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Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin.

Akimov SS, Krylov D, Fleischman LF, Belkin AM - J. Cell Biol. (2000)

Bottom Line: These effects are specific for tissue transglutaminase and are not shared by its functional homologue, a catalytic subunit of factor XIII.Adhesive function of tissue transglutaminase does not require its cross-linking activity but depends on its stable noncovalent association with integrins.Transglutaminase interacts directly with multiple integrins of beta1 and beta3 subfamilies, but not with beta2 integrins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, American Red Cross, Rockville, Maryland 20855, USA.

ABSTRACT
The protein cross-linking enzyme tissue transglutaminase binds in vitro with high affinity to fibronectin via its 42-kD gelatin-binding domain. Here we report that cell surface transglutaminase mediates adhesion and spreading of cells on the 42-kD fibronectin fragment, which lacks integrin-binding motifs. Overexpression of tissue transglutaminase increases its amount on the cell surface, enhances adhesion and spreading on fibronectin and its 42-kD fragment, enlarges focal adhesions, and amplifies adhesion-dependent phosphorylation of focal adhesion kinase. These effects are specific for tissue transglutaminase and are not shared by its functional homologue, a catalytic subunit of factor XIII. Adhesive function of tissue transglutaminase does not require its cross-linking activity but depends on its stable noncovalent association with integrins. Transglutaminase interacts directly with multiple integrins of beta1 and beta3 subfamilies, but not with beta2 integrins. Complexes of transglutaminase with integrins are formed inside the cell during biosynthesis and accumulate on the surface and in focal adhesions. Together our results demonstrate that tissue transglutaminase mediates the interaction of integrins with fibronectin, thereby acting as an integrin-associated coreceptor to promote cell adhesion and spreading.

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tTG amplifies integrin-mediated tyrosine phosphorylation of FAK and colocalizes with β1 integrins at focal adhesions. (A and B) tTG potentiates FAK phosphorylation. (A) Time course of FAK phosphorylation in REF52 cells expressing vector (vect.) or wild-type tTG (tTG), plated on Fn. Cells were either kept in suspension (susp.) or plated on Fn for 45, 90, or 180 min. (B) REF52 cells expressing vector (vect.) or wild-type tTG (tTG), were plated for 3 h on dishes coated with Fn, 110-kD, or 42-kD Fn fragments, polyclonal anti-tTG antibody or laminin (Ln). (A and B) The transfectants were plated on ECM proteins or anti-tTG antibody in serum-free medium in the presence of cycloheximide. The cells were lysed and FAK was immunoprecipitated from cell lysates followed by SDS-PAGE and immunoblotting of the immune complexes for phosphotyrosine with PY20 mAb (Belkin et al. 1996). (C–E) tTG colocalizes with β1 integrins on the cell surface of REF52 fibroblasts and causes enlargement of focal adhesions. (C) Live, nonpermeabilized cells transfected with vector (vect.) or tTG (tTG) were double stained for cell surface tTG with mAb CUB7402 and β1 integrins with hamster mAb HMβ1-1. Note codistribution of these proteins at focal adhesions and much larger size of these structures in tTG transfectants. (D) Formaldehyde-fixed, permeabilized cells transfected with vector or tTG, were double stained for vinculin and actin with mAb 7F9 and rhodamine-phalloidin. Note the increased size of focal adhesions and altered organization of actin bundles in the tTG transfectants. (E) Cells overexpressing tTG were plated for 1 or 2 h on 42-kD Fn fragment, fixed, and then double stained for surface tTG with mAb CUB7402 and β1 integrins with hamster mAb HMβ1-1. Arrows indicate focal adhesion sites. Bar, 20 μM.
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Figure 9: tTG amplifies integrin-mediated tyrosine phosphorylation of FAK and colocalizes with β1 integrins at focal adhesions. (A and B) tTG potentiates FAK phosphorylation. (A) Time course of FAK phosphorylation in REF52 cells expressing vector (vect.) or wild-type tTG (tTG), plated on Fn. Cells were either kept in suspension (susp.) or plated on Fn for 45, 90, or 180 min. (B) REF52 cells expressing vector (vect.) or wild-type tTG (tTG), were plated for 3 h on dishes coated with Fn, 110-kD, or 42-kD Fn fragments, polyclonal anti-tTG antibody or laminin (Ln). (A and B) The transfectants were plated on ECM proteins or anti-tTG antibody in serum-free medium in the presence of cycloheximide. The cells were lysed and FAK was immunoprecipitated from cell lysates followed by SDS-PAGE and immunoblotting of the immune complexes for phosphotyrosine with PY20 mAb (Belkin et al. 1996). (C–E) tTG colocalizes with β1 integrins on the cell surface of REF52 fibroblasts and causes enlargement of focal adhesions. (C) Live, nonpermeabilized cells transfected with vector (vect.) or tTG (tTG) were double stained for cell surface tTG with mAb CUB7402 and β1 integrins with hamster mAb HMβ1-1. Note codistribution of these proteins at focal adhesions and much larger size of these structures in tTG transfectants. (D) Formaldehyde-fixed, permeabilized cells transfected with vector or tTG, were double stained for vinculin and actin with mAb 7F9 and rhodamine-phalloidin. Note the increased size of focal adhesions and altered organization of actin bundles in the tTG transfectants. (E) Cells overexpressing tTG were plated for 1 or 2 h on 42-kD Fn fragment, fixed, and then double stained for surface tTG with mAb CUB7402 and β1 integrins with hamster mAb HMβ1-1. Arrows indicate focal adhesion sites. Bar, 20 μM.

