Tyrosine Phosphorylation of the beta 4 Integrin Cytoplasmic Domain Mediates Shc Signaling to Extracellular Signal-regulated Kinase and Antagonizes Formation of Hemidesmosomes*

Michael DansDagger, Laurent Gagnoux-Palacios§, Pamela Blaikie, Sharon Klein, Agnese Mariotti, and Filippo G. Giancotti||

From the Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021

Received for publication, September 21, 2000, and in revised form, October 16, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Ligation of the alpha 6beta 4 integrin induces tyrosine phosphorylation of the beta 4 cytoplasmic domain, followed by recruitment of the adaptor protein Shc and activation of mitogen-activated protein kinase cascades. We have used Far Western analysis and phosphopeptide competition assays to map the sites in the cytoplasmic domain of beta 4 that are required for interaction with Shc. Our results indicate that, upon phosphorylation, Tyr1440, or secondarily Tyr1422, interacts with the SH2 domain of Shc, whereas Tyr1526, or secondarily Tyr1642, interacts with its phosphotyrosine binding (PTB) domain. An inactivating mutation in the PTB domain of Shc, but not one in its SH2 domain, suppresses the activation of Shc by alpha 6beta 4. In addition, mutation of beta 4 Tyr1526, which binds to the PTB domain of Shc, but not of Tyr1422 and Tyr1440, which interact with its SH2 domain, abolishes the activation of ERK by alpha 6beta 4. Phenylalanine substitution of the beta 4 tyrosines able to interact with the SH2 or PTB domain of Shc does not affect incorporation of alpha 6beta 4 in the hemidesmosomes of 804G cells. Exposure to the tyrosine phosphatase inhibitor orthovanadate increases tyrosine phosphorylation of beta 4 and disrupts the hemidesmosomes of 804G cells expressing recombinant wild type beta 4. This treatment, however, exerts a decreasing degree of inhibition on the hemidesmosomes of cells expressing versions of beta 4 containing phenylalanine substitutions at Tyr1422 and Tyr1440, at Tyr1526 and Tyr1642, or at all four tyrosine phosphorylation sites. These results suggest that beta 4 Tyr1526 interacts in a phosphorylation-dependent manner with the PTB domain of Shc. This event is required for subsequent tyrosine phosphorylation of Shc and signaling to ERK but not formation of hemidesmosomes.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Basement membranes regulate the survival, proliferation, and differentiation of cells through ligation of integrin receptors (1-4). The alpha 6beta 4 integrin is a major receptor for the basement membrane component laminin-5 and is expressed in a variety of epithelial cells, in Schwann cells, in certain endothelial cells, and in CD4- CD8- T cells (5, 6). The cytoplasmic domain of beta 4, which is unusually long and dissimilar in amino acid sequence from the corresponding portions of other integrin beta  subunits, enables alpha 6beta 4 to recruit the adaptor protein Shc as well as to promote the assembly of hemidesmosomes (5, 6).

Shc is an SH2/PTB1 domain adaptor protein that couples a variety of receptor and nonreceptor tyrosine kinases, cytokine receptors, immune receptors, and adhesion receptors to Ras signaling (7, 8). In most cases, Shc binds to upstream tyrosine phosphorylated molecules through its SH2 domain, PTB domain, or both. It is then phosphorylated on tyrosine and recruits the Grb2/SOS complex, which can subsequently activate Ras. By this mechanism, Shc participates in mediating the proliferative functions of many receptors. In addition, it regulates cell migration (9-11). Although many receptor tyrosine kinases must interact directly with Shc to induce its tyrosine phosphorylation and activation, the EGF receptor is capable of signaling through Shc even when prevented from binding to it (12, 13). In addition, whereas alpha 6beta 4 can interact directly with Shc (14), a subset of beta 1 and alpha v integrins recruit Shc indirectly through Src family kinases (15, 16).

Dominant negative studies suggest that Shc is required to couple the alpha 6beta 4 integrin to Ras and thereby both the Raf-ERK and Rac-JNK signaling cascades. Through these pathways, alpha 6beta 4 cooperates with growth factor receptors to promote immediate-early gene expression and progression through the G1 phase of the cell cycle (17). Mice carrying a targeted deletion of the cytoplasmic domain of beta 4 display proliferative defects in the skin and gastro-intestinal tract, suggesting that signaling pathways activated by the cytoplasmic domain of beta 4, probably through Shc, are required for optimal epithelial cell proliferation in vivo (18).

In addition to its signaling function, the cytoplasmic domain of beta 4 plays a crucial role in the assembly of hemidesmosomes (18, 19). The hemidesmosomes are adhesive junctions that mediate stable attachment of stratified and transitional epithelia to the basement membrane. They differ from focal adhesions because they are linked to the keratin instead of the actin cytoskeleton. In accordance with the role of hemidesmosomes in mediating strong adhesion to the basement membrane, defects in hemidesmosome integrity cause epidermal fragility and skin blistering (6, 20). The assembly of hemidesmosomes is likely to require interaction of the cytoplasmic domain of beta 4 with HD1/plectin and BPAG2 (21-23).

Recent studies have indicated that ligation of alpha 6beta 4 promotes phosphorylation of the beta 4 cytoplasmic domain through activation of an integrin-associated Src family kinase.2,3 Because Src kinases are known to activate Shc (24), it is possible that alpha 6beta 4 activates Shc through the integrin-associated Src kinase independently of direct binding of Shc to the cytoplasmic domain of beta 4. We have thus examined whether alpha 6beta 4-mediated Shc signaling requires direct binding of Shc to the beta 4 cytoplasmic domain. Our results indicate that the SH2 and PTB domain of Shc bind to separate phosphotyrosines in the cytoplasmic domain of beta 4. The interaction mediated by the PTB domain is essential for Shc signaling to ERK in vivo, whereas that mediated by the SH2 domain is dispensable. In addition, we provide evidence that phosphorylation of the Shc binding sites in beta 4 antagonizes formation of hemidesmosomes.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Immunochemical Reagents-- The rabbit polyclonal antibody and the mAb against the extracellular domain of beta 4 (3E1) were characterized previously (17, 25). The rabbit polyclonal antibodies to GST were affinity purified on a GST-Sepharose column from sera of rabbits immunized with GST fusion proteins. The biotinylated anti-phosphotyrosine mAb 4G10 and protein A-agarose were purchased from Upstate Biotechnologies. The recombinant anti-phosphotyrosine antibody RC20 was from Transduction Laboratories. The anti-Myc tag mAb 9E10 was obtained from the Sloan-Kettering Hybridoma Facility and horseradish peroxidase (HRP)-conjugated mAb 9E10 from Roche Molecular Biochemicals. The mouse monoclonal antibody M2 to FLAG tag was from Kodak. The rabbit polyclonal antibodies to phospho-ERK were purchased from New England Biolabs. The rabbit polyclonal antibodies to ERK2 and the Myc tag were purchased from Santa Cruz. Unconjugated rabbit and horse anti-mouse secondary antibodies for cross-linking and protein G-agarose were from Pierce. FITC-conjugated anti-mouse IgGs were purchased from Molecular Probes. HRP-conjugated protein A and streptavidin were purchased from Amersham Pharmacia Biotech.

