1 Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
2 Departments of Medicine and Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
3 Veterans Affairs Hospital, Nashville, TN 37232, USA
¶ Author for correspondence (e-mail: graham.carpenter{at}vanderbilt.edu)
Accepted 11 November 2004
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Summary |
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Key words: PLC-1, Fibronectin, Adhesion, Migration
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Introduction |
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All PLC isozymes contain X and Y domains that fold together to form the catalytic site. The unique feature of the PLC- subfamily, which includes two isozymes, is the presence of a large linker between the X and Y domains that contains two SH2 domains, one SH3 domain and two sites of tyrosine phosphorylation (Carpenter and Ji, 1999
). The SH2 domains mediate the association of PLC-
with tyrosine-phosphorylated proteins, especially activated growth-factor-receptor tyrosine kinases. Nearly all growth factors induce the tyrosine phosphorylation and activation of PLC-
1 and thereby stimulate phosphatidylinositol-4,5-bisphosphate turnover. SH2-domain-mediated binding of PLC-
1 to activated receptors leads to the tyrosine phosphorylation of PLC-
1 at three tyrosine residues: Tyr771, Tyr783 and Tyr1254. Biochemical studies have revealed that tyrosine phosphorylation activates PLC-
1 enzyme activity (Nishibe et al., 1990
) and site-directed mutagenesis (Kim et al., 1991
) implicates Tyr783 as essential for PLC-
1 activation by growth factors.
Integrins are cell-surface receptors that mediate interactions between cells and the extracellular matrix (ECM) and have a crucial role in cellular functions such as cell adhesion, migration, proliferation and apoptosis. Integrins are heterodimeric molecules comprising an and a ß subunit, which combine in a restricted manner to form various dimers, each of which exhibits different ligand-binding properties (Hynes, 2002
; Plow et al., 2000
). The extracellular domains of integrin subunits are large and constitute the ligand-binding domain(s) of these receptors, whereas the short cytoplasmic tails play a crucial role in the promotion of cell anchorage. The cytoplasmic domains interact with cytoskeletal proteins to facilitate the connection of integrins to the cytoskeleton (van der Flier and Sonnenberg, 2001
). In addition, the cytoplasmic domains also recruit cytoplasmic tyrosine kinases such as focal-adhesion kinase (FAK), integrin-linked kinase and Src-family kinases, to propagate signals from the engaged integrins to the inside of the cell (van der Flier and Sonnenberg, 2001
).
Integrin-dependent cell adhesion to ECM results in integrin clustering and the formation of adhesion complexes, which provide an intersection where mechanical forces, cytoskeletal organization, biochemical signals and adhesion meet (Juliano, 2002). Several groups have previously demonstrated that PLC-
1 is present in adhesion complexes (Plopper et al., 1995
; Miyamoto et al., 1995
; Cybulsky et al., 1996
). Integrin ligation by ligand (Clark and Brugge, 1995
) or antibody (Kanner et al., 1993
) activates phospholipase C leading to IP3-dependent Ca2+ mobilization, suggesting that PLC isozymes have a functional role in integrin-dependent function. Recently, it has been demonstrated that PLC-
1 associates with
1ß1-integrin cytoplasmic domains in an adhesion- and time-dependent manner, and tyrosine phosphorylation of PLC-
1 is not required for this association to occur (Vossmeyer et al., 2002
). Furthermore, Vossmeyer et al. demonstrate that inhibition of PLC activation by a chemical inhibitor leads to reduced integrin
1ß1-integrin-dependent cell adhesion to collagen. Antibody cross-linking of ß2-integrins has been shown to elicit tyrosine phosphorylation of PLC-
1 (Kanner et al., 1993
).
In this report, PLC-1-deficient cells are shown to have an adhesion, spreading and migration defect. In addition, data are presented to show that integrin engagement by fibronectin induces tyrosine phosphorylation of PLC-
1 at Tyr783 and that this signaling event requires Src activity. Mutagenesis of Tyr783 abrogates the capacity of PLC-
1 to facilitate adhesion.
