Src Phosphorylates Tyr284 in TGF-β Type II Receptor and Regulates TGF-β Stimulation of p38 MAPK during Breast Cancer Cell Proliferation and Invasion (original) (raw)

Cell, Tumor, and Stem Cell Biology| April 17 2007

Department of Pharmacology, University of Colorado Health Sciences Center, Aurora, Colorado

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Department of Pharmacology, University of Colorado Health Sciences Center, Aurora, Colorado

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Requests for reprints: William P. Schiemann, Department of Pharmacology, University of Colorado Health Sciences Center, Room L18-6110, RC1 South Tower, 12801 East 17th Avenue, P.O. Box 6511, Aurora, CO 80045. Phone: 303-724-1541; Fax: 303-724-3663; E-mail: Bill.Schiemann@uchsc.edu.

Received: October 17 2006

Revision Received: December 22 2006

Accepted: February 02 2007

Online ISSN: 1538-7445

Print ISSN: 0008-5472

2007

Cancer Res (2007) 67 (8): 3752–3758.

Article history

Received:

October 17 2006

Revision Received:

December 22 2006

Accepted:

February 02 2007

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Abstract

Genetic and epigenetic events often negate the cytostatic function of transforming growth factor-β (TGF-β) in mammary epithelial cells (MEC), which ultimately enables malignant MECs to proliferate, invade, and metastasize when stimulated by TGF-β. The molecular mechanisms underlying this phenotypic conversion of TGF-β function during mammary tumorigenesis remain poorly defined. We previously established αvβ3 integrin and Src as essential mediators of mitogen-activated protein kinase (MAPK) activation, invasion, and epithelial-to-mesenchymal transition stimulated by TGF-β in normal and malignant MECs. Mechanistically, β3 integrin interacted physically with the TGF-β type II receptor (TβR-II), leading to its tyrosine phosphorylation by Src and the initiation of oncogenic signaling by TGF-β. We now show herein that Src phosphorylated TβR-II on Y284 both in vitro and in vivo. Interestingly, although the expression of Y284F-TβR-II mutants in breast cancer cells had no effect on TGF-β stimulation of Smad2/3, this TβR-II mutant completely abrogated p38 MAPK activation by TGF-β. Accordingly, Src-mediated phosphorylation of Y284 coordinated the docking of the SH2 domains of growth factor receptor binding protein 2 (Grb2) and Src homology domain 2 containing (Shc) TβR-II, thereby associating these adapter proteins to MAPK activation by TGF-β. Importantly, Y284F-TβR-II mutants also abrogated breast cancer cell invasion induced by αvβ3 integrin and TGF-β as well as partially restored their cytostatic response to TGF-β. Our findings have identified a novel αvβ3 integrin/Src/Y284/TβR-II signaling axis that promotes oncogenic signaling by TGF-β in malignant MECs and suggest that antagonizing this signaling axis may one day prove beneficial in treating patients with metastatic breast cancers. [Cancer Res 2007;67(8):3752–8]

Introduction

Transforming growth factor-β (TGF-β) is a powerful suppressor of breast cancer formation and progression. Quite dichotomously, mammary tumorigenesis frequently subverts the tumor-suppressing function of TGF-β, leading to its conversion from an inhibitor to a stimulator of breast cancer growth, invasion, and metastasis (1–4). The duality of TGF-β during breast cancer progression is evidenced by the fact that (a) neutralizing TGF-β antibodies inhibit breast cancer tumorigenicity in mice (5); (b) transgenic expression of dominant-negative TGF-β type II receptor (TβR-II) enhances mammary tumorigenesis in mice (6); and (c) TGF-β expression is elevated at the invading face of primary human breast carcinomas as well as in lymph node metastases (3). Moreover, transgenic expression (7) of a soluble Fc:TβR-II fusion protein, which antagonizes TGF-β signaling by binding and sequestering TGF-β, inhibited the survival, motility, and metastasis of mammary tumors in mice. Recently, TGF-β signaling, including that by Smad2 and Smad3 (8, 9), was shown to inhibit the tumorigenicity of normal, premalignant, and malignant breast cancer cells, while stimulating that of highly invasive and metastatic breast cancer cells (10). In addition, a large body of evidence indicates that TGF-β suppresses mammary tumorigenesis primarily through signals activated by Smad2/3, which promote cell cycle arrest (11). Conversely, the ability of TGF-β to promote mammary tumorigenesis is thought to occur through the activation and integration of signals arising not only from Smad2/3 but also those activated in response to β1 integrin, RhoA, mitogen-activated protein kinases [MAPK; e.g., extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 MAPK], and phosphatidylinositol 3-kinase (8, 12–16). Thus, breast cancer cells clearly have evolved a number of strategies effective in promoting oncogenic signaling by TGF-β.

