FA disappears or remains within the cell region of interest. Scale is 1 µm.
Figure 9.
Cortactin tyrosine to phenylalanine (3YF) mutation inhibits FAK-mediated cell migration and FA turnover.
FAK−/− MEFs reconstituted with GFP-FAK WT, GFP-FAK R454, or GFP-FAK A712/713 were co-transfected with RFP-cortactin WT, or RFP-cortactin Y421, Y466, and Y482 mutated to phenylalanine (3YF) or mutated to glutamic acid (3YE). (A) In MEFs over-expressing the indicated RFP-cortactin constructs, FA lifetime was determined by counting the number of sequential frames with GFP-FAK FA-associated fluorescence above background. Data from live-cell spinning disc confocal microscopy after growth media supplemented with 50 ng/ml EGF stimulation (2 min intervals from a 60 min time-lapse) represents the mean lifetime of at least 50 leading edge-associated FAs ± SEM from at least 12 different cells within the indicated GFP-FAK reconstituted FAK−/− MEFs (***p<0.001). (B) Cell migration speed in µm/sec was determined by cell tracking (n = 12 per group, values are means ±SD, ***p<0.001).
Figure 10.
Model of FA turnover through FAK and cortactin association.
1) FAK and cortactin associate under anchorage-independent conditions. 2) Upon transmembrane integrin receptor clustering by cell replating on FN, cytoplasmic FAK is recruited and activated at newly forming focal adhesions (FAs). Cortactin SH3 domain binding to FAK C-terminal PRR2 and PRR3 facilitates transient cortactin localization to FAs resulting in alterations in FA-associated f-actin. 3) Direct or FAK-enhanced cortactin tyrosine phosphorylation results in FAK-cortactin complex dissociation associated with FA turnover and 4) the formation of other signaling complexes (such as Arp2/3 or the Nck adaptor protein) with tyrosine-phosphorylated cortactin.