Constructs-- cDNAs encoding human alpha 6 and wild type and mutant versions of beta 4 were subcloned into the EcoRI site of the pRK5 expression vector. The generation of cDNAs encoding the canonical form of wild-type beta 4, A, and the mutant versions C, E (17, 19), G, Y1422F, Y1440F, and Y1422F/Y1440F (14) were described previously. beta 4 Y1422F/Y1440F without the Gap was generated by replacing the BssHII fragment containing the Gap with the BssHII fragment from wild type beta 4. The Gap construct was generated by replacing the BssHII fragment from wild type beta 4 with the BssHII fragment containing the Gap. The beta 4 construct I was generated by digesting pRC/CMV beta 4 A with NotI and SacI followed by blunt end ligation. The beta 4 construct J was generated by digesting pRC/CMV beta 4 G with NotI and XbaI followed by blunt end ligation, which created an in-frame stop codon. The beta 4 construct M was generated by digesting pRC/CMV beta 4 E with NotI and XbaI followed by blunt end ligation, resulting in an in-frame stop codon. pRK5 beta 4 Y1526F and Y1642F were generated by two-step polymerase chain reaction of fragments comprised between the NotI and EcoRV or EcoRV and XbaI sites of pRK5 beta 4 A. Double and quadruple tyrosine to phenylalanine mutants of beta 4 were then generated by shuffling fragments of NotI/EcoRV, EcoRV/XbaI, or NotI/XbaI. For stable transfection in 804G cells, beta 4 cDNAs were subcloned into the EcoRI site of pcDNA3. Expression vectors encoding Myc-tagged wild type and mutant murine p52 Shc proteins containing mutations in either the PTB domain (F198V) or SH2 domain (R397K) or both domains (R397K and F198V), were provided by Dr. Ben Margolis and were described previously (13). Expression vectors encoding FLAG-tagged dominant negative Shc (Y 239/317 F) were described previously (16). Plasmids encoding GST-Shc SH2 or PTB (residues 1-209) domains were described previously (26). Mutant versions of the GST-Shc SH2 (R397K) and PTB (F198V) domains were kindly provided by Dr. Ben Margolis. Correctness of all newly generated vectors was verified by sequencing.

Cell Culture and Transfections-- 293T cells were provided by Dr. David Levy (New York University School of Medicine) and cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) on dishes coated with gelatin and transfected by the calcium phosphate method with equimolar amounts of pRK5 alpha 6 and beta 4. Immortalized human keratinocytes (HaCat) were cultured in DMEM with 10% FBS. Human umbilical vein endothelial cells (HUVECs) were purchased from VEC Technologies and cultured on gelatin-coated dishes in endothelial cell SFM (Life Technologies, Inc.) supplemented with 20% FBS (Life Technologies, Inc.), 10 ng/ml EGF, 20 ng/ml basic fibroblast growth factor, and 1 µg/ml Heparin (Intergen). HUVECs were electroporated at 300 V and 450 microfarads with equimolar amounts of pRK5 alpha 6 and beta 4 supplemented with pBS to a total of 30 µg of DNA. HeLa cells were purchased from American Type Culture Collection and cultured in DMEM with 10% FBS. For transient transfection, 4.5 × 106 cells were plated on 15-cm-diameter plates for 24 h and then transfected with 20 µg of DNA and 100 µl of Lipofectin (Life Technologies, Inc.) in a total of 10 ml of DMEM for 5 h, according to manufacturer's recommendations. Cells were allowed to recover for 24 h before serum starvation. Rat bladder carcinoma 804G cells (27) were cultured in DMEM with 10% FBS and transfected by electroporation at 260 V and 975 microfarads in 300 µl of PBS, 1.25% Me2SO with 10 µg of pcDNA3 beta 4 and 10 µg of pBS. They were then replated onto gelatin-coated dishes in DMEM 10% FBS containing 1.25% Me2SO and cultured for additional 24 h. Stably transfected cells were selected with 400 µg/ml G418 (Life Technologies, Inc.) and maintained in 200 µg/ml G418. Clones were pooled and beta 4 expressing cells were selected by detaching cells with PBS/2 mM EDTA until cells rounded followed by brief treatment with 0.2% trypsin/EDTA and collection in DMEM, 10% FBS. Cells were then panned for 5 min at 37 °C over dishes coated overnight at 4 °C with 20 µg/ml 3E1 mAb followed by blocking with 1% heat-inactivated BSA. Adherent cells were allowed to grow until confluency. Cells expressing equivalent levels of human recombinant beta 4 were obtained by fluorescence-activated cell sorting with the 3E1 mAb.

Fusion Proteins-- GST fusion proteins were expressed in BL21-RES cells (Stratagene) treated with 0.1 mM isopropyl-1-thio-beta -D-galactopyranoside for 3 h at 37 °C and purified on glutathione-agarose as described previously (28). Proteins were eluted by incubating glutathione-agarose beads three times for 5 min at room temperature with an equal volume of 100 mM Tris, pH 8.5, 120 mM sodium chloride, 0.1% Triton X-100, and freshly added 20 mM glutathione. Aliquots were stored frozen at -20 °C until use.

