Limits...
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.

Show MeSH

Related in: MedlinePlus

Interaction of TAP20 with β5 integrin subunit. (A) Western analysis of proteins from ECV lysate precipitated by immobilized GST (control) or GST-TAP20 fusion protein. Antibodies used for the blots are indicated. Lane 1, ECV lysate alone; lane 2, cell proteins coprecipitated with GST alone; lane 3, cell proteins coprecipitated with GST-TAP20 fusion protein. Arrowheads indicate the proteins detected. (B) Western analysis of TAP20 protein in ECV cell lysate precipitated by GST-integrin tail fusion protein, using TAP20 antibody. Lane 1, the lysate of ECV cells transfected with TAP20 + GFP; lane 2, protein precipitated by GST–β3 tail fusion protein–coated beads from the cell lysate described in lane 1; lane 3, protein precipitated by the GST–β5 tail fusion protein–coated beads from the cell lysate described in lane 1. (C) Western analysis of TAP20 protein precipitated by GST-integrin tail fusion protein, using TAP20 antibody. Lane 1, lysate from E. coli BL21 cells expressing GST-TAP20 fusion protein; lane 2, purified GST-TAP20 fusion protein digested with thrombin, precipitated by the GST–β3 cytoplasmic domain fusion protein–coated beads for 20 h; lane 3, TAP20 protein precipitated by GST–β5 cytoplasmic domain fusion protein–coated beads at 4°C for 20 h.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169340&req=5

Figure 7: Interaction of TAP20 with β5 integrin subunit. (A) Western analysis of proteins from ECV lysate precipitated by immobilized GST (control) or GST-TAP20 fusion protein. Antibodies used for the blots are indicated. Lane 1, ECV lysate alone; lane 2, cell proteins coprecipitated with GST alone; lane 3, cell proteins coprecipitated with GST-TAP20 fusion protein. Arrowheads indicate the proteins detected. (B) Western analysis of TAP20 protein in ECV cell lysate precipitated by GST-integrin tail fusion protein, using TAP20 antibody. Lane 1, the lysate of ECV cells transfected with TAP20 + GFP; lane 2, protein precipitated by GST–β3 tail fusion protein–coated beads from the cell lysate described in lane 1; lane 3, protein precipitated by the GST–β5 tail fusion protein–coated beads from the cell lysate described in lane 1. (C) Western analysis of TAP20 protein precipitated by GST-integrin tail fusion protein, using TAP20 antibody. Lane 1, lysate from E. coli BL21 cells expressing GST-TAP20 fusion protein; lane 2, purified GST-TAP20 fusion protein digested with thrombin, precipitated by the GST–β3 cytoplasmic domain fusion protein–coated beads for 20 h; lane 3, TAP20 protein precipitated by GST–β5 cytoplasmic domain fusion protein–coated beads at 4°C for 20 h.

Mentions: The experimental evidence above suggested that the effects of TAP20 on cells were mediated by the αvβ5 integrin VN receptor. Immunoblotting with anti–β5 integrin antibody showed that the expression of the β5 integrin in TAP20 transfectants was similar to that in the control cells (Fig. 2 B). Therefore, we asked whether the effects of TAP20 could result from direct interaction with the αvβ5 integrin. We used a GST-TAP20 fusion protein purified with glutathione Sepharose beads to precipitate proteins from cell lysate, and probed proteins bound to the complexes with the antibodies against αv, β1, β3, and β5 integrin subunits. GST alone did not precipitate any protein recognized by these antibodies. Only the β5 integrin subunit was precipitated by the GST-TAP20 fusion protein (Fig. 7 A). To further confirm the TAP20–β5 integrin interaction, we used GST fusion proteins containing the cytoplasmic tail of either the β3 or β5 integrin subunit to precipitate TAP20 protein from lysate of ECV cells transfected with TAP20 + GFP. Immunoblotting with anti-TAP20 antibody demonstrated that TAP20 protein could coprecipitate with the GST–β5 tail fusion protein (Fig. 7 B). To rule out an indirect interaction between the GST-TAP20 protein and the β5 integrin complex, or between the GST–β5 tail and the TAP20 protein complex, we then used the GST-integrin tail fusion proteins to precipitate purified TAP20 protein. The GST-TAP20 fusion protein was first purified with glutathione Sepharose beads, and then was digested with thrombin. After incubation of the GST–integrin tail–coated glutathione Sepharose beads with TAP20 released by thrombin, GST–β5 tail beads were able to precipitate TAP20, as shown in immunoblotting with TAP20 antibody (Fig. 7 C).