Despite these recent advances, the precise molecular mechanisms whereby TGF-β mediates oncogenic signaling in malignant mammary epithelial cells (MECs) remain to be elucidated fully. We recently reported that the initiation of oncogenic signaling by TGF-β correlated with the ability of Src to phosphorylate the TβR-II (17). We further showed that in regulating MEC response to TGF-β, β3 integrin interacted physically with TβR-II, leading to its phosphorylation by Src (17). More importantly, we showed that measures capable of inhibiting Src expression or function prevented oncogenic signaling by TGF-β in normal and malignant MECs, particularly its ability to activate MAPKs and to induce MEC invasion and epithelial-to-mesenchymal transition (EMT; ref. 17). Based on these findings, we hypothesized that Src-mediated phosphorylation of TβR-II promotes oncogenic signaling by TGF-β in MECs and, as such, enhances their proliferation, invasion, and EMT in response to TGF-β. We now show herein that Src phosphorylated TβR-II both in vitro and in vivo on Y284, which then coordinated the docking of the SH2 domains of growth factor receptor binding protein 2 (Grb2) and Src homology domain 2 containing (Shc) to TβR-II. When expressed in breast cancer cells, TβR-IIs lacking Y284 failed to activate p38 MAPK and were unable to induce breast cancer cell invasion stimulated by αvβ3 integrin and TGF-β. Collectively, our findings have identified a novel β3 integrin/Src/TβR-II/Grb2-Shc signaling axis that functions in selectively coupling TGF-β to activation of p38 MAPK as well as in promoting oncogenic signaling by TGF-β in breast cancer cells.

Materials and Methods

Plasmids and transgene expression. Retroviral vectors encoding human WT-β3 or D119A-β3 integrins were described previously (17). A retroviral vector encoding human TβR-II was constructed by PCR amplification using oligonucleotides containing _Eco_RI (NH2 terminus) and _Xho_I (COOH terminus) restriction sites, which was ligated into identical sites immediately upstream of the IRES in the bicistronic retroviral vector pMSCV-IRES-GFP (17). Site-directed mutagenesis of TβR-II to replace Tyr259, Tyr284, Tyr336, Tyr424, and Tyr470 with Phe, either individually or in combination, or to replace Lys277 to Arg was done using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) as described previously (17). Control (i.e., pMSCV-IRES-GFP), wild-type (WT), and mutant TβR-II retroviral supernatants were produced and infected into murine 4T1 breast cancer cells as described previously (17). These same cDNA constructs also were transiently transfected into the TβR-II-null mink lung Mv1Lu epithelial cell derivatives, DR26 cells (18).

Recombinant His-tagged proteins containing the cytoplasmic domain of WT or mutant TβR-II derivatives were constructed by subcloning _Xho_I/_Eco_RI–digested PCR fragments into corresponding sites in pET-28a (Novagen, Madison, WI). His-tagged TβR-II fusion proteins were purified by passing 1% Triton X-100–solubilized bacterial cell extracts over a Ni2+-agarose column. Bound proteins were eluted with 6 column volumes of 200 mmol/L imidazole and concentrated against PBS (5-kDa cutoff; Sartorius, Goettingen, Germany).

All TβR-II inserts were sequenced in their entirety on an Applied Biosystems 377A DNA sequencing machine.