Far Western Analysis-- After overnight serum starvation, 293T cells were left untreated or treated for 5 min at 37 °C with 100 µM orthovanadate and 3 mM hydrogen peroxide, washed with ice-cold PBS, and lysed in lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EGTA, 5 mM EDTA) supplemented with phosphatase inhibitors (1 mM sodium orthovanadate, 5 mM sodium pyrophosphate, 25 mM sodium fluoride) and protease inhibitors (10 µg/ml pepstatin A, aprotinin, and leupeptin and 0.4 mM 4-(2-aminoethyl)-benzene sulfonylfluoride). Aliquots containing 1 mg of total proteins were immunoprecipitated for 3 h with 5 µg of 3E1 plus 30 µl of packed protein-G agarose. Immunocomplexes were separated by SDS-PAGE and transferred to nitrocellulose. Membranes were blocked with TBS, 0.1% Tween (TBST) containing 5% milk for 1 h at room temperature and incubated with 1 µg/ml GST Shc SH2 or PTB domain fusion proteins in TBST-2.5% milk, 1 mM dithiothreitol, 10 µg/ml aprotinin, leupeptin, and pepstatin A. After three washes with TBST, the membranes were incubated with 1 µg/ml anti-GST rabbit polyclonal antibody in TBST-2.5% milk, washed again, and then incubated with protein A-HRP in TBST-5% milk. After five washes with TBST and two washes with TBST-0.2% Triton X-100, the membranes were rinsed with TBS and incubated with ECL for 1 min before exposure to film. To assess phosphorylation of beta 4, the membranes were stripped for 30 min at 50 °C in 62.5 mM Tris, pH 6.7, 2% SDS, and 100 mM beta -mercaptoethanol, rinsed extensively with TBS, and then probed by immunoblotting with RC20 according to the manufacturer's recommendations. To control for equal levels of beta 4, membranes were stripped again, blocked with TBST, 5% milk and probed by immunoblotting with 1 µg/ml anti-beta 4 exo in TBS, 3% BSA followed by protein A-HRP.

Peptide Competition Assay-- HaCat cells were serum starved and stimulated as described above. beta 4 was immunoprecipitated with the 3E1 mAb from lysates containing 2 mg of total proteins and transferred to nitrocellulose. Membranes were cut, and Far Western blotting was performed as above in the absence or presence of various concentrations of peptides. Purified phosphorylated (pY) or nonphosphorylated peptides were produced by the Microchemistry Core Facility of Sloan-Kettering Institute. They included beta 4 pY1422 (LTRDpYNSLTRSE), beta 4 pY1440 (LPRDpYSTLTSVS), beta 4 pY1526 (DLLPNHSpYVFRV), beta 4 Y1526 (DLLPNHSYVFRV), and beta 4 pY1642 (GLSENVPpYKFKV). Phosphopeptides modeled after the high affinity binding site for the SH2 domain of Shc at Tyr579 in the platelet-derived growth factor receptor (DGHEpYIYVDPMQ) and after the high affinity binding site for the PTB domain of Shc in the middle T antigen (SLLSNPTpYSVMR) (29, 30) were used as controls.

Immunoprecipitation and Immmunoblotting-- To examine tyrosine phosphorylation of Shc, the cells were serum starved, detached with 2 mM EDTA, washed with DMEM, 0.2% BSA, resuspended at 107/ml in DMEM, 0.2% BSA, and subdivided in 300-µl aliquots. Cells were incubated on ice for 40 min with 10 µg of anti-beta 4 mAb 3E1, washed with 700 µl of cold DMEM, resuspended in 200 µl of cold DMEM containing 5 µg of horse anti-mouse IgG, and incubated at 37 °C for 5 min. After one wash with cold PBS, the cells were pelleted and lysed in 800 µl of lysis buffer with phosphatase and protease inhibitors. Recombinant Shc proteins were immunoprecipitated with a rabbit anti-Myc polyclonal antibody, separated by SDS-PAGE, and transferred to nitrocellulose. The membranes were probed by immunoblotting with 1 µg/ml of biotin-conjugated 4G10 followed by streptavidin-HRP. To examine tyrosine phosphorylation of beta 4, HUVECs were transfected with mutant versions of beta 4 as described above, allowed to recover for 24 h, serum starved overnight, and treated with 1 µM sodium orthovanadate and 3 mM hydrogen peroxide. After extraction in lysis buffer, beta 4 was immunoprecipitated with 5 µg of 3E1 mAb and 30 µl of protein G-agarose, and probed by immunoblotting with RC20.

Mitogen-activated Protein Kinase Assay-- Dishes were coated with 10 µg/ml rabbit anti-mouse IgGs for 2 h at room temperature, saturated with 0.5% heat-denatured BSA (fatty acid, globulin-free), and then incubated with 20 µg/ml 3E1 mAb overnight at 4 °C. Serum starved cells were detached with 2 mM EDTA, washed with DMEM, 0.2% BSA, and kept in suspension in DMEM, 0.2% BSA for 1 h at 37 °C. They were then plated onto 3E1-coated dishes for indicated times at 37 °C and lysed in RIPA buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 10% glycerol, 1 mM EGTA, 4 mM EDTA, and phosphatase and protease inhibitors). Aliquots containing 20 µg of total proteins were separated by SDS-PAGE and probed by immunoblotting with an antibodies to phospho-ERK.

Immunofluorescence-- 804G cells were cultured on glass coverslips for 24 h in DMEM, 10% FBS and then overnight in DMEM, 1% FBS in the presence of the indicated concentrations of sodium orthovanadate. The cells were fixed with methanol at -20 °C for 20 min and stained with 10 µg/ml 3E1 mAb followed by 2 µg/ml FITC-conjugated anti-mouse IgGs. Samples were examined with a Zeiss fluorescent microscope.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of the beta 4 Tyrosine Phosphorylation Sites Required for Interaction with the SH2 and the PTB Domain of Shc-- GST pull-down assays with SDS-denatured extracts have shown that the isolated SH2 and PTB domain of Shc can interact directly with the tyrosine phosphorylated beta 4 subunit in vitro (17). To identify the sequences of beta 4 cytoplasmic domain mediating the interaction with the SH2 and the PTB domain of Shc, we initially transfected 293T cells with the constructs encoding the wild type or deleted beta 4 subunits illustrated in Fig. 1. Antibody or laminin-5-mediated ligation of alpha 6beta 4 promotes tyrosine phosphorylation of beta 4 in vivo, but this event is rapidly reversed, presumably by tyrosine phosphatases. By contrast, treatment of the cells with the tyrosine phosphatase inhibitor pervanadate causes high level and persistent tyrosine phosphorylation of beta 4. We thus used this protocol to increase tyrosine phosphorylation of beta 4 in mapping experiments.



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Fig. 1.   Structure of integrin beta 4 constructs. The features of wild type (A) and mutant forms of human beta 4 used in this study are shown. TM, transmembrane domain; black box, type III Fn-like repeat; gray box, connecting segment; F, phenylalanine substitution. For brevity, some of the mutants are referred to in the text as indicated on the right.

