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Enhancement of endothelial cell migration and in vitro tube formation by TAP20, a novel beta 5 integrin-modulating, PKC theta-dependent protein.

Tang S, Gao Y, Ware JA - J. Cell Biol. (1999)

Bottom Line: A full-length cDNA encoding a novel 20-kD protein, whose expression was PKCtheta-dependent, was identified in endothelial cells, cloned, characterized, and designated as theta-associated protein (TAP) 20.An antiintegrin alphavbeta5 antibody prevented these TAP20 effects.The interaction between TAP20 and beta5 integrin cytoplasmic domain was demonstrated by protein coprecipitation and immunoblotting.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Division, Department of Medicine, Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA. tang@aecom.yu.edu

ABSTRACT
Migration, proliferation, and tube formation of endothelial cells are regulated by a protein kinase C isoenzyme PKCtheta. A full-length cDNA encoding a novel 20-kD protein, whose expression was PKCtheta-dependent, was identified in endothelial cells, cloned, characterized, and designated as theta-associated protein (TAP) 20. Overexpression of TAP20 decreased cell adhesion and enhanced migration on vitronectin and tube formation in three-dimensional culture. An antiintegrin alphavbeta5 antibody prevented these TAP20 effects. Overexpression of TAP20 also decreased focal adhesion formation in alphavbeta3-deficient cells. The interaction between TAP20 and beta5 integrin cytoplasmic domain was demonstrated by protein coprecipitation and immunoblotting. Thus, the discovery of TAP20, which interacts with integrin beta5 and modulates cell adhesion, migration, and tube formation, further defines a possible pathway to angiogenesis dependent on PKCtheta.

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TAP20 modulation of focal adhesion formation on MV3 cells. 24 h after transfection with the plasmids encoding GFP, TAP20 + GFP, or the fusion protein GFP-TAP20, respectively, human MV3 cells were seeded on 4-well glass chamber slides coated with VN (10 μg/ml) and incubated in a 37°C incubator for 16 h. After fixation, cells were immunostained with antibodies. (A) Cells transfected with GFP (panels a and b) or TAP20 + GFP (panels c and d) were stained with anti-TAP20 antibody. Cells were visualized with GFP fluorescence (panels a and c) and Cy-3 fluorescence (panels b and d). Cells expressing TAP20 were strongly stained. The weak stain in TAP20-negative cells may be caused by nonspecific staining by the antibody. (B) Cells expressing the GFP-TAP20 fusion protein were visualized with GFP fluorescence (panel a) or were stained with antivinculin antibody and visualized with Cy-3 fluorescence (panel b). (C) Cells expressing GFP (left panels) or TAP20 + GFP (right panels) were stained with antibodies against vinculin (V), paxillin (P), or FAK (F). Arrowheads indicate focal adhesions. (D) Focal adhesions were quantified by counting four cells for each staining. A relative value was determined in which the number of focal adhesions in TAP20 + GFP cells is expressed as a percentage of those in control (GFP alone) cells, which were normalized at 100%. Data are expressed as the mean ± S.D. (error bars). Asterisk indicates P < 0.01, TAP20 + GFP transfectants (TAP20 + GFP) versus GFP alone transfected cells by t test.
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Figure 4: TAP20 modulation of focal adhesion formation on MV3 cells. 24 h after transfection with the plasmids encoding GFP, TAP20 + GFP, or the fusion protein GFP-TAP20, respectively, human MV3 cells were seeded on 4-well glass chamber slides coated with VN (10 μg/ml) and incubated in a 37°C incubator for 16 h. After fixation, cells were immunostained with antibodies. (A) Cells transfected with GFP (panels a and b) or TAP20 + GFP (panels c and d) were stained with anti-TAP20 antibody. Cells were visualized with GFP fluorescence (panels a and c) and Cy-3 fluorescence (panels b and d). Cells expressing TAP20 were strongly stained. The weak stain in TAP20-negative cells may be caused by nonspecific staining by the antibody. (B) Cells expressing the GFP-TAP20 fusion protein were visualized with GFP fluorescence (panel a) or were stained with antivinculin antibody and visualized with Cy-3 fluorescence (panel b). (C) Cells expressing GFP (left panels) or TAP20 + GFP (right panels) were stained with antibodies against vinculin (V), paxillin (P), or FAK (F). Arrowheads indicate focal adhesions. (D) Focal adhesions were quantified by counting four cells for each staining. A relative value was determined in which the number of focal adhesions in TAP20 + GFP cells is expressed as a percentage of those in control (GFP alone) cells, which were normalized at 100%. Data are expressed as the mean ± S.D. (error bars). Asterisk indicates P < 0.01, TAP20 + GFP transfectants (TAP20 + GFP) versus GFP alone transfected cells by t test.

