v-Src SH3-enhanced interaction with focal adhesion kinase at 1 integrin-containing invadopodia promotes cell invasion

with v-Src is by combined v-Src SH2 and gain-of-function v-Src SH3 domain binding to FAK. Here, assess the significance of the Arg-95 to Trp gain-of-function mutation in the v-Src SH3 domain through comparisons of Src-/- fibroblasts transformed with either Prague C v-Src or a point-mutant (v-Src-RT) containing a normal (Arg-95) SH3 domain. Both v-Src isoforms exhibited equivalent kinase activity, enhanced Src-/- cell motility, and stimulated cell in both low and soft agar. reconstituted Matrigel basement v-Src co-localized at exhibited a partial peri-nuclear and focal contact distribution in Src-/- cells. Adenoviral-mediated FAK the recruitment of v-Src-RT to invadopodia, facilitated the formation of a v-Src-RT-FAK signaling complex, and reversed the v-Src-RT invasion deficit. Adenoviral-mediated dominant-negative inhibition of FAK blocked v-Src-stimulated cell invasion. These studies establish that gain-of-function v-Src SH3 targeting interactions with FAK at b 1 integrin-containing invadopodia act to stabilize a v-Src-FAK signaling complex promoting cell Within FAK kinase activity, the SH2-mediated binding of Grb2 to phosphorylated FAK Tyr-925, and the enhanced tyrosine phosphorylation of FAK-associated proteins such p130Cas and paxillin p130Cas-null cells to transformation by Src the role of the FAK in a v-Src signaling complex Here, we show that a v-Src SH3 domain-stabilized signaling complex with is localized to b 1 integrin-containing invadopodia and functions to cell

INTRODUCTION c-Src is a modular protein-tyrosine kinase (PTK) consisting of a unique N-terminal segment, a Src homology (SH) 3 domain, a SH2 domain, and a kinase domain. Highly transforming strains of Rous sarcoma virus were found to contain v-Src isoforms with Cterminal truncations (1) and activating mutations within the kinase or SH3 domains of v-Src (2,3). Importantly, when v-Src SH3 domain substitutions (Trp at Arg-95 and Ile at Thr-96) were introduced into normal c-Src, these changes converted c-Src into a transforming protein (4).
Crystal structure analyses revealed that the v-Src SH3 domain mutations were within the SH3 domain RT loop and were near the surface ligand binding groove (5). Substitution of murine c-Src RT loop residues (Trp at Arg-97 and Ile at Thr-98) singly or in combination did not disrupt the binding of normal c-Src SH3 targets such as p130Cas (6). Instead, RT loop residue changes at Trp-97 promoted the binding of additional target proteins such as connexin 43 and FAK to the c-Src SH3 domain (6,7). In FAK, the v-Src SH3 domain binding sites were mapped to three proline-rich motifs conforming to a PXXPXXφ consensus where φ is a hydrophobic or proline residue (6). This extended PXXPXXφ motif differs from c-Src class I or class II SH3 binding motifs (5) and is conserved in other v-Src SH3 domain binding proteins (7,8). Since the v-Src SH3 domain binds to additional targets compared to the c-Src SH3 domain, the RT loop substitutions can be considered gain-of-function mutations.
FAK is a non-receptor PTK that associates with transmembrane integrins to promote matrix-initiated signaling. In normal cells, a transient c-Src-FAK PTK signaling complex is formed by c-Src SH2 binding to FAK in an integrin-stimulated manner (9,10). This PTK complex is linked to increased cell motility, cell cycle progression, and cell survival signals (reviewed in [11][12][13][14]. However, FAK was first identified as a v-Src substrate (15) and combined v-Src SH2 and SH3 binding to FAK stabilize an integrin-independent signaling complex (6,9,16). Within this complex, v-Src promotes elevated FAK kinase activity, the SH2-mediated binding of Grb2 to phosphorylated FAK Tyr-925, and the enhanced tyrosine phosphorylation of 4 EXPERIMENTAL PROCEDURES Cells, DNA constructs, and retroviruses-Large T immortalized Src-/-fibroblasts were maintained in DMEM containing 10% fetal calf serum as described (10). Quikchange mutagenesis (Stratagene, La Jolla, CA) was used to change the codon for W-95 to R-95 within v-Src Prague C using the sense primer 5'-TACGACTACGAGTCGCGAACTGAAACGGACTTGTC-3'. A NruI site (underlined) was used for v-Src-RT screening, the full-length construct was verified by sequencing, and subcloned into pRetroOff (Clontech, Palo Alto, CA). The murine c-Src cDNA was used as described (6).
293 Phoenix-Eco packaging cells (from G. Nolan, Stanford) were transfected and the retroviruscontaining media was collected after 72 h as described (18). Src-/-cells were infected for 24 h in media containing 5 µg/ml polybrene and selected for growth in 3 µg/ml puromycin. Pooled populations of cells were used in all assays.
Cell growth and soft agar assays-1x10 4 serum starved cells were plated onto fibronectin (FN) coated (5 µg/ml) 60 mm dishes in media containing 0.5% or 10% serum. Every 24 h, cells were collected after trypsin treatment and counted. For measurement of anchorage-independent growth, 4x10 4 cells suspended in 0.3% agar were seeded onto a solidified base of growth medium containing 0.6% agar and overlaid with 1 ml of growth medium. Colonies were scored after 3 weeks. Values were determined in triplicate. Ordinary one-way ANOVA was used to determine significance within data groups. The Turkey-Kramer multiple comparisons t test was used to determine significance between groups. serum for invasion assays, respectively. After 4 h (haptotaxis) or 24 h (invasion) at 37°C, cells on the lower membrane surface were enumerated as described (19). Mean values were obtained from three chambers for each experimental point per assay.