Src phosphorylation and SH2 binding assays. Monitoring the ability of Src to phosphorylate recombinant WT and mutant TβR-II proteins was determined using an in vitro protein kinase assay as described previously (17). In some experiments, the ability of SH2 domain–containing proteins to bind Src-phosphorylated TβR-II was determined by performing in vitro binding assays. To do so, Src protein kinase reactions were stopped by addition of 600 μL leukemia inhibitory factor receptor immunoprecipitation buffer (19). The resulting mixtures were supplemented with 0.2 μg per tube of glutathione _S_-transferase (GST) fusion proteins containing the SH2 domains of either Nck1, Nck2, PLC-γ, Grb2, or Shc (ref. 20; provided by Dr. Tony Pawson, Mount Sinai Hospital, Ontario, Canada). The binding reactions were tumbled with Ni2+-agarose beads (30 μL per tube) for 30 min at 4°C. Bound proteins were eluted with 500 mmol/L Imidazole and fractionated through 12% SDS-PAGE before their electrophoretic transfer to nitrocellulose membranes. Immobilized proteins were probed sequentially with antibodies against phosphotyrosine (4G10 antibodies; Upstate, Charlottesville, VA) followed by those against GST (Invitrogen, Carlsbad, CA). Loading differences were monitored by reprobing stripped membranes with anti-TβR-II antibodies.

TβR-II coimmunoprecipitation assays. NMuMG and 4T1 cells were cultured onto six-well plates and subsequently stimulated with TGF-β1 (5 ng/mL) for varying times in the absence or presence of the Src inhibitors PP2 (10 μmol/L; Calbiochem, Temecula, CA) or SU6656 (10 μmol/L; EMD Biosciences, La Jolla, CA). Following agonist stimulation, the cells were washed twice in ice-cold PBS and lysed in Buffer H/Triton X-100 (19). The resulting detergent-solubilized whole-cell extracts were clarified by microcentrifugation and subjected to the following immunoprecipitation conditions: (a) anti–β3 integrin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) using 600 μg per tube of whole-cell extract; (b) anti-Grb2 antibodies (Santa Cruz Biotechnology) using 200 μg per tube of whole-cell extract; (c) anti–phosphotyrosine 4G10 antibodies (Upstate) using 600 μg per tube of whole-cell extract; or (d) anti-TβR-I or anti-TβR-II antibodies using 200 μg per tube of whole-cell extract as described previously (17). All immunoprecipitations were incubated for 16 h at 4°C with slow rotation. The resulting immunocomplexes were collected by microcentrifugation, washed several times in lysis buffer, and fractionated through 10% SDS-PAGE gels before electrophoretic immobilization of the proteins to nitrocellulose membranes, which subsequently were probed with anti–β3 integrin (1:1,000), anti-TβR-II (1:1,500; Santa Cruz Biotechnology), or anti–phosphotyrosine 4G10 (1:1,500; Upstate) antibodies.

Recombinant GST-Smad3 phosphorylation assay. The phosphorylation of recombinant Smad3 by activated TGF-β receptor complexes was done essentially as described previously (17). Briefly, 4T1 cells expressing WT-TβR-II, Y284F-TβR-II, and Y470-TβR-II were stimulated with TGF-β1 (5 ng/mL) for 30 min at 37°C. Cytokine stimulations were terminated by washing the cells twice in ice-cold PBS, which then were lysed and solubilized on ice in Buffer H/Triton X-100. The resulting cell extracts (600 μg per tube) were clarified by microcentrifugation and subsequently immunoprecipitated with anti-TβR-II antibodies for 2 h at 4°C. TβR-II immunocomplexes were recovered by brief microcentrifugation and washed twice in PBS. TGF-β receptor phosphotransferase activity against recombinant GST-Smad3 was measured for 30 min at 30°C in a final reaction volume of 40 μL, consisting of 30 μL TGF-β receptor immunocomplexes, 4 μg GST-Smad3, and 10 μL of 4× assay buffer (17). Phosphotransferase reactions were stopped by the addition of 4× sample buffer and boiled for 5 min before their fractionation through 10% SDS-PAGE. Fractionated proteins were immobilized electrophoretically to nitrocellulose membranes, which subsequently were probed with anti–phosphorylated Smad3 antibodies to visualize phosphorylated GST-Smad3. Differences in immunoprecipitation efficiency and loading were monitored by reprobing stripped membranes with anti-TβR-II antibodies.

