Identification of 'tissue' transglutaminase binding proteins in neural cells committed to apoptosis.
Overexpression of 'tissue' transglutaminase (tTG) in the human neuroblastoma cells increases spontaneous apoptosis and renders these cells highly susceptible to death induced by various stimuli. We used immunoprecipitation to identify cellular proteins that interact specifically with tTG in SK-N-BE(2) -derived stable transfectants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that tTG binding proteins have molecular masses of 110, 50, 22, 14, and 12 kDa. Microsequencing and computer search analyses allowed us to identify these polypeptides as the beta-tubulin (50 kDa), the histone H2B (14 kDa), and two GST P1-1-truncated forms (22 and 12 kDa). The specificity of the interaction between tTG and these proteins was confirmed by competing tTG binding with purified enzyme and by detecting tTG in immunoprecipitates obtained using beta-tubulin or GST P1-1 mAb's. Here we demonstrate that the GST P1-1 acts as an efficient acyl donor as well as acceptor tTG substrate both in cells and in vitro. The tTG-catalyzed polymerization of GST P1-1 leads to its functional inactivation and is competitively inhibited by GSH. By contrast, the tTG-beta-tubulin interaction does not result in the cross-linking of this cytoskeletal protein, which suggests that microtubules act as the anchorage site for tTG and GST P1-1 interaction.
Piredda
L
L
Department of Biology, University of Rome 'Tor Vergata' Rome, Italy.
Farrace
M G
MG
Lo Bello
M
M
Malorni
W
W
Melino
G
G
Petruzzelli
R
R
Piacentini
M
M
D000595:Amino Acid Sequence; D017209:Apoptosis; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase / Q000235:genetics* / Q000378:metabolism; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology; D009474:Neurons / Q000378:metabolism* / Q000473:pathology*; D011485:Protein Binding; D011506:Proteins / Q000235:genetics* / Q000378:metabolism*; D017421:Sequence Analysis; D013379:Substrate Specificity; D011503:Transglutaminases / Q000378:metabolism*; D014407:Tumor Cells, Cultured