After immunoprecipitation from cells treated with pervanadate, alpha 6beta 4 was separated by SDS-PAGE and probed by Far Western blotting with GST fusion proteins containing the SH2 or PTB domain of Shc or by immunoblotting with anti-phosphotyrosine antibodies. As shown in Fig. 2A, the SH2 domain of Shc bound to wild type beta 4 and to beta 4 mutants containing the connecting segment (E and M) in a tyrosine phosphorylation-dependent manner. It did not, however, bind to mutants lacking the connecting segment (G and I), despite the fact that they were still phosphorylated on tyrosine. We consistently observed that the mutants E and M, which lack the membrane proximal region of the cytoplasmic domain, were phosphorylated to a higher stoichiometry than wild type beta 4. It is possible that the segment deleted in E and M is necessary for efficient association with a tyrosine phosphatase able to reverse beta 4 phosphorylation. Alternatively, the deletion may bring some of the beta 4 tyrosine phosphorylation sites in closer proximity to the tyrosine kinase that phosphorylates them. The mutants truncated after the second fibronectin (Fn) type III repeat (J and C) did not become phosphorylated on tyrosine. Because these mutants can still associate with Src kinases,2 it is likely that the major tyrosine phosphorylation sites in beta 4 reside downstream of the second Fn type III module. Taken together, these observations suggest that the SH2 domain of Shc binds to phosphorylated tyrosines located within the connecting segment of beta 4.



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Fig. 2.   Far Western analysis indicates that beta 4 Tyr1440 is the major binding site for the SH2 domain of Shc and Tyr1526 is the major binding site for its PTB domain. 293T cells were transiently transfected with alpha 6 and either wild type (A) or the indicated mutant versions of beta 4. Cells were either left untreated (-) or treated with pervanadate (+). After immunoprecipitation, alpha 6beta 4 was separated by SDS-PAGE and transferred to nitrocellulose. Membranes were incubated with GST fusion proteins comprising the SH2 (A) or PTB domain of Shc (B), followed by immunoblotting with anti-GST antibodies (top panels). Membranes were stripped and reprobed with antibodies against phosphotyrosine (middle panels) and then stripped again and reprobed with antibodies against the extracellular domain of beta 4 (bottom panels).

The connecting segment contains two potential tyrosine phosphorylation sites that conform to the consensus for binding to the SH2 domain of Shc (31). We thus mutated these two tyrosines (Tyr1422 and Tyr1440) to phenylalanine either separately or in combination. The resulting versions of beta 4 were tested for their ability to bind the SH2 domain of Shc. Fig. 2A shows that the SH2 domain of Shc does not bind to beta 4 Y1422F/Y1440F and binds to a modest extent to beta 4 Y1440F. By contrast, the SH2 domain of Shc interacts effectively with beta 4 Y1422F. These results suggest that the SH2 domain of Shc binds primarily to phosphorylated Tyr1440 and secondarily to phosphorylated Tyr1422 in the connecting segment of beta 4.

The PTB domain of Shc binds to phosphorylated tyrosines in the context of NXXY motifs (24, 29). The cytoplasmic domain of beta 4 contains three NXXY motifs: one in the region between the transmembrane domain and the first Fn type III module and the other two downstream of the connecting segment, one in the third and the other in the fourth Fn type III repeat. As shown in Fig. 2B, the PTB domain of Shc bound to phosphorylated beta 4 mutant E as effectively as to phosphorylated wild type beta 4, but it did not interact with phosphorylated mutant M. This result suggests that the major binding site for the PTB domain of Shc in beta 4 resides downstream of the connecting segment and possibly corresponds to the NXXY motif in the third or that in the fourth Fn type III repeat. Therefore, versions of beta 4 containing phenylalanine substitutions of the tyrosines within each of these two NXXY sites (Tyr1526 and Tyr1642) were analyzed by Far Western blotting with the Shc PTB domain (Fig. 2B). Phenylalanine substitution of either Tyr1526 or Tyr1642 decreased only partially binding of the PTB domain to beta 4, with substitution of Tyr1526 resulting in a significantly greater reduction. Mutation of both sites in combination (Y1526F/Y1642F) completely prevented the binding of PTB domain to beta 4. These results suggest that the PTB domain of Shc binds primarily to Tyr1526 and secondarily to Tyr1642 in beta 4.

Phosphopeptide competition assays were performed to compare the relative affinities of the potential binding sites for the SH2 or PTB domain of Shc in beta 4 (see Table I for peptide sequences). For these experiments, we used HaCat keratinocytes, which express endogenous alpha 6beta 4. Cells were either left untreated or treated with pervanadate. After immunoprecipitation, beta 4 was transferred to nitrocellulose and probed with GST fusion proteins containing the SH2 or PTB domain of Shc in the absence or presence of tyrosine phosphorylated synthetic peptides modeled after the sequences surrounding Tyr1422, Tyr1440, Tyr1526, or Tyr1642 in beta 4 (Table I). As positive controls, we used tyrosine phosphorylated peptides reproducing the high affinity binding sites for the SH2 and PTB domain of Shc in the platelet-derived growth factor receptor and middle T antigen, respectively (29, 30).


                              
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Table I
Sequences of peptides used in competition studies

As shown in Fig. 3A, the peptides beta 4 pY1422 and beta 4 pY1440 inhibited the binding of the SH2 domain of Shc to wild type beta 4 to a similar extent, suggesting that Tyr1422 and Tyr1440 are both potential binding sites for the SH2 domain of Shc. It is likely that the SH2 domain of Shc binds preferentially to Tyr1440 (Fig. 2B) because this tyrosine is more efficiently phosphorylated than Tyr1422 in vivo (14, 17). Tyr1440 may thus be the main physiologic binding site for the SH2 domain of Shc in beta 4. Neither beta 4 Tyr(P)1526 nor a nonphosphorylated peptide (beta 4 Tyr1526) were able to inhibit the binding of the SH2 domain of Shc to beta 4.



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Fig. 3.   Phosphopeptides modeled after beta 4 sequences comprising Tyr1422 and Tyr1440 inhibit binding of beta 4 to the SH2 domain of Shc, whereas a phosphopeptide including Tyr1526 prevents interaction with the PTB domain. HaCat cells were either left untreated (-) or treated with pervanadate (+). After immunoprecipitation, alpha 6beta 4 was transferred to nitrocellulose. Pieces of membrane containing individual bands of phosphorylated beta 4 were incubated with GST-Shc-SH2 (A) or GST-Shc-PTB (B) in the absence or presence of 100 µM of the indicated phosphorylated and nonphosphorylated peptides (top panels). Membranes were stripped and reprobed with anti-phosphotyrosine antibodies (middle panels) and then stripped again and reprobed with antibodies against the extracellular domain of beta 4 (bottom panels).