Cell biological assays. The effect of WT or mutant TβR-II expression on various TGF-β–stimulated activities in DR26 cells and 4T1 MECs were determined as follows: (a) Cell proliferation using 5,000 cells per well in a [3H]thymidine incorporation assay as described (17); (b) Smad2/3, ERK1/2, and p38 MAPK phosphorylation was monitored by immunoblotting with phospho-specific antibodies as described (17); (c) cell invasion induced by 10% serum using 200,000 cells per well in a modified Boyden chamber coated with Matrigel matrices (diluted 1:50 in serum-free DMEM) as described (17); and (d) gene expression using 30,000 cells per well in a pSBE-luciferase reporter gene assay as described (17). In some experiments, 4T1 cells were rendered β3 integrin deficient using SMARTpool siRNA directed against β3 integrin exactly as described previously (17). Afterward, the effect of β3 integrin deficiency on 4T1 cell invasion was determined as above.

Results

Src phosphorylates TβR-II at Y284 both in vitro and in vivo. Our recently published study established β3 integrin and Src as an essential mediators of MEC proliferation, invasion, and EMT stimulated by TGF-β, doing so in part through Src-mediated phosphorylation of TβR-II (17). To extend these findings, we first sought to identify the tyrosine residue(s) in TβR-II that is phosphorylated by Src. We therefore queried the cytoplasmic TβR-II sequence in Scansite Motif Scanner, which identified Y284 and Y470 as potential Src phosphorylation sites. Thus, Y284 and Y470 were each converted to phenylalanine, as were the three TβR-II Tyr autophosphorylation sites: Y259, Y336, and Y424 (ref. 21; i.e., triple mutant). In addition, all Tyr → Phe substitutions were generated in a kinase-dead TβR-II background (i.e., K277R mutant) to eliminate potential confounding phosphorylation events mediated by TβR-II autophosphorylation. Figure 1A shows that increasing concentrations of Src (0.5 → 10 units per tube) tyrosine-phosphorylated TβR-II in a linear manner. Under these assay conditions, Src also readily phosphorylated the triple- and Y470F-TβR-II mutants, but not the Y284F-TβR-II mutant in vitro (Fig. 1B). More importantly, WT-TβR-II and Y470F-TβR-II proteins both were tyrosine phosphorylated and formed complexes with β3 integrin when expressed in 4T1 breast cancer cells (Fig. 1C). In stark contrast, isolated Y284F-TβR-II proteins failed become tyrosine phosphorylated in 4T1 cells despite their ability to form complexes with β3 integrins (Fig. 1C). Similar to our previous report (17), PP2 or SU6656 treatment of 4T1 cells to inhibit Src activity abrogated tyrosine phosphorylation of TβR-II in breast cancer cells (Fig. 1D). Collectively, these findings identified Y284 as the major residue in TβR-II that is phosphorylated by Src both in vitro and in vivo. These findings also show that the formation of β3 integrin/TβR-II complexes, which promote oncogenic signaling by TGF-β (17), occurs independently of tyrosine phosphorylation of TβR-II.

Figure 1.

Src phosphorylates TβR-II at Y284 both in vitro and in vivo. A, increasing concentrations of active recombinant Src (0.5–10 units per tube) were allowed to phosphorylate recombinant cytoplasmic K277R-TβR-II (0.5 μg per tube) for 30 min at 30°C. Src-mediated phosphorylation of TβR-II was visualized by Western blotting (WB) reaction mixtures with antibodies against phosphotyrosine (PY) followed by those against TβR-II. Immunoblots from a single experiment that was repeated three times with identical results. B, active recombinant Src (1 unit per tube) was incubated with WT or tyrosine-substituted K277R-TβR-II mutants (0.5 μg per tube) as indicated. Phosphorylation reactions were terminated after 30 min, and phosphotyrosine status of individual TβR-II proteins was determined by immunoblotting as above. Immunoblots from a single experiment that was repeated thrice with similar results. Triple, Tyr259, Tyr336, and Tyr424 were converted to Phe. C, 4T1 cells were engineered by bicistronic retroviral transduction to stably express either WT-TβR-II, Y284F-TβR-II, or Y470F-TβR-II. Upon reaching 90% confluency, the cells were harvested, and detergent-solubilized whole-cell extracts (W.C.E.) were prepared (600 μg per tube) for immunoprecipitation (IP) with either anti-phosphotyrosine or TβR-II antibodies followed by Western blotting with antibodies against either phosphotyrosine, TβR-II, or β3 integrin as indicated. Differences in protein loading were monitored by immunoblotting whole-cell extracts (50 μg per lane) for TβR-II. Data from a representative experiment that was repeated twice with identical results. D, 4T1 cells were incubated for 2 h in the absence or presence of the Src inhibitors, PP2 (10 μmol/L) or SU6656 (10 μmol/L), and subsequently stimulated with TGF-β1 (5 ng/mL) for 30 min at 37°C. Afterward, detergent-solubilized whole-cell extracts were prepared (1 mg per tube) for immunoprecipitation with anti-phosphotyrosine antibodies, followed by Western blotting with anti-TβR-II antibodies. Differences in protein loading were monitored by immunoblotting whole-cell extracts (50 μg/lane) for TβR-II.

