7 B)

7 B). integrin 5 and modulates cell adhesion, migration, and tube formation, further defines a possible pathway to angiogenesis dependent on PKC. BL21 cells and immobilized on glutathione Sepharose beads (Amersham Pharmacia Biotech) following the manufacturer’s instructions. To precipitate protein from EC lysate, the GST-TAP20 coated beads were incubated with the cell lysate in PBS for 20 h at 4C. To precipitate TAP20 with GST-integrin tail fusion protein, the GST-TAP20 beads were first incubated in thrombin containing buffer for 16 h at room temperature to release the TAP20 protein. The TAP20-containing solution was then incubated with GST-integrin tail fusion protein beads for 20 h at 4C. After three washes with X-376 PBS, the proteins bound to the Rabbit Polyclonal to MBD3 beads were eluted in 10 mM glutathione buffer and analyzed by Western blotting. The antibodies used included: GST polyclonal antibody (Amersham Pharmacia Biotech), v polyclonal antibody (Santa Cruz Biotechnology), 1 mAb and 3 mAb (Transduction Laboratories), and 5 polyclonal antibody (Chemicon). Results Cloning of TAP20 We investigated the expression of genes regulated X-376 by PKC 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 PKC (PKC-kn, a dominant negative inhibitor) or a constitutively active form of PKC (PKC-ca) were overexpressed (Tang et al. 1997). An RNA of 0.8 kb whose expression depended upon the presence of active PKC was identified. Northern transfer analysis showed that this 0.8-kb mRNA is highly expressed in PKC-ca RCE but is dramatically suppressed in PKC-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 PKC, as suggested by the Northern X-376 transfer analysis. In the PKC 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 X-376 PKC-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 3-endonexin (Shattil et al. X-376 1995; Eigenthaler et al. 1997; Kashiwagi et al. 1997), a 111-residue polypeptide that interacts with the 3 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 3-endonexin, we hypothesized that TAP20 is involved in integrin-mediated cell functions that are regulated by PKC. Open in a separate window Open in a separate window Open in a separate window 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 PKC activity levels. Total RNA (B) and RCE lysate (C) were prepared from wt RCE (wt).