Fig. 3B shows that the phosphorylated peptide beta 4 Tyr(P)1526, but not beta 4 Tyr(P)1642, inhibited the binding of the PTB domain of Shc to wild type beta 4. Unphosphorylated beta 4 Tyr1526 as well as phosphorylated beta 4 Tyr(P)1440 had no effect. Together with those of mutational analysis (Fig. 2B), these results indicate that Tyr1526 is the primary binding site for the PTB domain of Shc in beta 4.

Signaling by alpha 6beta 4 Requires the PTB, but Not SH2, Domain of Shc-- To examine whether Shc signaling by alpha 6beta 4 required direct interaction of the SH2 and/or PTB domain of Shc with the beta 4 cytoplasmic domain, we used versions of Shc carrying inactivating mutations in either the SH2 domain (R397K), PTB domain (F198V), or both domains. The SH2 domain mutation resides within the conserved FLVRES motif and prevents the interaction with phosphotyrosine, whereas the PTB domain mutation prevents interaction with the hydrophobic residue at position -5 and with the asparagine at position -3 in the Psi XNXXY motif (32). As shown in Fig. 4A, each of these mutations prevented the binding of a GST fusion protein containing the corresponding domain of Shc to tyrosine phosphorylated beta 4 in vitro (Fig. 4A).



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Fig. 4.   alpha 6beta 4-mediated phosphorylation of Shc requires an intact PTB, but not SH2, domain. A, mutations that inactivate the SH2 or PTB domain of Shc abolish binding of the corresponding domain to beta 4 in vitro. HaCat cells were either left untreated (-) or treated with pervanadate (+). After immunoprecipitation, alpha 6beta 4 was transferred to nitrocellulose. Pieces of membrane containing bands of phosphorylated beta 4 were probed with wild type (WT) or mutant (R397K) GST-Shc-SH2 domain or with wild type or mutant (F198V) GST-Shc-PTB domain (top panels). Membranes were then stripped and reprobed with anti-phosphotyrosine antibodies (middle panels) and then stripped again and reprobed with antibodies against the extracellular domain of beta 4 (bottom panels). B, an inactivating mutation in the PTB domain of Shc, but not one in its SH2 domain, prevents phosphorylation of Shc upon ligation of alpha 6beta 4. HeLa cells were transiently transfected with Myc-tagged versions of wild type Shc or versions of Shc carrying mutations in the SH2 domain (R397K), the PTB domain (F198V), or both (F198V/R397K). Cells were incubated in suspension with the anti-beta 4 mAb 3E1 followed by anti-mouse IgGs. Control cells (lanes C) were stimulated with secondary anti-mouse IgGs alone. After immunoprecipitation, the Myc-tagged recombinant proteins were probed with anti-phosphotyrosine antibodies (top panel). The membranes were then stripped and reprobed with anti-Myc antibodies (bottom panel). C, as a control, adherent cells were either left untreated (-) or were treated with 250 ng/ml EGF for 5 min (+). After immunoprecipitation, the Myc-tagged recombinant proteins were probed with anti-phosphotyrosine antibodies (top panel). The membranes were then stripped and reprobed with anti-Myc antibodies (bottom panel). EGF receptor-mediated phosphorylation of Shc is not prevented by mutation of the SH2 domain of Shc, its PTB domain, or both, as reported previously.

We examined the ability of alpha 6beta 4 to activate in vivo versions of Shc containing inactivating mutations in either or both the SH2 and PTB domains. HeLa cells were transfected with Myc-tagged wild type or mutant versions of Shc. HeLa cells were chosen because they are easily transfectable, express endogenous alpha 6beta 4, and were used previously to study alpha 6beta 4 signaling (17) as well as EGF receptor activation of the same Shc mutants (13). To specifically ligate alpha 6beta 4, cells were incubated in suspension with the anti-beta 4 mAb 3E1, followed by an anti-mouse secondary antibody. The recombinant Shc proteins were immunoprecipitated with an anti-Myc antibody and analyzed by immunoblotting with anti-phosphotyrosine antibodies. As shown in Fig. 4B, both wild type and SH2 mutant Shc (R397K) were efficiently phosphorylated on tyrosine in response to ligation of alpha 6beta 4. By contrast, the PTB mutant (F198V) and double mutant (F198V/R397K) versions of Shc were not phosphorylated on tyrosine upon alpha 6beta 4 stimulation. All four versions of Shc were efficiently phosphorylated upon treatment of the cells with EGF (Fig. 4C), as shown previously (13). These results indicate that a functional PTB domain is necessary for alpha 6beta 4-mediated Shc signaling and imply that Shc has to bind to beta 4 through this domain to become phosphorylated on tyrosine by the alpha 6beta 4-associated kinase.

Signaling by alpha 6beta 4 Requires Interaction with the PTB, but Not SH2, Domain of Shc-- In HeLa cells, alpha 6beta 4 signaling to ERK proceeds through Shc (17). We thus examined whether the binding of Shc to beta 4 was required also for activation of ERK. To test this hypothesis, we chose to use primary HUVECs because they do not express alpha 6beta 4 and can therefore be transfected with various mutant versions of beta 4. These cells offer a better model for examining alpha 6beta 4 signaling than fibroblastic cells because alpha 6beta 4 is expressed in certain endothelial cells in vivo (5, 6). In addition, because ERK is often constitutively activated in established cell lines, nonimmortalized cells such as the HUVECs enable a more accurate assessment of the activation of ERK.

To verify that alpha 6beta 4 activated ERK in a Shc-dependent manner in HUVECs, these cells were transfected with constructs encoding alpha 6beta 4 alone or in combination with increasing doses of a FLAG-tagged version of dominant negative Shc carrying phenylalanine permutations at both potential Grb2 binding sites. After serum starvation, the cells were detached and replated onto dishes coated with the anti-beta 4 mAb 3E1. As shown in Fig. 5A, dominant negative Shc inhibited alpha 6beta 4-induced activation of ERK in a dose-dependent manner, suggesting that alpha 6beta 4 signaling to ERK proceeds through Shc also in HUVECs.