Figure 1.

Close modal

Y284 in TβR-II is not required for TGF-β–mediated activation of Smad2/3. Our results thus far identified Y284 as the major residue in TβR-II that was phosphorylated by Src both in vitro and in vivo (Fig. 1). We next sought to determine whether Y284F-TβR-II proteins remained competent to respond to TGF-β when expressed in cells. To do so, cDNAs encoding for WT-TβR-II, Y284F-TβR-II, or Y470F-TβR-II were transiently transfected into DR26 cells, which are chemically mutagenized derivatives of mink lung Mv1Lu epithelial cells that fail to respond to TGF-β due to their loss of TβR-II (18). Importantly, restoration of TGF-β responsiveness is readily reestablished in DR26 cells by re-expression of TβR-II (18). As expected, TGF-β treatment of parental DR26 cells failed to induce luciferase expression driven by the synthetic Smad-binding element (SBE) promoter (Fig. 2A). In contrast, DR26 cells engineered to express WT-TβR-II, Y284F-TβR-II, or Y470F-TβR-II proteins readily and indistinguishably induced SBE-driven luciferase expression when stimulated by TGF-β (Fig. 2A). Similar TGF-β–mediated SBE promoter activation was observed when these same TβR-II constructs were stably introduced into 4T1 breast cancer cells (Fig. 2B). Thus, the loss of Y284 in TβR-II fails to effect TGF-β stimulation of Smad2/3. Accordingly, TGF-β stimulation of 4T1 cells expressing WT-TβR-II, Y284F-TβR-II, or Y470F-TβR-II induced Smad2 and Smad3 phosphorylation in an indistinguishable manner (Fig. 2C). Furthermore, TGF-β receptor complexes isolated from 4T1 cells expressing WT-TβR-II, Y284F-TβR-II, and Y470-TβR-II all phosphorylated recombinant Smad3 to similar extents in vitro (Fig. 2D), suggesting that the coupling to and activation of TβR-I by Y284F-TβR-II is similar to that mediated by its WT counterpart. Collectively, these findings suggest that Y284F-TβR-II proteins are expressed to the cell surface and remain competent to normally activate Smad2/3 signaling in response to TGF-β.

Figure 2.

Y284 in TβR-II is not required for TGF-β–mediated activation of Smad2/3. cDNAs encoding for WT-TβR-II, Y284F-TβR-II, or Y470F-TβR-II together with pSBE-luciferase and pCMV-β-gal were transiently transfected in either DR26 (A) or 4T1 (B) cells as indicated. Afterward, the cells were stimulated with TGF-β1 (5 ng/mL) for 24 h and subsequently processed to measure luciferase and β-galactosidase (β-gal) activities contained in detergent-solubilized cell extracts. Columns, mean (n = 3); bars, SE. C, WT-TβR-II–, Y284F-TβR-II–, or Y470F-TβR-II–expressing 4T1 cells were serum starved for 4 h before TGF-β1 stimulation (5 ng/mL) for 0 to 120 min as indicated. Top, activation of Smad2 and Smad3 was determined by immunoblotting using phospho-specific antibodies as indicated. Bottom, parental and 4T1 cells expressing WT-TβR-II, Y284F-TβR-II, or Y470F-TβR-II were harvested (100 μg per lane) and immunoblotted for β3 integrin and TβR-II as indicated. Differences in protein loading were monitored using anti–pan Smad2/3 antibodies. Images from a single experiment that was repeated twice with similar results. D, 4T1 cells were stimulated with TGF-β1 (5 ng/mL) for 30 min and subsequently lysed and immunoprecipitated (600 μg per tube) with anti-TβR-II antibodies. The resulting immunocomplexes were used to phosphorylate recombinant GST-Smad3, which was visualized by immunoblotting with anti–phosphorylatyed Smad3 (pSmad3) antibodies. Data from a representative experiment that was repeated twice with similar results.