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)

Interaction of TAP20 with β5 integrin subunit. (A) Western analysis of proteins from ECV lysate precipitated by immobilized GST (control) or GST-TAP20 fusion protein. Antibodies used for the blots are indicated. Lane 1, ECV lysate alone; lane 2, cell proteins coprecipitated with GST alone; lane 3, cell proteins coprecipitated with GST-TAP20 fusion protein. Arrowheads indicate the proteins detected. (B) Western analysis of TAP20 protein in ECV cell lysate precipitated by GST-integrin tail fusion protein, using TAP20 antibody. Lane 1, the lysate of ECV cells transfected with TAP20 + GFP; lane 2, protein precipitated by GST–β3 tail fusion protein–coated beads from the cell lysate described in lane 1; lane 3, protein precipitated by the GST–β5 tail fusion protein–coated beads from the cell lysate described in lane 1. (C) Western analysis of TAP20 protein precipitated by GST-integrin tail fusion protein, using TAP20 antibody. Lane 1, lysate from E. coli BL21 cells expressing GST-TAP20 fusion protein; lane 2, purified GST-TAP20 fusion protein digested with thrombin, precipitated by the GST–β3 cytoplasmic domain fusion protein–coated beads for 20 h; lane 3, TAP20 protein precipitated by GST–β5 cytoplasmic domain fusion protein–coated beads at 4°C for 20 h.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169340&req=5

Figure 7: Interaction of TAP20 with β5 integrin subunit. (A) Western analysis of proteins from ECV lysate precipitated by immobilized GST (control) or GST-TAP20 fusion protein. Antibodies used for the blots are indicated. Lane 1, ECV lysate alone; lane 2, cell proteins coprecipitated with GST alone; lane 3, cell proteins coprecipitated with GST-TAP20 fusion protein. Arrowheads indicate the proteins detected. (B) Western analysis of TAP20 protein in ECV cell lysate precipitated by GST-integrin tail fusion protein, using TAP20 antibody. Lane 1, the lysate of ECV cells transfected with TAP20 + GFP; lane 2, protein precipitated by GST–β3 tail fusion protein–coated beads from the cell lysate described in lane 1; lane 3, protein precipitated by the GST–β5 tail fusion protein–coated beads from the cell lysate described in lane 1. (C) Western analysis of TAP20 protein precipitated by GST-integrin tail fusion protein, using TAP20 antibody. Lane 1, lysate from E. coli BL21 cells expressing GST-TAP20 fusion protein; lane 2, purified GST-TAP20 fusion protein digested with thrombin, precipitated by the GST–β3 cytoplasmic domain fusion protein–coated beads for 20 h; lane 3, TAP20 protein precipitated by GST–β5 cytoplasmic domain fusion protein–coated beads at 4°C for 20 h.
Mentions: The experimental evidence above suggested that the effects of TAP20 on cells were mediated by the αvβ5 integrin VN receptor. Immunoblotting with anti–β5 integrin antibody showed that the expression of the β5 integrin in TAP20 transfectants was similar to that in the control cells (Fig. 2 B). Therefore, we asked whether the effects of TAP20 could result from direct interaction with the αvβ5 integrin. We used a GST-TAP20 fusion protein purified with glutathione Sepharose beads to precipitate proteins from cell lysate, and probed proteins bound to the complexes with the antibodies against αv, β1, β3, and β5 integrin subunits. GST alone did not precipitate any protein recognized by these antibodies. Only the β5 integrin subunit was precipitated by the GST-TAP20 fusion protein (Fig. 7 A). To further confirm the TAP20–β5 integrin interaction, we used GST fusion proteins containing the cytoplasmic tail of either the β3 or β5 integrin subunit to precipitate TAP20 protein from lysate of ECV cells transfected with TAP20 + GFP. Immunoblotting with anti-TAP20 antibody demonstrated that TAP20 protein could coprecipitate with the GST–β5 tail fusion protein (Fig. 7 B). To rule out an indirect interaction between the GST-TAP20 protein and the β5 integrin complex, or between the GST–β5 tail and the TAP20 protein complex, we then used the GST-integrin tail fusion proteins to precipitate purified TAP20 protein. The GST-TAP20 fusion protein was first purified with glutathione Sepharose beads, and then was digested with thrombin. After incubation of the GST–integrin tail–coated glutathione Sepharose beads with TAP20 released by thrombin, GST–β5 tail beads were able to precipitate TAP20, as shown in immunoblotting with TAP20 antibody (Fig. 7 C).

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