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Fig. 5.   alpha 6beta 4-mediated activation of ERK requires phosphorylation of beta 4 Tyr1526, which mediates interaction with the PTB domain of Shc. A, dominant negative Shc inhibits activation of ERK by alpha 6beta 4 in HUVECs. HUVECs were transiently transfected with wild type alpha 6 and beta 4 in combination with increasing amounts of vector encoding FLAG-tagged dominant negative (Dn) Shc (7.5, 15, and 30 µg). Cells were detached and replated for 60 min on dishes coated with the anti-beta 4 mAb 3E1. Because dishes were post-coated with BSA, only the cells expressing alpha 6beta 4 (approximately 30%) attached. Total proteins from cells in suspension (lane S) or adhering to the 3E1 mAb (3E1) were probed with antibodies against phosphorylated ERK (top panel) or the FLAG epitope (middle panel). Blots were stripped and reprobed with antibodies against total ERK (bottom panel) as control. B, phenylalanine substitution of Tyr1440 and Tyr1526 inhibits vanadate-induced phosphorylation of beta 4. HUVECs were transiently transfected with alpha 6 and either wild type (A) or the indicated mutant versions of beta 4 (Y1422F, Y1440F, Y1526F, Y1642F, and 4F). Cells were either left untreated (-) or treated with pervanadate (+) and lysed. After immunoprecipitation, alpha 6beta 4 was separated by SDS-PAGE and probed by immunoblotting with antibodies against phosphotyrosine (top panel) and then stripped and reprobed with antibodies against the extracellular domain of beta 4 (bottom panel). C, a version of beta 4 containing phenylalanine substitutions at the PTB domain binding sites (Y1526F/Y1642F) does not activate ERK as efficiently as wild type beta 4 or a version with phenylalanine substitutions at the SH2 domain binding sites (Y1422F/Y1440F). HUVECs were transfected with alpha 6 together with wild type (A) or the indicated mutant forms of of beta 4 (Y1526F/Y1642F and Y1422F/Y1440F). Total proteins from cells in suspension or plated for the indicated times on dishes coated with the 3E1 mAb were probed by immunoblotting with antibodies against beta 4 (top panel) or phosphorylated ERK (middle panel). The blot was stripped and re-probed with an antibody to total Erk2 (bottom panel) as control. D, phenylalanine substitution of the primary PTB domain binding site in beta 4 prevents Erk activation. HUVECs transfected with alpha 6 together with wild type (A) or the indicated mutant versions of beta 4 (Y1526F, Y1642F, Y1526F/Y1642F, L) were plated on 3E1-coated dishes for 60 min. Total proteins were probed by immunoblotting with antibodies against beta 4 (top panel) or phosphorylated ERK (middle panel). The blot was then stripped and reprobed with an antibody recognizing total Erk2 (bottom panel).

We next analyzed the relative efficiency of phosphorylation of the beta 4 tyrosines involved in binding to the SH2 and PTB domain of Shc. Previous studies using phosphopeptide mapping had identified Tyr1440 as a major tyrosine phosphorylation site in beta 4 and revealed that beta 4 is phosphorylated at several additional tyrosines in vivo (14, 17). HUVECs were transfected with mutant forms of beta 4 carrying phenylalanine substitutions at each one or all four tyrosines (Tyr1422, Tyr1440, Tyr1526, and Tyr1642) capable of binding to Shc and stimulated with pervanadate (Fig. 5B). The phosphorylation of beta 4 was analyzed by immunoblotting with antibodies against phosphotyrosine. Phenylalanine substitution of either Tyr1440 or Tyr1526 resulted in a significant decrease in total phosphorylation of beta 4, whereas phenylalanine substitutions at Tyr1422 or Tyr1642 had a negligible effect. Mutation of all four tyrosines to phenylalanine (4F) resulted in an almost complete block in tyrosine phosphorylation. With the caveat that the anti-phosphotyrosine antibodies may not interact with equal affinity with all phosphorylated tyrosines in beta 4, these results suggest that Tyr1440 and Tyr1526 are the major phosphorylation sites in beta 4.

To examine whether the interaction of Shc with beta 4 was required for signaling to ERK, the HUVECs were transfected with constructs encoding alpha 6 in combination with versions of beta 4 unable to bind to either the SH2 domain (Y1422F/Y1440F) or the PTB domain (Y1526F/Y1642F) of Shc. Cells were then replated onto 3E1-coated dishes and analyzed by immunoblotting with anti-phospho-ERK antibodies. As shown in Fig. 5C, ligation of beta 4 Y1422F/Y1440F caused activation of ERK, although slightly less efficiently than ligation of wild type beta 4, whereas ligation of beta 4 Y1526F/Y1642F did not induce activation of ERK. Immunoblotting with a polyclonal antibody against the extracellular domain of beta 4 (Fig. 5C, top panel) and fluorescence-activated cell sorting analysis (data not shown) confirmed that the different versions of beta 4 were expressed at comparable levels. These results provide evidence that alpha 6beta 4 signaling to ERK requires interaction of the PTB domain of Shc with beta 4. This finding is consistent with the observation that alpha 6beta 4 signaling to ERK proceeds through Shc (Fig. 5B) and the activation of Shc by alpha 6beta 4 requires a functional PTB domain (Fig. 4B).

Finally, we examined the relative importance of the two potential PTB binding sites in beta 4 for activation of ERK. As shown in Fig. 5D, ligation of beta 4 Y1526F did not cause activation of ERK, whereas ligation of beta 4 Y1642F induced activation of ERK, although slightly less efficiently than wild type beta 4. These results indicate that beta 4 Tyr1526, the primary binding site for the PTB domain of Shc, is required for activation of ERK.

Mutation of the Major Tyrosine Phosphorylation Sites Does Not Prevent Incorporation of the Canonical Form A of beta 4 into Hemidesmosome-like Adhesions-- The cytoplasmic domain of beta 4, and in particular the segment comprising the first two Fn type III modules and the connecting segment, is required for formation of hemidesmosomes (18, 19). Our initial studies had suggested that Tyr1422 and Tyr1440 (which resemble a tyrosine-based activation motif or TAM) were necessary for incorporation of recombinant beta 4 into the hemidesmosome-like adhesions formed by 804G cells in culture (14). Subsequent studies have, however, indicated that residues C-terminal to residue 1355 in the beta 4 cytoplasmic domain are not required for the formation of hemidesmosome-like adhesions by cultured cells (23, 33). To resolve this discrepancy, we have performed a number of additional studies. Among them, an analysis of the entire coding sequence of the constructs used in our previous study revealed that the beta 4 mutant Y1422F/Y1440F (called the TAM mutant or YZ in (14)) originated from a version of beta 4 that contained an in-frame deletion of the sequences encoding amino acids 880-886 (DHTIVDT). Because this sequence is located in the membrane proximal portion of the cytoplasmic domain of beta 4, which previous studies had indicated to be dispensable for incorporation in hemidesmosomes-like adhesion structures (19, 23), we had not covered it during our initial sequence reanalysis. The nature and origin of the variant cDNA lacking amino acids 880-886 remains to be examined. As shown below, this deletion (termed the Gap) in combination with the Y1422F/Y1440F mutation prevents incorporation of beta 4 into hemidesmosomes in 804G cells, in agreement with our original result.

