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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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Molecular cloning and analysis of TAP20. (A) Nucleotide and deduced amino acid sequence of TAP20. The deduced amino acid sequence (single letter code) is shown under the nucleotide sequence. The solid line indicates the sequence used as the reverse primer to screen the rat PC12 library. The box indicates the sequence of peptide used as an immunogen to induce TAP20 antibody formation. (B and C) Differential expression of TAP20 in RCE with varying PKCθ activity levels. Total RNA (B) and RCE lysate (C) were prepared from wt RCE (wt) or cells stably expressing control plasmid (v), PKCθ-ca (ca1 and ca2), or PKCθ-kn (kn1 and kn2). The full-length TAP20 cDNA was used as probe on Northern blot (B), and the TAP20 antibody was used for Western analysis (C). (D) Amino acid sequence comparison of TAP20 with β3-endonexin. Identical amino acids are shown by the letters in gray boxes. Lower case letters indicate additional 59 amino acids of the longer form of β3-endonexin, and the boxed sequence indicates the immunogen peptide for TAP20.
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Figure 1: Molecular cloning and analysis of TAP20. (A) Nucleotide and deduced amino acid sequence of TAP20. The deduced amino acid sequence (single letter code) is shown under the nucleotide sequence. The solid line indicates the sequence used as the reverse primer to screen the rat PC12 library. The box indicates the sequence of peptide used as an immunogen to induce TAP20 antibody formation. (B and C) Differential expression of TAP20 in RCE with varying PKCθ activity levels. Total RNA (B) and RCE lysate (C) were prepared from wt RCE (wt) or cells stably expressing control plasmid (v), PKCθ-ca (ca1 and ca2), or PKCθ-kn (kn1 and kn2). The full-length TAP20 cDNA was used as probe on Northern blot (B), and the TAP20 antibody was used for Western analysis (C). (D) Amino acid sequence comparison of TAP20 with β3-endonexin. Identical amino acids are shown by the letters in gray boxes. Lower case letters indicate additional 59 amino acids of the longer form of β3-endonexin, and the boxed sequence indicates the immunogen peptide for TAP20.

Mentions: We investigated the expression of genes regulated by PKCθ with the mRNA display method (Liang and Pardee 1992) using mRNAs from clonal populations of rat capillary endothelial cells (RCE), in which either the kinase-negative PKCθ (PKCθ-kn, a dominant negative inhibitor) or a constitutively active form of PKCθ (PKCθ-ca) were overexpressed (Tang et al. 1997). An RNA of ∼0.8 kb whose expression depended upon the presence of active PKCθ was identified. Northern transfer analysis showed that this 0.8-kb mRNA is highly expressed in PKCθ-ca RCE but is dramatically suppressed in PKCθ-kn RCE (Fig. 1 B). The partial sequence of this gene obtained from differential display was used to screen a rat PC12 cDNA library by the PCR method. The cDNA generated from the PCR was cloned, and the nucleotide sequence was determined (Fig. 1 A). The open reading frame encodes a novel protein of 175 residues with a calculated molecular mass of ∼20 kD. An antibody raised against the COOH terminus of this putative protein recognized a protein of 20 kD (Fig. 1 C), as assessed by immunoblotting of an RCE cell lysate, which confirmed that the expression of this protein depended upon functional PKCθ, as suggested by the Northern transfer analysis. In the PKCθ kinase-negative RCE, TAP20 expression was significantly decreased at the protein level. This anti-TAP20 antibody also recognized a 20-kD band in TAP20 transfected human cells (Fig. 1 C and Fig. 2), suggesting that TAP20 can be expressed as a 20-kD, full-length protein in the cells. Accordingly, this novel PKCθ-associated protein was designated TAP20. By searching the GeneBank database, we found that a partial 3′ end sequence of TAP20 had been reported previously (Kerr et al. 1994), and the first 110 amino acid residues of TAP20 share 55% homology with human β3-endonexin (Shattil et al. 1995; Eigenthaler et al. 1997; Kashiwagi et al. 1997), a 111-residue polypeptide that interacts with the β3 integrin subunit (Fig. 1 D). TAP20 also has an additional 66-residue COOH terminus. Thus, although the differences suggest that TAP20 may have functional properties that differ from those of β3-endonexin, we hypothesized that TAP20 is involved in integrin-mediated cell functions that are regulated by PKCθ.