Mentions: Since tTG mediates binding of integrins to Fn, we next analyzed whether tTG functionally collaborates with integrins in adhesion-dependent signal transduction. Analysis of integrin-mediated tyrosine phosphorylation of FAK was performed with REF52 cells overexpressing tTG (Fig. 9A and Fig. B). Tyrosine phosphorylation of FAK was very low in cells in suspension regardless of the levels of tTG expression (Fig. 9 A). A gradual time-dependent increase in FAK phosphorylation was observed in both types of transfectants during adhesion and spreading on Fn, but the phosphotyrosine content of FAK was consistently higher in the tTG-transfected cells (Fig. 9 A). We also compared the extent of FAK phosphorylation in the transfectants plated on different substrata for 3 h (Fig. 9 B). Unlike in the case of Fn, no difference was seen for the two cell lines adhering to 110-kD Fn fragment, despite the substantial levels of FAK phosphorylation in the transfectants. However, adhesion on 42-kD Fn fragment or anti-tTG antibody caused a much greater increase in FAK phosphorylation in cells overexpressing tTG compared with vector-transfected controls. In contrast, the levels of FAK phosphorylation were similar in these cell lines after adhesion on laminin, an ECM protein that does not bind tTG (Aeschlimann and Paulsson 1991). Enzymatically inactive mutant tTG[C277→S] had similar effects on integrin signaling (data not shown). These results demonstrate that association of integrin-bound tTG with the 42-kD Fn fragment is sufficient to trigger tyrosine phosphorylation of FAK even in the absence of direct integrin–ligand interaction. tTG also potentiates integrin-mediated FAK phosphorylation in cells adhering on Fn, showing cooperativity with integrins in outside-in signal transduction.


Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin.

Akimov SS, Krylov D, Fleischman LF, Belkin AM - J. Cell Biol. (2000)