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Materials and Methods |
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A monoclonal antibody against phosphotyrosine (PY20) and a monoclonal antibody to the N-terminus of PLC-1 were purchased from Transduction Laboratories. Monoclonal antibodies to Src kinase and polyclonal antibodies to FAK were obtained from Oncogene, polyclonal antibodies to phosphorylated PLC-
1 (Tyr783) from Santa Cruz Biotechnology. Antibodies againts phosphorylated Y771, Y783 and Y1253 in the PLC-
1 sequence were a gift from S. G. Rhee (National Institutes of Health, Bethesda, MD) (Sekiya et al., 2004
). Rabbit antiserum against PLC-
1 has been described previously (Arteaga et al., 1991
). Antibodies against integrins
1,
2,
5,
6,
V, ß1 and ß4 were from BD Biosciences. Rat antibody against integrin ß1 (used for immunofluorescence) and the antibody against
3 integrin were purchased from Chemicon. Horseradish peroxidase (HRP)-conjugated protein A was from Zymed Laboratories and secondary goat anti-mouse IgG antibody was from Transduction Laboratories. Vaccinia virus containing wild-type and site-specific mutants (Y771F, Y783F, Y1253F) of PLC-
1 (Kim et al., 1991
) was obtained from S. G. Rhee. Null cells expressing PLC-
1 N- and C-terminal SH2 domain loss-of-function mutations have been described elsewhere (Ji et al., 1999
).
Immunoprecipitation and immunoblotting
For ligand coating, cell-culture dishes were incubated overnight with 10 µg ml-1 fibronectin, unless otherwise indicated, in PBS at 4°C. Nonspecific binding sites were blocked with heat-denatured 1% bovine serum albumin (BSA) in PBS for 1 hour at 37°C. Subconfluent cells were incubated for 12 hours in DMEM containing 0.5% FCS. Subsequently, the cells were trypsinized, washed with DMEM containing 0.5 mg ml-1 trypsin inhibitor and then washed with DMEM. The cells were then kept in suspension for 30 minutes or allowed to adhere to fibronectin-coated dishes for the indicated periods of time. Subsequently the cells were treated with lysis buffer (1% Nonidet P-40, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 2 mM Na3VO4, 100 µM phenylmethylsulfonyl fluoride and 10 µg ml-1 each aprotinin and leupeptin) for 2 minutes, scraped and the insoluble material was removed by centrifugation (15,000 g for 2 minutes) at 4°C.
For immunoprecipitation, cells lysates were mixed with antibodies (generally 1 µg) for 2hours followed by the addition of 40 µl 50% protein-A/Sepharose or protein-G/Sepharose beads (Zymed Laboratories) for an additional 2 hours at 4°C. The immunoprecipitates were then washed three times with lysis buffer and boiled in 40 µl 2xLaemmli's buffer. Subsequently, the samples were subjected to 7.5% SDS-PAGE and electrotransferred to polyvinylidene difluoride membranes (Millipore).
The membranes were probed with the indicated primary antibodies, washed in Tris buffer saline (TTBS) (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% Tween 20) followed by HRP-conjugated secondary antibodies or HRP-conjugated protein A. Membranes were washed and visualized by an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech). When necessary, membranes were stripped by incubation in stripping buffer (62.5 mM Tris-HCl, pH 5.7, 100 mM 2-mercaptoethanol, 2% SDS) for 1 hour at 70°C with constant agitation, washed and then reprobed with other antibodies as indicated.
Adhesion assays
24-well plates were coated with fibronectin overnight at 4°C. Nonspecific binding sites were blocked with heat-denatured 1% BSA in PBS for 1 hour at 37°C. The cells were trypsinized, washed with DMEM containing 0.5 mg ml-1 trypsin inhibitor and then washed with DMEM. Then, the cells were counted (using a Coulter Counter) and 105 cells were added to each well and allowed to attach for the indicated times at 37°C. The cells were subsequently washed with PBS and the attached cells were fixed with 3.7% formaldehyde in PBS for 1 hour and stained with crystal violet. Crystal violet was eluted with 10% acetic acid. Cell adhesion was calculated by measuring the absorbance of eluted dye at 595 nm with an enzyme-linked immunosorbent assay (ELISA) reader. The result was calculated as an optical density (OD) percentage ratio [(ODtest - ODblank)/(ODpositive control - ODblank)]x100, where ODtest was the measured OD of cells that adhered to ligand, ODblank was the OD of cells that were allowed to adhere to BSA-blocked wells and ODpositive control was the optical density of cells that were allowed to adhere in the presence of 10% FCS.