Figure 2.

Close modal

Y284 in TβR-II is essential for TGF-β–mediated activation of p38 MAPK. Our previous report showed that the expression and activity of β3 integrin and Src both are essential for the TGF-β to induce oncogenic signaling and EMT in normal and malignant MECs (17). This study also suggested that β3 integrin and Src both enhanced the coupling of TGF-β to activate MAPKs, particularly that of p38 MAPK (17). As such, we hypothesized that Src-mediated phosphorylation of Y284 in TβR-II enables TGF-β stimulation of p38 MAPK in malignant MECs. We tested the above hypothesis in 4T1 cells, which selectively up-regulate their expression of the αv and β3 integrin subunits in response to TGF-β (Fig. 3A), an event essential for oncogenic signaling by TGF-β in normal and malignant MECs (17). Figure 3B shows that WT-TβR-II– and Y470F-TβR-II–expressing 4T1 cells readily activated p38 MAPK when stimulated by TGF-β, whereas those expressing Y284F-TβR-II were unable to recapitulate this response to TGF-β. This finding suggests that phosphorylation of or events occurring at Y284 are necessary to couple TGF-β and TβR-II to activation of p38 MAPK. Further support of this supposition is provided in Fig. 3C, which shows that expression of WT-TβR-II or Y470F-TβR-II proteins in DR26 cells readily recapitulated p38 MAPK activation by TGF-β. In stark contrast, Y284F-TβR-II–expressing DR26 cells failed to activate p38 MAPK when stimulated by TGF-β (Fig. 3C). Collectively, these findings and those presented in Fig. 2 show a novel and important function of Y284 in coupling TGF-β and TβR-II to activation of p38 MAPK in malignant MECs. In addition, these findings also identify the first signaling specificity determinant in TβR-II that dissociates the ability of TGF-β to activate Smad2/3 from that of MAPKs.

Figure 3.

Y284 in TβR-II is essential for TGF-β–mediated activation of p38 MAPK. A, quiescent 4T1 cells were stimulated with TGF-β1 (5 ng/mL) for 24 h at 37°C. Afterward, detergent-solubilized whole-cell extracts (50 μg per lane) were prepared and subjected to immunoblot analysis to detect differential expression of the β3, αv, or β1 integrin subunits, of TβR-II, and of focal adhesion kinase (FAK), and focal adhesion kinase phosphorylation (pFAK). Differences in protein loading were monitored by reprobing stripped blots with anti-β-actin antibodies. (B) WT-TβR-II–, Y284F-TβR-II–, or Y470F-TβR-II–expressing 4T1 cells were serum starved for 4 h before TGF-β stimulation (5 ng/mL) for 0 to 120 min as indicated. The activation status of p38 MAPK was monitored by immunoblotting using phospho-specific p38 MAPK antibodies. Differences in protein loading were monitored using anti-p38 antibodies. Images from a single experiment that was repeated twice with similar results. C, DR26 cells were transiently transfected with cDNAs encoding WT-TβR-II, Y284F-TβR-II, or Y470F-TβR-II proteins as indicated. Afterward, the transfectants were serum starved 4 h before TGF-β1 simulation (5 ng/mL) for 0 to 60 min as indicated. The phosphorylation of p38 MAPK was determined by immunoblotting with phospho-specific p38 MAPK antibodies. Differences in protein loading were monitored using anti–p38 MAPK, whereas TβR-II expression levels were determined by immunoblotting with anti-TβR-II antibodies. Images from a single experiment that was repeated twice with similar results.

Figure 3.