Migration and invasion assays-
In vitro kinase (IVK) assays-Assays were performed on immuno-isolated proteins at 32°C for 15 min as described (19). Briefly, (γ-32 P)ATP was added in the presence (Src IVK) or absence (FAK IVK) of a Src substrate (GST-FAK-CT), labeled proteins were visualized by autoradiography, and the equal recovery of the immuno-isolated kinase was verified by blotting as described (18). Adenovirus production and infection-β-galactosidase (LacZ) and FRNK-expressing adenoviruses were used as described (19). HA-tagged murine FAK was subcloned into pShuttle-CMV to prepare recombinant E1-deleted adenovirus (Stratagene). All viruses were propagated in 293 cells, clonally isolated, and titered using agar plaque assays as described (19). Src-/-cells were infected at a matched multiplicity of infection (MOI) between 30 and 100 plaque forming units/cell and analyzed for protein expression or invasion activity after 2 days.

Comparisons of v-Src Isoforms with Either Trp-95 or Arg-95 Residues in the SH3
Domain-To test the biological significance of gain-of-function v-Src SH3 binding interactions, comparisons were made between Src-/-fibroblasts stably reconstituted with murine c-Src, Prague C v-Src with a Trp-95-containing SH3 domain, and Prague C v-Src-RT with a normal c-Src-like Arg-95-containing SH3 domain (Fig. 1A). Pooled Src-/-cell populations equally expressed either c-Src, v-Src, or v-Src RT (Fig. 1A). Analyses of Src-associated in vitro kinase (IVK) activity from serum-starved cells revealed that both v-Src and v-Src RT possessed higher activity compared to normal c-Src (Fig. 1B) and repeated assays did not reveal significant differences between v-Src and v-Src-RT autophosphorylation or IVK activity toward GST-FAK-CT ( Fig. 1B) or acid-denatured enolase as substrates (data not shown). showing that v-Src phosphorylation of integrins inhibited in vitro cell migration compared to 8 normal cells (22). Since a fully-transformed cell phenotype encompasses changes in the growth, motility, and invasive property of cells, the reconstituted Src-/-cells were analyzed for the acquisition of an invasive phenotype. Whereas c-Src readily promoted Src-/-motility, c-Srcreconstituted cells did not penetrate through a Matrigel basement membrane barrier (Fig. 2B).