The 804G cells are a rat bladder carcinoma cell line that expresses endogenous alpha 6beta 4 and forms hemidesmosome-like adhesions in culture. Immunofluorescence staining of hemidesmosomal components reveal that the hemidesmosome-like adhesions of these cells are arranged in a "Swiss cheese" pattern (34). We transfected 804G cells with constructs encoding human versions of either the canonical (A) or various variant forms of beta 4 (Y1422F/Y1440F, Y1526F/Y1642F, 4F, Gap or Gap-Y1422F/Y1440F). Pools of cells stably expressing these recombinant forms of beta 4 (see "Materials and Methods") were stained with the 3E1 mAb that recognizes human but not rat beta 4. As shown in Fig. 6A, the beta 4 variants Y1422F/Y1440F, Y1526F/Y1642F, 4F, and Gap localized to hemidesmosomes as efficiently as wild type beta 4 (A), whereas the beta 4 mutant Gap- Y1422F/Y1440F displayed a significantly decreased ability to localize to hemidesmosomes. Thus, mutation of the potential beta 4 TAM impairs localization of beta 4 to hemidesmosomes only in the context of the Gap version of beta 4. These results indicate that the integrity of Tyr1422 and Tyr1440 is not required for incorporation of beta 4 in hemidesmosome-like adhesions, as shown previously by others (23). However, the synergy between the TAM mutation and the Gap suggests that Tyr1422 and Tyr1440 may play a role in assembly of hemidesmosomes. It is possible that a full assessment of this role requires examination of the assembly of bona fide hemidesmosomes in skin organ culture systems or in vivo.



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Fig. 6.   Tyrosine phosphorylation of the beta 4 cytoplasmic domain antagonizes assembly of hemidesmosomes. A, 804G cells expressing either wild type (A) or the indicated mutant versions of beta 4 (Y1422/1440F, Y1526/1642F, 4F, Gap, Gap-Y1422/1440F) were cultured on coverslips for 2 days, fixed, and stained with the anti-beta 4 mAb 3E1 followed by FITC-conjugated anti-mouse IgGs. B, phenylalanine substitution of the major tyrosine phosphorylation sites in beta 4 antagonizes the disruption of hemidesmosomes caused by orthovanadate. 804G cells expressing either wild type (A) or the indicated mutant versions of beta 4 (Y1422/1440F, Y1526/1642F, and 4F) were cultured for 24 h and then serum starved overnight in the presence of 20 or 100 µM orthovanadate. Cells were fixed and stained with 3E1 mAb followed by FITC-conjugated anti-mouse IgGs.

Phosphorylation of the beta 4 Tyrosines Involved in the Recruitment of Shc Antagonizes Assembly of Hemidesmosomes-- Hemidesmosomes are structures that provide stable adhesion of epithelial cells to the underlying basement membranes and are therefore disassembled during cell migration (34, 35). Because phenylalanine substitution of the tyrosines that bind to Shc does not prevent incorporation of the canonical form of beta 4 in hemidesmosomes, it is very unlikely that the phosphorylation of these sites participates in the assembly of hemidesmosomes (Ref. 23 and Fig. 6A).

We have previously shown that treatment with EGF induces tyrosine phosphorylation of beta 4 and disassembly of hemidesmosomes in keratinocytes, although we did not establish a cause-effect relationship (36). To examine whether tyrosine phosphorylation of beta 4 causes disassembly of hemidesmosomes, we used 804G cell lines expressing wild type beta 4 (A) or the beta 4 mutants Y1422F/Y1440F, Y1526F/Y1642F, or 4F. Because these cells express very low levels of EGF receptor, they were treated with either 20 or 100 µM orthovanadate for 15 h to increase tyrosine phosphorylation of beta 4. When orthovanadate is added in the absence of hydrogen peroxide, it behaves as a competitive inhibitor of tyrosine phosphatases, whereas in the presence of hydrogen peroxide it is converted to the irreversible inhibitor pervanadate (37). We used orthovanadate for these experiments because prolonged exposure to pervanadate causes cell toxicity. Treatment with 20 µM orthovanadate induced tyrosine phosphorylation of wild type beta 4 but not of the mutant 4F in 804G cells (data not shown). As shown in Fig. 6B, 20 µM orthovanadate caused disruption of hemidesmosomes in 804G cells expressing wild type beta 4 (A) but not in those expressing any of the mutant versions of beta 4 (Y1422F/Y1440F, Y1526F/Y1642F, or 4F). In the presence of 100 µM orthovanadate, most 804G cells expressing wild type beta 4 (A) rounded up and almost completely detached (>90% detached), whereas cells expressing the mutant versions of beta 4 were protected from disruption of hemidesmosomes to varying degrees. Those expressing the beta 4 mutant 4F were the most protected (~20% detached, some remaining hemidesmosomes), whereas those expressing the beta 4 mutant Y1526F/Y1642F were protected to a lesser degree (~30% detached), and those expressing the beta 4 mutant Y1422F/Y1440F were the least protected (~60% detached). Therefore, phosphorylation of these tyrosines can lead to a reduction of hemidesmosomes and may be a physiologic mechanism for regulating hemidesmosome turnover. Treatment with 100 ng/ml EGF for 15 h resulted in a modest and equivalent disruption of hemidesmosomes in all four transfectants (data not shown), suggesting that the partial disassembly of hemidesmosomes caused by EGF in 804G cells may also involve an additional mechanism, as suggested previously (38).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Ligation of the alpha 6beta 4 integrin induces tyrosine phosphorylation of the cytoplasmic domain of beta 4, recruitment of Shc, and activation of Ras-dependent mitogen-activated protein kinase cascades (14, 17). Biochemical and genetic evidence implies that alpha 6beta 4-dependent signaling promotes proliferation of both keratinocytes and intestinal epithelial cells (17, 18). To assess the specific role of Shc in these processes, it is necessary to identify the mechanism by which Shc binds to beta 4 and determine whether this binding is required for activation of downstream signaling pathways. The results of this study provide evidence that the PTB domain of Shc interacts in a phosphorylation-dependent manner with Tyr1526 in beta 4, whereas the SH2 domain binds to Tyr1440. Both phenylalanine substitution of Tyr1526 in beta 4 and inactivation of the PTB domain in Shc suppress alpha 6beta 4-mediated phosphorylation of Shc and signaling to ERK. By contrast, mutation of both the primary and secondary binding site for the Shc SH2 domain in beta 4 or inactivation of the SH2 domain itself exert only a minor effect on Shc signaling to ERK. These observations suggest that the binding of the PTB domain of Shc to Tyr1526 in beta 4 is crucial for subsequent phosphorylation of Shc by the alpha 6beta 4-associated kinase and thus activation of Ras-dependent pathways. It remains, however, possible and indeed likely that the interaction mediated by the SH2 domain of Shc contributes to some extent to the stability of the association of Shc with beta 4 in vivo.