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)

Molecular cloning and analysis of TAP20. (A) Nucleotide and deduced amino acid sequence of TAP20. The deduced amino acid sequence (single letter code) is shown under the nucleotide sequence. The solid line indicates the sequence used as the reverse primer to screen the rat PC12 library. The box indicates the sequence of peptide used as an immunogen to induce TAP20 antibody formation. (B and C) Differential expression of TAP20 in RCE with varying PKCθ activity levels. Total RNA (B) and RCE lysate (C) were prepared from wt RCE (wt) or cells stably expressing control plasmid (v), PKCθ-ca (ca1 and ca2), or PKCθ-kn (kn1 and kn2). The full-length TAP20 cDNA was used as probe on Northern blot (B), and the TAP20 antibody was used for Western analysis (C). (D) Amino acid sequence comparison of TAP20 with β3-endonexin. Identical amino acids are shown by the letters in gray boxes. Lower case letters indicate additional 59 amino acids of the longer form of β3-endonexin, and the boxed sequence indicates the immunogen peptide for TAP20.
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Related In: Results  -  Collection

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Figure 1: Molecular cloning and analysis of TAP20. (A) Nucleotide and deduced amino acid sequence of TAP20. The deduced amino acid sequence (single letter code) is shown under the nucleotide sequence. The solid line indicates the sequence used as the reverse primer to screen the rat PC12 library. The box indicates the sequence of peptide used as an immunogen to induce TAP20 antibody formation. (B and C) Differential expression of TAP20 in RCE with varying PKCθ activity levels. Total RNA (B) and RCE lysate (C) were prepared from wt RCE (wt) or cells stably expressing control plasmid (v), PKCθ-ca (ca1 and ca2), or PKCθ-kn (kn1 and kn2). The full-length TAP20 cDNA was used as probe on Northern blot (B), and the TAP20 antibody was used for Western analysis (C). (D) Amino acid sequence comparison of TAP20 with β3-endonexin. Identical amino acids are shown by the letters in gray boxes. Lower case letters indicate additional 59 amino acids of the longer form of β3-endonexin, and the boxed sequence indicates the immunogen peptide for TAP20.
Mentions: We investigated the expression of genes regulated by PKCθ with the mRNA display method (Liang and Pardee 1992) using mRNAs from clonal populations of rat capillary endothelial cells (RCE), in which either the kinase-negative PKCθ (PKCθ-kn, a dominant negative inhibitor) or a constitutively active form of PKCθ (PKCθ-ca) were overexpressed (Tang et al. 1997). An RNA of ∼0.8 kb whose expression depended upon the presence of active PKCθ was identified. Northern transfer analysis showed that this 0.8-kb mRNA is highly expressed in PKCθ-ca RCE but is dramatically suppressed in PKCθ-kn RCE (Fig. 1 B). The partial sequence of this gene obtained from differential display was used to screen a rat PC12 cDNA library by the PCR method. The cDNA generated from the PCR was cloned, and the nucleotide sequence was determined (Fig. 1 A). The open reading frame encodes a novel protein of 175 residues with a calculated molecular mass of ∼20 kD. An antibody raised against the COOH terminus of this putative protein recognized a protein of 20 kD (Fig. 1 C), as assessed by immunoblotting of an RCE cell lysate, which confirmed that the expression of this protein depended upon functional PKCθ, as suggested by the Northern transfer analysis. In the PKCθ kinase-negative RCE, TAP20 expression was significantly decreased at the protein level. This anti-TAP20 antibody also recognized a 20-kD band in TAP20 transfected human cells (Fig. 1 C and Fig. 2), suggesting that TAP20 can be expressed as a 20-kD, full-length protein in the cells. Accordingly, this novel PKCθ-associated protein was designated TAP20. By searching the GeneBank database, we found that a partial 3′ end sequence of TAP20 had been reported previously (Kerr et al. 1994), and the first 110 amino acid residues of TAP20 share 55% homology with human β3-endonexin (Shattil et al. 1995; Eigenthaler et al. 1997; Kashiwagi et al. 1997), a 111-residue polypeptide that interacts with the β3 integrin subunit (Fig. 1 D). TAP20 also has an additional 66-residue COOH terminus. Thus, although the differences suggest that TAP20 may have functional properties that differ from those of β3-endonexin, we hypothesized that TAP20 is involved in integrin-mediated cell functions that are regulated by PKCθ.

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