Close modal

SH2 domains of Grb2 and Shc bind phosphorylated Y284 in TβR-II in vitro and in vivo. Phosphotyrosine residues often function as docking sites for SH2 domain–containing proteins, several of which promote the activation of MAPKs (22, 23). We therefore hypothesized that Src-mediated phosphorylation of Y284 coordinates the docking of SH2 domain–containing proteins to TβR-II. Figure 4A shows that Src-phosphorylated TβR-II selectively bound the SH2 domains of Grb2 and Shc but not to those of Nck1, Nck2, or PLC-γ. More importantly, the ability of the SH2 domains of Grb2 and Shc to dock TβR-II absolutely required Src to phosphorylate Y284 (Fig. 4B). Grb2 binding to TβR-II also occurred readily in NMuMG and 4T1 cells following their stimulation with TGF-β (Fig. 4C). Consistent with our in vitro findings (Fig. 4B), Grb2 readily bound WT-TβR-II proteins expressed in 4T1 cells but not to their Y284F-TβR-II counterparts (Fig. 4D), which bound TβR-I analogous to that by WT-TβR-II (Fig. 4D). Collectively, these results show that Src-mediated phosphorylation of Y284 coordinates the docking of the SH2 domains of Grb2 and Shc to TβR-II in vitro and of Grb2 in vivo.

Figure 4.

SH2 domains of Grb2 and Shc bind phosphorylated Y284 in TβR-II in vitro and in vivo. Active recombinant Src (1 unit per tube) was used to phosphorylate either (A) K277R-TβR-II or (B) WT-TβR-II, Y284F-TβR-II, or Y470F-TβR-II (0.5 μg per tube) as indicated. Phosphorylation reactions were terminated after 30 min by dilution into binding buffer (600 μL) and immediately supplemented with recombinant GST-SH2 domains (0.2 μg per tube) as indicated. TβR-II protein complexes were captured by Ni2+-agarose affinity chromatography and bound proteins visualized by sequential immunoblotting with anti-GST, anti-PY, and anti-TβR-II antibodies as indicated. Data from a representative experiment that was repeated thrice with similar results. Tested SH2 domains were GST-Nck1, GST-Nck2, GST-PLCγ, GST-Grb2, and GST-Shc. C, NMuMG and 4T1 cells were stimulated with TGF-β1 (5 ng/mL) for 36 h at 37°C. Afterward, detergent-solubilized whole-cell extracts were prepared (200 μg per tube) for immunoprecipitation with anti-Grb2 antibodies followed by Western blotting with antibodies against TβR-II. Differences in protein loading were monitored by immunoblotting whole-cell extracts (40 μg per lane) for β-actin. Data from a representative experiment that was repeated twice with identical results. D, detergent-solubilized whole-cell extracts (200 μg per tube) were prepared from 4T1 cells expressing either WT-TβR-II or Y284F-TβR-II and subsequently immunoprecipitated with antibodies against either Grb2 or TβR-I followed by Western blotting with anti-TβR-II antibodies. Differences in protein loading were monitored by immunoblotting whole-cell extracts (40 μg per lane) for β-actin. Data from a representative experiment that was repeated twice with identical results.

Figure 4.

Close modal

Y284 is essential for αvβ3 integrin– and TGF-β–stimulated invasion of 4T1 cells. TGF-β prevents MEC proliferation primarily via signals induced by Smad2/3, whereas its ability to promote MEC invasion transpires via integrated signaling inputs induced by Smad2/3 in conjunction with those by MAPKs (16, 24). The selective defect of Y284F-TβR-II proteins to activate MAPKs suggested that this mutant might also impair the ability of TGF-β to regulate MEC proliferation and/or invasion. We tested these possibilities by first measuring the changes in 4T1 cell proliferation mediated by their expression of WT-TβR-II, Y284F-TβR-II, or Y470F-TβR-II proteins. In accord with a previous report (25), we too find 4T1 cells to be resistant to TGF-β–mediated growth arrest (i.e., WT-TβR-II and Y470F-TβR-II), a cytostatic response that was partially restored by Y284F-TβR-II expression (Fig. 5A). Thus, phosphorylation at Y284 seems to override the growth-inhibitory function of TGF-β in MECs.

Figure 5.

Y284 is essential for αvβ3 integrin– and TGF-β–stimulated invasion of 4T1 cells. A, murine 4T1 cells expressing WT-TβR-II, Y284F-TβR-II, or Y470F-TβR-II were stimulated with TGF-β1 (5 ng/mL) for 48 h at 37°C. Cellular DNA was radiolabeled with [3H]thymidine and quantified by scintillation counting. Columns, mean (n = 4); bars, SE. *, P < 0.05. Murine 4T1 cells engineered to express (B) TβR-II derivatives, (C) β3 integrin derivatives, or (D) TβR-II and β3 integrin derivatives in combination were allowed to invade through Matrigel matrices in the absence or presence of TGF-β1 (5 ng/mL) for 30 h at 37°C. Columns, mean (n = 3); bars, SE. * and **, P < 0.05.