tTG amplifies integrin-mediated tyrosine phosphorylation of FAK and colocalizes with β1 integrins at focal adhesions. (A and B) tTG potentiates FAK phosphorylation. (A) Time course of FAK phosphorylation in REF52 cells expressing vector (vect.) or wild-type tTG (tTG), plated on Fn. Cells were either kept in suspension (susp.) or plated on Fn for 45, 90, or 180 min. (B) REF52 cells expressing vector (vect.) or wild-type tTG (tTG), were plated for 3 h on dishes coated with Fn, 110-kD, or 42-kD Fn fragments, polyclonal anti-tTG antibody or laminin (Ln). (A and B) The transfectants were plated on ECM proteins or anti-tTG antibody in serum-free medium in the presence of cycloheximide. The cells were lysed and FAK was immunoprecipitated from cell lysates followed by SDS-PAGE and immunoblotting of the immune complexes for phosphotyrosine with PY20 mAb (Belkin et al. 1996). (C–E) tTG colocalizes with β1 integrins on the cell surface of REF52 fibroblasts and causes enlargement of focal adhesions. (C) Live, nonpermeabilized cells transfected with vector (vect.) or tTG (tTG) were double stained for cell surface tTG with mAb CUB7402 and β1 integrins with hamster mAb HMβ1-1. Note codistribution of these proteins at focal adhesions and much larger size of these structures in tTG transfectants. (D) Formaldehyde-fixed, permeabilized cells transfected with vector or tTG, were double stained for vinculin and actin with mAb 7F9 and rhodamine-phalloidin. Note the increased size of focal adhesions and altered organization of actin bundles in the tTG transfectants. (E) Cells overexpressing tTG were plated for 1 or 2 h on 42-kD Fn fragment, fixed, and then double stained for surface tTG with mAb CUB7402 and β1 integrins with hamster mAb HMβ1-1. Arrows indicate focal adhesion sites. Bar, 20 μM.
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Figure 9: tTG amplifies integrin-mediated tyrosine phosphorylation of FAK and colocalizes with β1 integrins at focal adhesions. (A and B) tTG potentiates FAK phosphorylation. (A) Time course of FAK phosphorylation in REF52 cells expressing vector (vect.) or wild-type tTG (tTG), plated on Fn. Cells were either kept in suspension (susp.) or plated on Fn for 45, 90, or 180 min. (B) REF52 cells expressing vector (vect.) or wild-type tTG (tTG), were plated for 3 h on dishes coated with Fn, 110-kD, or 42-kD Fn fragments, polyclonal anti-tTG antibody or laminin (Ln). (A and B) The transfectants were plated on ECM proteins or anti-tTG antibody in serum-free medium in the presence of cycloheximide. The cells were lysed and FAK was immunoprecipitated from cell lysates followed by SDS-PAGE and immunoblotting of the immune complexes for phosphotyrosine with PY20 mAb (Belkin et al. 1996). (C–E) tTG colocalizes with β1 integrins on the cell surface of REF52 fibroblasts and causes enlargement of focal adhesions. (C) Live, nonpermeabilized cells transfected with vector (vect.) or tTG (tTG) were double stained for cell surface tTG with mAb CUB7402 and β1 integrins with hamster mAb HMβ1-1. Note codistribution of these proteins at focal adhesions and much larger size of these structures in tTG transfectants. (D) Formaldehyde-fixed, permeabilized cells transfected with vector or tTG, were double stained for vinculin and actin with mAb 7F9 and rhodamine-phalloidin. Note the increased size of focal adhesions and altered organization of actin bundles in the tTG transfectants. (E) Cells overexpressing tTG were plated for 1 or 2 h on 42-kD Fn fragment, fixed, and then double stained for surface tTG with mAb CUB7402 and β1 integrins with hamster mAb HMβ1-1. Arrows indicate focal adhesion sites. Bar, 20 μM.
Mentions: Since tTG mediates binding of integrins to Fn, we next analyzed whether tTG functionally collaborates with integrins in adhesion-dependent signal transduction. Analysis of integrin-mediated tyrosine phosphorylation of FAK was performed with REF52 cells overexpressing tTG (Fig. 9A and Fig. B). Tyrosine phosphorylation of FAK was very low in cells in suspension regardless of the levels of tTG expression (Fig. 9 A). A gradual time-dependent increase in FAK phosphorylation was observed in both types of transfectants during adhesion and spreading on Fn, but the phosphotyrosine content of FAK was consistently higher in the tTG-transfected cells (Fig. 9 A). We also compared the extent of FAK phosphorylation in the transfectants plated on different substrata for 3 h (Fig. 9 B). Unlike in the case of Fn, no difference was seen for the two cell lines adhering to 110-kD Fn fragment, despite the substantial levels of FAK phosphorylation in the transfectants. However, adhesion on 42-kD Fn fragment or anti-tTG antibody caused a much greater increase in FAK phosphorylation in cells overexpressing tTG compared with vector-transfected controls. In contrast, the levels of FAK phosphorylation were similar in these cell lines after adhesion on laminin, an ECM protein that does not bind tTG (Aeschlimann and Paulsson 1991). Enzymatically inactive mutant tTG[C277→S] had similar effects on integrin signaling (data not shown). These results demonstrate that association of integrin-bound tTG with the 42-kD Fn fragment is sufficient to trigger tyrosine phosphorylation of FAK even in the absence of direct integrin–ligand interaction. tTG also potentiates integrin-mediated FAK phosphorylation in cells adhering on Fn, showing cooperativity with integrins in outside-in signal transduction.

Bottom Line: These effects are specific for tissue transglutaminase and are not shared by its functional homologue, a catalytic subunit of factor XIII.Adhesive function of tissue transglutaminase does not require its cross-linking activity but depends on its stable noncovalent association with integrins.Transglutaminase interacts directly with multiple integrins of beta1 and beta3 subfamilies, but not with beta2 integrins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, American Red Cross, Rockville, Maryland 20855, USA.

ABSTRACT
The protein cross-linking enzyme tissue transglutaminase binds in vitro with high affinity to fibronectin via its 42-kD gelatin-binding domain. Here we report that cell surface transglutaminase mediates adhesion and spreading of cells on the 42-kD fibronectin fragment, which lacks integrin-binding motifs. Overexpression of tissue transglutaminase increases its amount on the cell surface, enhances adhesion and spreading on fibronectin and its 42-kD fragment, enlarges focal adhesions, and amplifies adhesion-dependent phosphorylation of focal adhesion kinase. These effects are specific for tissue transglutaminase and are not shared by its functional homologue, a catalytic subunit of factor XIII. Adhesive function of tissue transglutaminase does not require its cross-linking activity but depends on its stable noncovalent association with integrins. Transglutaminase interacts directly with multiple integrins of beta1 and beta3 subfamilies, but not with beta2 integrins. Complexes of transglutaminase with integrins are formed inside the cell during biosynthesis and accumulate on the surface and in focal adhesions. Together our results demonstrate that tissue transglutaminase mediates the interaction of integrins with fibronectin, thereby acting as an integrin-associated coreceptor to promote cell adhesion and spreading.

Show MeSH
Related in: MedlinePlus