Migration assays
Polycarbonate transwells (6.5 mm diameter, 8 µm pore diameter) were coated on the underside with 10 µg ml-1 fibronectin overnight at 4°C. Nonspecific binding sites were blocked with heat-denatured 1% BSA in PBS for 1 hour at 37°C. Cells were then trypsinized, washed with DMEM containing 0.5 mg ml-1 trypsin inhibitor and washed with DMEM. The cells were counted and 5x104 cells were added to each transwell and allowed to attach and migrate for 3 hours at 37°C. Afterwards, the top of each chamber was cleaned with a cotton swab to remove all cells. The remaining cells were fixed and stained with crystal violet as described above and nine randomly chosen fields from triplicate wells were counted at 200xmagnification.
Fluorescence imaging
To visualize actin-filament distribution, trypsinized cells were plated on fibronectin-coated glass coverslips for different times. After the indicated time, adherent cells were briefly washed in PBS and fixed with 3.7% formaldehyde in PBS for 15 minutes room temperature. Actin distribution was visualized by staining the cells with tetramethyl rhodamine isothiocyanate (TRITC)-phalloidin for 30 minutes at room temperature followed by washing with PBS. For integrin-ß1 staining, cells were fixed, permeabilized in 0.1% Triton X-100 in PBS and blocked with 10% goat preimmune serum. After that, cells were incubated with primary anti-integrin-ß1 antibody, washed and incubated with goat anti-rat antibody conjugated to TRITC. Glass coverslips were analysed on a Zeiss Axiovert 135 confocal microscope.
Flow cytometry
Cells were disassociated from tissue-culture dishes and exposed to diluted monoclonal antibodies of the appropriate integrin, followed by appropriate secondary antibodies (fluorescein-isothiocyanate-coupled rabbit anti-rat or anti-hamster immunoglobulin). Flow cytometry was performed with a FACScan instrument (Becton Dickinson).
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Results |
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Based on these integrin expression profiles, fibronectin (a well characterized ligand for integrin 5ß1) was used to examine the differences in cell adhesion between the Null and Null+ cell lines. The two cell types were plated on increasing concentrations of fibronectin and cell adhesion was measured after 30 minutes. As shown in Fig. 2A, at low concentrations of fibronectin (1-5 µg ml-1), adhesion of the Null cells is significantly diminished relative to Null+ cells; however, at higher concentrations (5-20 µg ml-1), the adhesion of both cell lines to fibronectin is equivalent. Similar results were obtained when fibrinogen was used as the extracellular matrix (data not shown), although the cell adhesion for both cell lines was significantly less than on fibronectin. The data in Fig. 2B demonstrate that the observed differences in adhesion between Null and Null+ cells at low fibronectin concentrations is reflected in a reduced rate of adhesion. At high fibronectin concentrations, the rate of adhesion is equivalent between the two cell types.
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Because the Null cells adhered less than did the Null+ cells at low concentrations of fibronectin, we investigated whether there were differences in cell spreading and alterations in the cell morphology at low fibronectin concentrations. The cells were plated onto fibronection-coated coverslips for 60 minutes or 120 minutes and than stained with TRITC-phalloidin. At 60 minutes, the Null cells had a more rounded morphology compared with the elongated shape of the spread Null+ cells. By 120 minutes, the Null+ cells had developed lamellipodia at their leading edges. At this time point, the Null cells were spread and had a polygonal shape but there was no evidence of lamellipodia (Fig. 3A), suggesting that PLC-1 might influence cell polarity and/or cytoskeleton dynamics.
