Interaction of syndecan and alpha6beta4 integrin cytoplasmic domains: regulation of ErbB2-mediated integrin activation

Abstract

The alpha6beta4 integrin is a laminin 332 (LN332) receptor central to the formation of hemidesmosomes in epithelial layers. However, the integrin becomes phosphorylated by keratinocytes responding to epidermal growth factor in skin wounds or by squamous cell carcinomas that overexpress/hyperactivate the tyrosine kinase ErbB2, epidermal growth factor receptor, or c-Met. We show here that the beta4-dependent signaling in A431 human squamous carcinoma cells is dependent on the syndecan family of matrix receptors. Yeast two-hybrid analysis identifies an interaction within the distal third (amino acids 1473-1752) of the beta4 cytoplasmic domain and the conserved C2 region of the syndecan cytoplasmic domain. Via its C2 region, Sdc1 forms a complex with the alpha6beta4 integrin along with the receptor tyrosine kinase ErbB2 and the cytoplasmic kinase Fyn in A431 cells. Engagement of LN332 or clustering of the alpha6beta4 integrin with integrin-specific antibodies causes phosphorylation of ErbB2, Fyn, and the beta4 subunit as well as activation of phosphatidylinositol 3-kinase and Akt and their assimilation into this complex. This leads to phosphatidylinositol 3-kinase-dependent cell spreading and Akt-dependent protection from apoptosis. This is disrupted by RNA interference silencing of Sdc1 but can be rescued by mouse Sdc1 or Sdc4 but not by syndecan mutants lacking their C-terminal C2 region. This disruption does not prevent the phosphorylation of ErbB2 or Fyn but blocks the Fyn-mediated phosphorylation of the beta4 tail. We propose that syndecans engage the distal region of the beta4 cytoplasmic domain and bring it to the plasma membrane, where it can be acted upon by Src family kinases.

FIGURE 1.

Interaction of syndecan cytoplasmic C2 region with β4 integrin cytoplasmic domain in a yeast two-hybrid assay. A, yeast strain AH109 was transformed with p53 (Gal4-binding domain bait vector) and T-antigen (Gal4 activation domain prey vector as a positive control) or with Lamin C (bait) and T antigen (prey) or empty vector (bait) and β4 integrin sequence encoding amino acids 1473–1752 (prey) as negative controls. The yeast were spread on SD-leu-trp-his-ade plates containing x-α-gal to visualize positive (blue) colonies. B and C, the β4 fragment (prey) was transformed into yeast with bait vector containing the cytoplasmic domains of either Sdc1, Sdc2, Sdc3, or Sdc4 (B) or Sdc1 lacking its C1 and C2 regions (Sdc1ΔC1/C2), its C1 region (Sdc1ΔC1), or its C2 region (Sdc1ΔC2) (C). D, quantification of data shown in A–C. After 9 days, the number of blue transformants growing on the SD-leu-trp-his-ade plates was counted and divided by the total number of transformants grown on SD-leu-trp plates to calculate the percentage of colonies with positive interactions. All percentages represent the average percentage of three independent transformations. Any strain with a percentage greater than 2% is shown by a plus sign in the β4 interaction column. No positive colonies were obtained for syndecan fragments co-transformed with a plasmid bearing empty prey vector. The syndecan cytoplasmic domains are depicted containing the conserved region 1 (C1), variable region (V), and conserved region 2 (C2) and expressed as a fusion with the Gal-4-binding domain. E, schematic diagram showing the α6β4 integrin and its transmembrane (TM) region, FNIII repeats I–IV, and cytoplasmic tyrosines. The region containing the syndecan binding site (amino acids 1473–1752) identified by yeast two-hybrid analysis is shown.

FIGURE 2.