Mentions: The results of adhesion with the integrin-blocking antibodies suggested that TAP20 affects cell function through the αvβ5 integrin. To further clarify the role of TAP20 on αvβ5 integrin, we next examined the effect of TAP20 on focal adhesion formation in MV3 cells (van Muijen et al. 1991), which are αvβ3-negative human melanoma cells and therefore have the αvβ5 integrin as the principal surface receptor for VN. Thus, when the cells are cultured on VN-coated plates, the focal adhesion formation is caused chiefly by the interaction of αvβ5 integrin with VN. We first investigated the cellular localization of TAP20. After 16 h of adhesion to VN, MV3 cells expressing either GFP alone (Fig. 4 A, panels a and b) or TAP20 + GFP (Fig. 4 A, panels c and d) were stained with anti-TAP20 antibody that was visualized with a Cy-3–conjugated secondary antibody (Fig. 4 A, panels b and d). The strong staining in Fig. 4 A, panel d, suggests that TAP20 exists in transfected cells as a full-length protein. The TAP20 fluorescence was observed diffusely in the cytoplasm and the nucleus, which was also demonstrated by GFP fluorescence of the cells expressing the GFP-TAP20 fusion protein (Fig. 4 B, panel a). When these cells were stained with antifocal adhesion component antibodies (antivinculin shown in Fig. 4 B, panel b) to visualize the focal adhesions, no strong colocalization of GFP-TAP20 with focal adhesions was observed, indicating a possibility that TAP20 might dissociate from or interrupt focal adhesion during the cell adhesion process. To characterize the effect of TAP20 on focal adhesions, MV3 cells were transfected with either control GFP plasmid or TAP + GFP plasmid and plated on VN-coated chamber slides. GFP transfectants formed numerous focal adhesions, as demonstrated by antibodies against vinculin, paxillin, or FAK (Fig. 4 C). In TAP20 + GFP transfected cells, the number of focal adhesions was significantly reduced by 60–70% (Fig. 4 D), compared with those in wt or GFP alone transfected MV3 cells.


Enhancement of endothelial cell migration and in vitro tube formation by TAP20, a novel beta 5 integrin-modulating, PKC theta-dependent protein.

Tang S, Gao Y, Ware JA - J. Cell Biol. (1999)