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Based on the decreased cell adhesion and spreading observed in the Null cells relative to Null+ cells, an assessment was made of differences in cell migration using transwells precoated with increasing concentrations of fibronectin. The results (Fig. 3B) demonstrate that Null cells migrate significantly less than Null+ cells at all fibronectin concentrations, including those concentrations at which differences in adhesion were not observed.
That Null cells are deficient in adhesion in a fibronectin-concentration-dependent manner might suggest that PLC-1 influences integrin clustering. To test this, Null and Null+ cells were allowed to adhere to low and high concentrations of fibronectin for various times and then stained with an integrin-ß1 antibody. As seen in Fig. 4A, although there are some differences in cell morphology, the pattern of integrin staining was similar in Null and Null+ cells plated on a high level (10 µg ml-1) of fibronectin. This pattern was also present at low levels (2.5 µg ml-1) of fibronectin, which produces clear differences in adhesion between the two cell lines. In addition, Null and Null+ cells were plated on fibronectin for 0 minutes, 30 minutes or 60 minutes and blotted with antibodies against phosphorylated FAK or phosphorylated extracellular-signal-regulated kinase (ERK) to determine whether there were differences in integrin-dependent FAK and ERK activation following cell adhesion. No difference was observed in the activation of either of these kinases (Fig. 4B,C).
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Cell adhesion to fibronectin induces PLC-1 tyrosine phosphorylation
PLC-1 activation caused by tyrosine phosphorylation is thought to mediate cellular signaling induced by growth factors (Carpenter and Ji, 1999
). To determine whether PLC-
1 tyrosine phosphorylation occurred following integrin clustering, Null+ cells were allowed to adhere to fibronectin for 15 minutes and 30 minutes, and then the cells were lysed and PLC-
1 was immunoprecipitated and blotted with site-specific antibodies against phosphorylated PLC-
1. As shown in Fig. 5A, fibronectin induces tyrosine phosphorylation of PLC-
1 in the Null+ cells, particularly at Tyr783 and to a lesser extent at Tyr1254. At Tyr771, there was a low level of phosphorylation that was not altered by the presence of fibronectin.
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The fibronectin-dependent tyrosine phosphorylation of PLC-1 predicts that loss of the SH2 domains of PLC-
1, which facilitate requisite delivery to tyrosine kinases, should abrogate tyrosine phosphorylation under these conditions. This was tested and it was found (Fig. 5B) that fibronectin fails to induce PLC-
1 tyrosine phosphorylation in a PLC-
1 mutant in which both SH2 domains are disabled by point mutation of a crucial lysine residue in each SH2 domain (Ji et al., 1999
).
The data in Fig. 5 suggest that phosphorylation of PLC-1 at Tyr783 and Tyr1253 might be significant for adhesion to fibronectin. This was tested by expressing tyrosine mutants (Y771F, Y783F, Y1253F) or wild-type PLC-
1 in Null cells and measuring adhesion. The results in Fig. 6A show that an equivalent level of each PLC-
1 construct was expressed and demonstrate that only Tyr783 is crucial for adhesion. Although the Y771F and Y1254F mutants were equivalent to wild-type PLC-
1 in this adhesion assay, the Y783F mutant showed the same level of deficiency as the complete absence of PLC-
1 in the Null cells. Similarly, the loss of PLC-
1 SH2 domains abrogates the capacity of PLC-
1 to mediate fibronectin-dependent adhesion (Fig. 6B).
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In an attempt to identify the tyrosine kinase that induces PLC-1 tyrosine phosphorylation under these conditions, the most likely candidates were screened. It is has been shown that the engagement of integrins can lead to activation of the epidermal growth factor (EGF) receptor (Moro et al., 2002
) and we have observed that A431 cell adhesion to fibronectin can induce EGF-receptor tyrosine phosphorylation (data not shown). To assess whether the kinase activity of the EGF receptor was required for PLC-
1 tyrosine phosphorylation, Null+ cells were pretreated with the EGF-receptor tyrosine kinase inhibitor AG1478 before adhesion to fibronectin. As shown in Fig. 7A, this inhibitor had no effect on fibronectin-induced PLC-
1 tyrosine phosphorylation but completely blocked EGF-induced tyrosine phosphorylation of PLC-
1.