Requirement of Sdc1 for A431 cell spreading on LN332. Shown are A431 cells plated on slides coated with 10 μg/ml LN332 without treatment (A), with blocking antibody 3E1 to α6β4 (B), with prior treatment with heparinase II. (C), or with prior treatment with siRNA-specific for human Sdc1 (D). E, quantification of spread cells plated on LN332 and treated with blocking antibody to β1 integrin (mAb13, 30 μg/ml) or control IgG (20 μg/ml), 3E1 blocking antibody (20 μg/ml) to β4 integrin, heparinase II (0.4 conventional unit/ml), or human Sdc1 siRNA (100 nm). Data represent triplicate experiments ± S.D. F, flow cytometry of mock-transfected A431 cells stained with hSdc1-specific antibody B-A38 or control IgG and hSdc1 siRNA-transfected cells stained for hSdc1. *, p < 0.05. Bar, 40 μm.

FIGURE 3.

Syndecan-dependent spreading of A431 cells on β4 antibody 3E1. A431 cells were plated on 3 μg/ml 3E1 for 1 h at 37 °C and then stained with rhodamine-conjugated phalloidin to visualize the spread lamellipodia. Shown are parental cells (A), parental cells pretreated with hSdc1-specific siRNA (B), siRNA-treated cells expressing mSdc1 (C), and siRNA-treated cells expressing mSdc1ΔC2 mutant (D). E, the cell surface expression of mSdc1 or mSdc1ΔC2 (mAb 281.2) and mSdc4 or mSdc4ΔC2 (mAb KY 8.2) is compared with control rat IgG by flow cytometry. F, quantification of spread cells with or without pretreatment with hSdc1-specific siRNA and stable expression of mouse Sdc1 or Sdc4 constructs. Data represent triplicate experiments ± S.D. *, p < 0.05. Bar, 40 μm.

FIGURE 4.

Interaction of Sdc1 and Sdc4 with α6β4 integrin in HaCat and A431 cells. A, HaCat cells treated with or without hSdc1 siRNA are plated on 3 μg/ml 3E1 as described in the legend to Fig. 3. B, hSdc1, hSdc4, or β4 integrin subunit was immunoprecipitated from A431 or HaCat cells and probed for precipitation of either hSdc1 or hSdc4. C, staining of HaCat cells for expression of hSdc1 (mAb B-A38), hSdc4 (mAb 150.9), or hSdc4 after permeabilization (hSdc4 perm). D, staining of A431 cells for hSdc1, hSdc4 (with and without permeabilization), and α6β4 integrin (with (mAb GoH3) and without (mAb 439–9B) permeablization). Mouse IgG (mIgG) and rat IgG (rIgG) staining controls are shown. Bar, 20 μm. IP, immunoprecipitation; IB, immunoblot.

FIGURE 5.

Inhibition of α6β4-mediated spreading by PI3K, SFK, and ErbB2 inhibitors. A431 cells were plated for 1 h on substrata coated with 3 μg/ml mAb 3E1 to engage the α6β4 integrin in the presence of PI3K inhibitor LY294002 (60 μm) or wortmannin (5 μm), SFK inhibitor PP2 (1 μm), or ErbB2 tyrphostin inhibitor AG825 (5 μm). Data are shown as percentage of spread cells compared with control in triplicate experiments ± S.D. *, p < 0.05.

FIGURE 6.

Sdc1 immunoprecipitates in a complex containing α6β4 integrin, ErbB2, Fyn, and PI3K. A, A431 cells cultured in serum were extracted and subjected to immunoprecipitation (IP) using mAb B-A38 to hSdc1, 3E1 to β4 integrin subunit, Ab-15 to ErbB2, FYN15 to Fyn, or AB6 to the p85α subunit of PI3K. The immunoprecipitates were analyzed on Western blots by probing with these antibodies. *, note that 4-fold less of the Fyn immunoprecipitate was analyzed compared with the other samples. B, A431 cells transfected with mSdc1, mSdc1ΔC2, or mSdc4 were similarly analyzed by immunoprecipitation of hSdc1 with B-A38, mouse Sdc1, or Sdc1ΔC2 with mAb 281.2 or mSdc4 with mAb KY 8.2. The blots were then probed with the same antibodies to confirm syndecan precipitation and for co-precipitation of α6β4 using mAb 3E1 or ErbB2 using Ab-15.