Figure 5.

Close modal

We also compared the ability of individual TβR-II derivatives to alter the invasion of 4T1 cells through synthetic basement membranes. Figure 5B shows that Y284F-TβR-II–expressing 4T1 cells were minimally invasive when stimulated by TGF-β compared with their WT-TβR-II– or Y470F-TβR-II–expressing counterparts. In accordance with our previous report (17), loss of β3 integrin expression or function significantly inhibited 4T1 cell invasion stimulated by TGF-β (Fig. 5C). More importantly, coexpression of β3 integrin with either WT-TβR-II or Y470F-TβR-II significantly enhanced the ability of TGF-β to promote 4T1 cell invasion, a response that was abrogated by expression of Y284F-TβR-II (Fig. 5D). Collectively, these findings identify Y284 of TβR-II in mediating oncogenic signaling by TGF-β and its stimulation of MEC invasion.

Discussion

How TGF-β both suppresses and promotes tumorigenesis remains an unknown and fundamental question that directly affects the ability of science and medicine to effectively target the TGF-β signaling system during the treatment of metastatic breast cancers. We previously established β3 integrin and Src as essential mediators of oncogenic signaling by TGF-β in MECs (17). We now show that (a) Src phosphorylates TβR-II at Y284 both in vitro and in vivo (Fig. 1); (b) Y284 of TβR-II is essential for TGF-β stimulation of p38 MAPK (Fig. 3), but not Smad2 and Smad3 (Fig. 2); (c) Src-mediated phosphorylation of Y284 coordinates the docking of Grb2 and Shc to TβR-II both in vitro and in vivo (Fig. 4); and (d) Y284 is essential for αvβ3 integrin and TGF-β stimulation of malignant MEC invasion and for counteracting the cytostatic function of TGF-β (Fig. 5). Our findings also identify Y284 as a novel TβR-II signaling specificity determinant that enables MECs to interpret cytostatic (i.e., unphosphorylated) versus oncogenic (i.e., Src phosphorylated) signals by TGF-β.

Our previous findings (17) and those presented herein highlight the essential function of αvβ3 integrin and Src in mediating oncogenic signaling by TGF-β in MECs. In this manner, up-regulated expression of αvβ3 integrin has been observed in cancer cells and localizes to the leading invasive edge where it enhances tumor metastasis (26). Moreover, TGF-β stimulation of αvβ3 integrin expression enhances breast cancer cell metastasis to bone and lung (27, 28). Our findings also indicate that Src-mediated TβR-II phosphorylation and formation of Grb2 and Shc signaling complexes may play an essential role in promoting oncogenic signaling by TGF-β. In support of this supposition, nearly 70% of human breast cancers exhibit elevated Src expression or activity (29), an aberrant event that also correlates with the appearance of systemic relapse in patients with breast cancer (30). Src also associates with and promotes the formation of Shc/Grb2/Gab1 complexes in developing mammary tumors driven by ErbB2/3 and by polyoma middle T expression (31). Importantly, the ability of polyoma middle T to induce mammary tumorigenesis absolutely requires Src and β1 integrin function, both of which promote the formation of ShcA/Grb2/Gab1 signaling complexes (32). These findings are strikingly reminiscent of our own that show the role of Src and β3 integrin in regulating oncogenic signaling by TGF-β (17). Indeed, we propose that oncogenic signaling by TGF-β in MECs is evolutionarily and functionally redundant with that mediated by growth factor receptors, and as such, that the development of pharmacologic interventions designed to antagonize and/or circumvent this oncogenic TGF-β signaling axis may one day prove effective in treating metastatic breast cancer.

Acknowledgments

Grant support: NIH grants CA095519 and CA114039 and Concern Foundation (W.P. Schiemann).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank R&D Systems, Inc. (Minneapolis, MN) for generously providing TGF-β1 and members of the Schiemann Laboratory for critical reading of the article.

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Src Phosphorylates Tyr284 in TGF-β Type II Receptor and Regulates TGF-β Stimulation of p38 MAPK during Breast Cancer Cell Proliferation and Invasion (original) (raw)

Abstract

Introduction

Materials and Methods

Results

Discussion

Acknowledgments

References

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