TAP20 modulation of focal adhesion formation on MV3 cells. 24 h after transfection with the plasmids encoding GFP, TAP20 + GFP, or the fusion protein GFP-TAP20, respectively, human MV3 cells were seeded on 4-well glass chamber slides coated with VN (10 μg/ml) and incubated in a 37°C incubator for 16 h. After fixation, cells were immunostained with antibodies. (A) Cells transfected with GFP (panels a and b) or TAP20 + GFP (panels c and d) were stained with anti-TAP20 antibody. Cells were visualized with GFP fluorescence (panels a and c) and Cy-3 fluorescence (panels b and d). Cells expressing TAP20 were strongly stained. The weak stain in TAP20-negative cells may be caused by nonspecific staining by the antibody. (B) Cells expressing the GFP-TAP20 fusion protein were visualized with GFP fluorescence (panel a) or were stained with antivinculin antibody and visualized with Cy-3 fluorescence (panel b). (C) Cells expressing GFP (left panels) or TAP20 + GFP (right panels) were stained with antibodies against vinculin (V), paxillin (P), or FAK (F). Arrowheads indicate focal adhesions. (D) Focal adhesions were quantified by counting four cells for each staining. A relative value was determined in which the number of focal adhesions in TAP20 + GFP cells is expressed as a percentage of those in control (GFP alone) cells, which were normalized at 100%. Data are expressed as the mean ± S.D. (error bars). Asterisk indicates P < 0.01, TAP20 + GFP transfectants (TAP20 + GFP) versus GFP alone transfected cells by t test.
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Figure 4: TAP20 modulation of focal adhesion formation on MV3 cells. 24 h after transfection with the plasmids encoding GFP, TAP20 + GFP, or the fusion protein GFP-TAP20, respectively, human MV3 cells were seeded on 4-well glass chamber slides coated with VN (10 μg/ml) and incubated in a 37°C incubator for 16 h. After fixation, cells were immunostained with antibodies. (A) Cells transfected with GFP (panels a and b) or TAP20 + GFP (panels c and d) were stained with anti-TAP20 antibody. Cells were visualized with GFP fluorescence (panels a and c) and Cy-3 fluorescence (panels b and d). Cells expressing TAP20 were strongly stained. The weak stain in TAP20-negative cells may be caused by nonspecific staining by the antibody. (B) Cells expressing the GFP-TAP20 fusion protein were visualized with GFP fluorescence (panel a) or were stained with antivinculin antibody and visualized with Cy-3 fluorescence (panel b). (C) Cells expressing GFP (left panels) or TAP20 + GFP (right panels) were stained with antibodies against vinculin (V), paxillin (P), or FAK (F). Arrowheads indicate focal adhesions. (D) Focal adhesions were quantified by counting four cells for each staining. A relative value was determined in which the number of focal adhesions in TAP20 + GFP cells is expressed as a percentage of those in control (GFP alone) cells, which were normalized at 100%. Data are expressed as the mean ± S.D. (error bars). Asterisk indicates P < 0.01, TAP20 + GFP transfectants (TAP20 + GFP) versus GFP alone transfected cells by t test.
Mentions: The results of adhesion with the integrin-blocking antibodies suggested that TAP20 affects cell function through the αvβ5 integrin. To further clarify the role of TAP20 on αvβ5 integrin, we next examined the effect of TAP20 on focal adhesion formation in MV3 cells (van Muijen et al. 1991), which are αvβ3-negative human melanoma cells and therefore have the αvβ5 integrin as the principal surface receptor for VN. Thus, when the cells are cultured on VN-coated plates, the focal adhesion formation is caused chiefly by the interaction of αvβ5 integrin with VN. We first investigated the cellular localization of TAP20. After 16 h of adhesion to VN, MV3 cells expressing either GFP alone (Fig. 4 A, panels a and b) or TAP20 + GFP (Fig. 4 A, panels c and d) were stained with anti-TAP20 antibody that was visualized with a Cy-3–conjugated secondary antibody (Fig. 4 A, panels b and d). The strong staining in Fig. 4 A, panel d, suggests that TAP20 exists in transfected cells as a full-length protein. The TAP20 fluorescence was observed diffusely in the cytoplasm and the nucleus, which was also demonstrated by GFP fluorescence of the cells expressing the GFP-TAP20 fusion protein (Fig. 4 B, panel a). When these cells were stained with antifocal adhesion component antibodies (antivinculin shown in Fig. 4 B, panel b) to visualize the focal adhesions, no strong colocalization of GFP-TAP20 with focal adhesions was observed, indicating a possibility that TAP20 might dissociate from or interrupt focal adhesion during the cell adhesion process. To characterize the effect of TAP20 on focal adhesions, MV3 cells were transfected with either control GFP plasmid or TAP + GFP plasmid and plated on VN-coated chamber slides. GFP transfectants formed numerous focal adhesions, as demonstrated by antibodies against vinculin, paxillin, or FAK (Fig. 4 C). In TAP20 + GFP transfected cells, the number of focal adhesions was significantly reduced by 60–70% (Fig. 4 D), compared with those in wt or GFP alone transfected MV3 cells.

Bottom Line: A full-length cDNA encoding a novel 20-kD protein, whose expression was PKCtheta-dependent, was identified in endothelial cells, cloned, characterized, and designated as theta-associated protein (TAP) 20.An antiintegrin alphavbeta5 antibody prevented these TAP20 effects.The interaction between TAP20 and beta5 integrin cytoplasmic domain was demonstrated by protein coprecipitation and immunoblotting.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Division, Department of Medicine, Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA. tang@aecom.yu.edu

ABSTRACT
Migration, proliferation, and tube formation of endothelial cells are regulated by a protein kinase C isoenzyme PKCtheta. A full-length cDNA encoding a novel 20-kD protein, whose expression was PKCtheta-dependent, was identified in endothelial cells, cloned, characterized, and designated as theta-associated protein (TAP) 20. Overexpression of TAP20 decreased cell adhesion and enhanced migration on vitronectin and tube formation in three-dimensional culture. An antiintegrin alphavbeta5 antibody prevented these TAP20 effects. Overexpression of TAP20 also decreased focal adhesion formation in alphavbeta3-deficient cells. The interaction between TAP20 and beta5 integrin cytoplasmic domain was demonstrated by protein coprecipitation and immunoblotting. Thus, the discovery of TAP20, which interacts with integrin beta5 and modulates cell adhesion, migration, and tube formation, further defines a possible pathway to angiogenesis dependent on PKCtheta.

Show MeSH
Related in: MedlinePlus