v-Src and v-Src-RT Transform Src-/-cells-
Notably, Src-/-cells transformed by v-Src but not v-Src-RT possessed invasive activity in 24 h assays (Fig. 2B). Although v-Src-RT are able to penetrate the Matrigel barrier at low numbers after 48 h (data not shown), our results support the conclusion that gain-of-function v-Src SH3 domain binding interactions promote enhanced cell invasion activity that is independent of changes in cell growth. Localization of v-Src, FAK, and 1 Integrin to Invadopodia-To evaluate whether differences in v-Src-stimulated cell invasion and FAK phosphorylation were associated with either altered cell morphology or v-Src-RT localization, Src-/-cells expressing v-Src or v-Src-RT were triple-stained for v-Src, FAK, and β1 integrin (Fig. 3). When plated onto FN-coated slides, v-Src-expressing cells formed fine cell extensions that were enriched with v-Src (Fig. 3A, arrowheads). Staining for FAK revealed a co-localization with v-Src at cell extensions (Fig. 3B) and with v-Src in ventral focal contact sites (data not shown). v-Src-RT expressing cells formed reduced numbers of long cell extensions and instead formed short pointed projections around the cell periphery (Fig. 3A). v-Src-RT and FAK co-localized at pointed cell projections (Fig. 3B), however, v-Src-RT also exhibited strong peri-nuclear staining that was not detected in v-Srcexpressing cells (Fig. 3A) β1 integrin staining of v-Src-RT cells showed a strong perimeter distribution with minimal staining in the central or ventral region of cells (Fig. 3C). v-Src-RT was co-localized with β1 integrin only in a subset of cell perimeter pointed projections (Fig. 3D). Whereas SH3 domain integrity is important for targeting both c-Src and v-Src to focal contact sites (25,26), our studies suggest that specific gain-of-function v-Src SH3 interactions with targets such as FAK may enhance the formation of invadopodia. To determine the potential mechanism of increased HA-FAK phosphorylation, v-Src-RT-associated in vitro kinase assays were performed (Fig. 4D). Compared to non-infected cells, HA-FAK overexpression facilitated the formation of an active signaling complex with v-Src-RT and also promoted the co-immunoprecipitation of the integrin-associated protein paxillin with v-Src-RT (Fig. 4D).

Modulation of FAK Expression or Activity Alters v-Src-stimulated Cell Invasion-
Since FAK overexpression promoted increased numbers of v-Src-RT but not control Src-/-cell invasion through Matrigel after 24 h as visualized by crystal violet staining of invasive cells on the lower Millicell membrane surface (Fig. 5A), indirect immunofluorescence staining of Millicell membranes was used to evaluate whether FAK functioned to recruit v-Src-RT to invadopodia during Matrigel invasion (Fig. 5B). In the absence of FAK overexpression, no v-Src-RT cell protrusions were present at 12 h and after 18 h, partial co-localization of v-Src and endogenous FAK were detected at the low numbers of emerging invadopodia (Fig. 5B). In To support the importance of FAK in mediating v-Src-stimulated cell invasion, the FAK C-terminal domain termed FRNK, that functions as a specific dominant-negative inhibitor of FAK activity (11)(12)(13), was expressed in v-Src-transformed Src-/-cells by recombinant adenovirus infection ( Fig. 4B and C). FRNK potently blocked v-Src-stimulated cell invasion ( Fig. 4B) and FRNK inhibited v-Src-stimulated endogenous FAK tyrosine phosphorylation (data not shown). Together, these results support the conclusion that v-Src co-localization with FAK at invadopodia and signals generated by the v-Src-FAK signaling complex function to promote cell invasion.
In summary, our results show that the introduction of a gain-of-function v-Src SH3 domain point-mutation results in the activation of an invasion-promoting signaling pathway in Src-/-cells. Notably, this cell invasion activity was independent of changes in either v-Srcstimulated haptotaxis motility or v-Src-stimulated cell growth in low serum and soft agar. FAK signaling complex, increased FAK tyrosine phosphorylation, and the co-localization of v-Src with FAK and β1 integrin at invadopodia cell projections. By using adenovirus to either overexpress FAK or a dominant-negative inhibitor of FAK function, our results support the hypothesis that FAK acts to promote invadopodia formation and to increase cell invasion through the recruitment of v-Src into an activated signaling complex also containing integrinassociated proteins such as paxillin. Since FAK functions to promote normal cytotrophoblastmediated invasion of the uterus during placental formation (20) and there is a strong correlation between the acquisition of an invasive phenotype and FAK overexpression in human tumor cells (19,27), future studies using v-Src-transformed cells may yield important insights into the molecular mechanisms and signals through which FAK promotes cell invasion.