The primary beta 4 binding site for the PTB domain of Shc, Tyr1526, is located in the third Fn type III module. Interestingly, all known type III Fn repeats contain a tyrosine at this position. Fn type III repeats are found not only in extracellular matrix proteins (fibronectin and tenascin) and cell surface receptors (for growth hormone, prolactin, and insulin) but also in cytoplasmic components (twitchin, titin, and the beta 4 cytoplasmic domain) (39). To our knowledge, this study is the first to identify a physiologically relevant phosphorylation site in a Fn type III module. Because several of these repeats have been crystallized, including the N-terminal pair in beta 4, it is possible to predict the conformation of beta 4 Tyr1526 and surrounding amino acids. The residues N-terminal to tyrosine 1526 that are also important for interaction with the PTB domain of Shc, namely the asparagine and leucine at the -3 and -5 positions, are predicted to be in a loop between the E and F strands of the beta -sandwich. Based on its location in a loop between beta  strands, this sequence motif is likely to be exposed to solvent in the intact molecule. This position would facilitate its phosphorylation and subsequent interaction with the PTB domain of Shc. In addition, there is evidence suggesting that the C-terminal portion of the beta 4 tail binds to a more proximal segment comprising the second Fn type III repeat and the connecting segment in vitro (23). If this intramolecular interaction occurs also in vivo, it may bring the third Fn type III repeat closer to the plasma membrane and thus facilitate the interaction of the Src family kinase with Tyr1526 in beta 4 and thereby the recruitment of Shc. This mechanism would also enable Shc to position Grb2/SOS in closer proximity to its target Ras.

The SH2 domain of Shc interacts in a phosphorylation-dependent manner primarily with Tyr1440 and secondarily with Tyr1422 in beta 4. In agreement with the observation that these two sites are homologous to one another and both conform well to the consensus for binding to the SH2 domain of Shc (YXXL), our results show that phosphopeptides encompassing both sites inhibit to a similar extent the interaction of the SH2 domain of Shc with beta 4. It is likely that Tyr1440 is more important in both in vitro and in vivo experiments because it is more efficiently phosphorylated (see also Refs. 14 and 17). Based on the observation that phenylalanine substitutions at Tyr1422 and Tyr1440 prevent incorporation of beta 4 in the hemidesmosome-like adhesions of 804G cells, we had made the hypothesis that phosphorylation of both tyrosines, which encompass a potential TAM, be required for assembly of hemidesmosomes (14). This hypothesis now has to be reevaluated. Together with the results of studies by others (21, 23), our current observations indicate that the phosphorylation of Tyr1422 and Tyr1444, as well as that of Tyr1526 and Tyr1642, disrupts hemidesmosomes or inhibits their assembly. The simplest hypothesis is that these tyrosines of beta 4 interact with a component, such as BPAG2 (21, 23) that confers stability to hemidesmosomes. Phosphorylation of these tyrosines would interfere with the association of beta 4 with this component and thus destabilize hemidesmosomes. This model is attractive also because it potentially explains why phenylalanine substitution may exert an effect similar to, although much smaller than, phosphorylation of Tyr1440 and Tyr1422. In particular, a previous study has shown that phenylalanine substitution of these tyrosines reduces association of BPAG2 with hemidesmosomes (21, 23), and we have shown here that it prevents incorporation of a form of beta 4 lacking amino acids 880-886 in the hemidesmosome-like adhesions of 804G cells. Further experiments will be required to test this model.

Our results indicate that tyrosine phosphorylation of beta 4 induces Shc signaling but antagonizes formation of hemidesmosomes, suggesting that these two processes may be mutually exclusive. It has been reported that processing of the alpha 6beta 4 ligand laminin-5 inhibits assembly of hemidesmosomes and promotes cell migration (40-42). It will be interesting to determine whether this process also affects alpha 6beta 4 signaling to Shc. If so, differential processing of laminin-5 may help direct whether alpha 6beta 4 ligation leads to tyrosine phosphorylation and Shc signaling or hemidesmosome formation.

The finding that the region of the beta 4 cytoplasmic domain required for Shc signaling is distinct from that required for hemidesmosome formation may enable further studies on the role of alpha 6beta 4 signaling. For example, analysis of knock-in mice expressing a version of beta 4 unable to signal through Shc but still able to promote assembly of hemidesmosomes will enable a more comprehensive understanding of the physiologic role of alpha 6beta 4 signaling.


    ACKNOWLEDGEMENTS

We thank Ben Margolis and David Levy for reagents and members of our laboratory for discussion. We are grateful to Paul Tempst for assistance in our attempts to map the major tyrosine phosphorylation sites of beta 4 by mass spectrometry.


    FOOTNOTES

* This work was supported by National Institutes of Health Grants R01-CA58976 and P30-CA08748.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Student in the M.D.-Ph.D. Program of New York University School of Medicine.

§ Recipient of a fellowship from INSERM (France).

Supported by National Institutes of Health Postdoctoral Fellowship F32-CA79516.

|| Established Investigator of the American Heart Association. To whom correspondence should be addressed: Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, Box 216, 1275 York Ave., New York, NY 10021. Tel.: 212-639-6998; Fax: 212-794-6236; E-mail: F-Giancotti@ski.mskcc.org.

Published, JBC Papers in Press, October 23, 2000, DOI 10.1074/jbc.M008663200

2 L. Gagnoux-Palacios, M. Dans, W. van t'Hoff, M. Resh, and F. G. Giancotti, manuscript in preparation.

3 A. Mariotti, P. Kedeshian, M. Dans, A.M. Curatola, L. Gagnoux-Palacios, and F. G. Giancotti, manuscript in preparation.


    ABBREVIATIONS

The abbreviations used are: SH2, Src homology 2; PTB, phosphotyrosine binding; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; mAb, monoclonal antibody; GST, glutathione S-transferase; HRP, horseradish peroxidase; FITC, fluorescein isothiocyanate; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; HUVEC, human umbilical vein endothelial cell; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; Fn, fibronectin; TAM, tyrosine-based activation motif.


    REFERENCES
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ABSTRACT
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RESULTS
DISCUSSION
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