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Because the activation of Src kinase in response to integrin engagement is also well established, we used the selective Src kinase inhibitor PP2 to test the role of Src kinases in PLC-1 phosphorylation. As shown in Fig. 7B, the presence of PP2 significantly reduces PLC-
1 tyrosine phosphorylation following fibronectin-induced integrin activation. These results indicate that one or more of the Src kinase family members mediate PLC-
1 phosphorylation.
To investigate further whether Src kinase family members are required for the integrin-dependent phosphorylation of PLC-1, a triple-knockout fibroblast cell line lacking Src, Yes and Fyn expression (SYF-/-) was tested. When these cells adhered to fibronectin, PLC-
1 phosphorylation is significantly reduced compared with Null+ cells (Fig. 8A), although PLC-
1 tyrosine phosphorylation in both these cell types is equivalent following stimulation by EGF (data not shown). We have also assessed whether FAK is required for PLC-
1 phosphorylation during fibronectin-dependent adhesion. The data in Fig. 8B show that the absence of FAK (FAK-/- cells) does not inhibit PLC-
1 phosphorylation.
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PLC-1 associates with Src kinase in the absence of FAK
To determine the mechanism whereby Src modulates PLC-1 phosphorylation, we assessed whether these two proteins associate with each other. For these experiments, Null+ cells were allowed to adhere to fibronectin, following which Src was immunoprecipitated and subsequently blotted with PLC-
1 antibodies. Because it is known that FAK is a substrate for Src, the Src immunoprecipitates were also tested for the presence of FAK. As shown in Fig. 9A, both PLC-
1 and FAK are detected in Src immunoprecipitates from cells that adhere to fibronectin for 30 minutes. It seems likely that the capacity of Src to associate with and phosphorylate PLC-
1 will depend on presence of functional SH2 domains in PLC-
1. The data in Fig. 9B show that, when a PLC-
1 mutant with loss-of-function mutation in each SH2 domain was tested, there was no association of PLC-
1 with Src following fibronectin-mediated adhesion. The SH2 domains of PLC-
1 are probably required to facilitate association with a Src phosphotyrosine residue as a prelude to tyrosine phosphorylation of PLC-
1.
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The association of PLC-1 with Src raised the question as to whether this interaction is mediated by FAK. It has been reported that the Src-FAK interaction is one of the first and most crucial steps in integrin signaling (Thomas et al., 1998
; Hanks et al., 2003
) and PLC-
1 associates with FAK after integrin engagement (Zhang et al., 1999
). The data in Fig. 9C show that, when Null+ cells were plated on fibronectin, increasing amounts of PLC-
1 immunoprecipitated with FAK over time. To test whether the PLC-
1 interaction with Src involves FAK, a FAK-/- mouse embryonic fibroblast cell line with a tetracycline-inducible FAK construct was used (Owen et al., 1999
). These cells, designated TFW-46 cells, were grown in the presence of tetracycline (Tet+) to keep them in the uninduced state. FAK expression was then induced by replacing the growth medium with fresh medium lacking tetracycline (Fig. 10A). As shown in Fig. 10B, PLC-
1 immunoprecipitated with Src kinase in the presence or absence of FAK expression, which supports the conclusion that the PLC-
1/Src interaction does not require FAK.
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To determine whether the PLC-1/FAK interaction depends on Src, SYF-/- and Null+ cells were plated on fibronectin-coated dishes for different periods of time. The cells were lysed and the lysates were precipitated with anti-FAK antibody and then blotted with PLC-
1 antibody. The data in Fig. 10C show that PLC-
1 precipitates with FAK in a fibronectin-inducible manner in both SYF-/- and Null+ cells. Taken together, these data indicate that PLC-
1 interactions with FAK and Src are not dependent on each other after integrin engagement.