FIGURE 7.

Sdc1 is necessary for Fyn-mediated phosphorylation of α6β4 integrin. A, suspended A431 cells are treated with PBS alone or PBS containing 1 μg/ml LN111 or LN332. Cell lysates were split between two Western blots stained for β4 subunit with mAb 3E1 and phosphotyrosine with mAb PY20. B and C, suspended A431 cells are treated with or without mAb 3E1 and an anti-mouse IgG secondary (2°) antibody to induce clustering of α6β4 integrin in the presence of 5 μm AG825 to inhibit ErbB2 or 1 μm PP2 to inhibit SFK or 5 μm wortmannin or 40 μm LY294002 to inhibit PI3K. B, ErbB2 was precipitated with mAb Ab-15, Fyn with FYN15, and β4 with 3E1, and the immunoprecipitates were probed using these same antibodies, using PY20 to detect phosphotyrosine, and using anti-phospho-Src family (Tyr-416) mAb to detect pY416 specific for Fyn activation. C, lysates were probed on Western blots for Akt and for phosphorylation of the activation-specific Ser-473 in Akt using anti-phospho-AKT (Ser-473) mAb. D, A431 cells or A431 cells expressing mSdc1 were pretreated with Lipofectamine with or without hSdc1-specific siRNA and then suspended and treated with or without 3E1 plus anti-mouse IgG (10 min) to induce β4 clustering. ErbB2, Fyn, and β4 integrin subunit were immunoprecipitated, and phosphorylation of ErbB2 and β4 was determined by blotting with PY20, phosphorylation of Fyn by blotting with mAb anti-phospho-Src family (Tyr-416), and co-precipitation of PI3K with β4 by blotting with mAb AB6. Note that the Fyn blot was stripped and reprobed for the double staining. IP, immunoprecipitation.

FIGURE 8.

Sdc1-dependent signaling by α6β4 prevents apoptosis in A431 cells. A, A431 cells were grown in serum-containing medium for 5, 30, and 55 h following pretreatment with Lipofectamine with or without hSdc1-specific siRNA. The number of apoptotic cells was determined by staining with Alexa488-conjugated Annexin V and expressed as a percentage of the total cells. B, A431 cells expressing either empty vector or vector encoding mSdc1 or mSdc1ΔC2 mutant were treated with or without siRNA and then plated for 55 h in serum-containing medium followed by staining with Annexin V. C, A431 cells were pretreated with Lipofectamine alone or Lipofectamine with hSdc1-specific siRNA and then were plated in serum-containing medium for 24 h in 20 μg/ml mAb 3E1 to block ligand binding to the α6β4 integrin or treated with mAb 3E1 plus anti-mouse IgG to cluster the α6β4 integrin to rescue the block to integrin signaling. Cells were suspended and stained with Alexa488-conjugated Annexin V. D, quantification of apoptosis monitored by Annexin V staining under the conditions defined in C. Data are from triplicate experiments ± S.D. *, p < 0.05.

FIGURE 9.

Speculative model of syndecan and integrin interaction. The α6β4 integrin is shown assembled in a complex with ErbB2, Fyn, and Sdc1. Note that although Sdc1 predominates in this mechanism in the A431 cells, other syndecan family members may function in this role in other cells. Sdc1 binds the β4 subunit within the distal portion of its cytoplasmic domain containing the third and fourth FNIII domains and the C terminus. Clustering of these complexes (ErbB2 from a second complex is shown after clustering) causes transphosphorylation of ErbB2 (step 1), docking and autophosphorylation of Fyn (step 2), and Fyn-dependent phosphorylation of the β4 cytoplasmic domain (step 3). The phosphorylated β4 cytoplasmic domain recruits signaling proteins that lead to the activation of PI3K, cell adhesion and spreading, and resistance to apoptosis.