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Discussion |
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Using Plcg1-null cells, this report extends the observations of many others who have shown that inhibitors of PLC activity decrease cell adhesion (Schottelndreier et al., 1999). However, the PLC-selective basis of these pharmacological inhibitors is unclear. Published data showing that Plcg2-/- platelets have a spreading defect following adhesion to collagen, fibrinogen or vonWillebrand factor (Asselin et al., 1997
; Goncalves et al., 2003
; Inoue et al., 2003
; Wonerow et al., 2003
; Rathore et al., 2004
) concurs with our data. In contrast to our data, Plcg2-/- platelets do not show deficits in cell adhesion. However, the concentrations of extracellular matrix ligand used in those studies was high and not varied. It is possible that lower concentrations of those extracellular matrix molecules might have revealed a PLC-
2 requirement for platelets in the adhesion process. Plcg2-/- platelet adhesion to a collagen surface was decreased relative to wild-type platelets, when a shear force was applied (Suzuki-Inoue et al., 2003
).
In contrast to the adhesion results, a defect in cell migration was present in Null cells at both low and high concentrations of fibronectin. These data indicate that the decrease in migration of the Null cells is not due to just the adhesion defects. PLC-1 is important in growth-factor-induced migration, in which it is thought to play a role in modulating events related to cytoskeleton organization at the leading edge of migrating cells (Piccolo et al., 2002
).
FAK is a crucial component in the transduction of signaling pathways following integrin ligation (Sieg et al., 1999). The fact that FAK phosphorylation was similar in Null and Null+ cells suggests that activation of this protein is not dependent on the presence of PLC-
1. In addition, although FAK associates with PLC-
1, FAK is not required for integrin-induced phosphorylation of PLC-
1. These results support the report that, although FAK autophosphorylation site Tyr397 mediates a direct interaction with PLC-
1, FAK does not directly phosphorylate PLC-
1 (Zhang et al., 1999
).
It has been demonstrated that integrins associate with EGF receptors in a macromolecular complex during the early phases of cell adhesion, which results in phosphorylation of the EGF receptor (Moro et al., 2002). Data showing that an EGF-receptor kinase inhibitor does not inhibit cell-adhesion-dependent PLC-
1 tyrosine phosphorylation suggests that integrin-dependent PLC-
1 activation does not require EGF-receptor tyrosine kinase activity. By contrast, addition of the Src-kinase inhibitor PP2 resulted in decreased PLC-
1 tyrosine phosphorylation, implying that this kinase is required for fibronectin-dependent PLC-
1 phosphorylation. Results using the SYF-/- cells, which are deficient in Src-family kinases, confirm the need for Src for PLC-
1 phosphorylation. Src has been demonstrated to activate PLC-
1, resulting in Ca2+ release in fertilization-induced egg activation (Sato et al., 2003
). This result contrasts with the growth-factor literature, in which no differences are reported in PLC-
1 activation following platelet-derived-growth-factor stimulation of SYF-/- cells (Klinghoffer et al., 1999
).
The interaction of Src-family members with FAK is well established (Hanks et al., 2003; Zhang et al., 1999
). In addition, it has been demonstrated that Src can phosphorylate PLC-
1 in vitro in the absence of FAK (Zhang et al., 1999
) and at the Y783 site (Sekiya et al., 2004
). The data presented in Fig. 9 demonstrate that Src associates with PLC-
1 following integrin-dependent cell adhesion. FAK-null cells were used to show that the capacity of Src to phosphorylate PLC-
1 is independent of Src association with FAK. In some models, such as CAS phosphorylation, it has been proposed that FAK is required to mediate a productive ternary complex of FAK-CAS-Src (Hanks et al., 2003
). Based on our data, this model would not be applicable to PLC-
1 phosphorylation by Src.
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Acknowledgments |
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Footnotes |
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Present address: MediCity Research Laboratory Tykistokatu 6A, Turku, Finland
Present address: Department of Technology Development, Biology, Discover Research, GlaxoSmithKline, 5 Moore Drive, Research Triangle Park, NC 27709, USA
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