RACK1, a new ADAM12 interacting protein. Contribution to liver fibrogenesis.
ADAM12 belongs to a disintegrin-like and metalloproteinase-containing protein family that possesses multidomain structures composed of a pro-domain, a metalloprotease, disintegrin-like, cysteine-rich, epidermal growth factor-like, and transmembrane domains, and a cytoplasmic tail. Overexpression of several ADAMs has been reported in human cancer, and we recently described the involvement of ADAM12 in liver injury (Le Pabic, H., Bonnier, D., Wewer, U. M., Coutand, A., Musso, O., Baffet, G., Clement, B., and Theret, N. (2003) Hepatology 37, 1056-1066). In this study, we used a yeast two-hybrid screening of a cDNA library from human hepatocellular carcinoma to analyze binding partners of ADAM12. We identify RACK1, a receptor for activated protein kinase C (PKC), as a new ADAM12 interacting protein. RACK1 is up-regulated in patients with hepatocellular carcinoma and is highly expressed by activated hepatic stellate cells. We demonstrate the involvement of RACK1 in mediating the PKC-dependent translocation of ADAM12 to membranes of activated hepatic stellate cells. In particular, treatment of cells with phorbol esters enhances ADAM12 immunostaining in the membrane fractions and the co-immunoprecipitation of ternary complexes containing RACK1, ADAM12, and PKC. By using RNA interference, we demonstrate that inhibition of RACK1 expression diminishes the phorbol 12-myristate 13-acetate-dependent translocation of ADAM12 to membranes of hepatic stellate cells. Finally, hepatic stellate cells cultured on coated type I collagen induces relocalization of ADAM12 in the membrane, suggesting that this major matrix component in liver cancer and fibrogenesis might stimulate ADAM12 translocation to the cell membrane where its shedding activity takes place.
Bourd-Boittin
Katia
K
INSERM U620, IFR140, University of Rennes 1, 35000 Rennes, France.
Le Pabic
Hélène
H
Bonnier
Dominique
D
L'Helgoualc'h
Annie
A
Théret
Nathalie
N
D051722:ADAM Proteins / Q000378:metabolism*; D000072199:ADAM12 Protein; D000595:Amino Acid Sequence; D019921:Biotinylation; D002462:Cell Membrane / Q000378:metabolism; D005337:Fibrin / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D019012:Integrin beta1 / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000378:metabolism* / Q000473:pathology*; D008565:Membrane Proteins / Q000378:metabolism*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry / Q000502:physiology*; D014176:Protein Biosynthesis; D021381:Protein Transport; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D020798:Two-Hybrid System Techniques
D019012:Integrin beta1; D008565:Membrane Proteins; D009363:Neoplasm Proteins; C419368:RACK1 protein, human; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D005337:Fibrin; D051722:ADAM Proteins; D000072199:ADAM12 Protein; C000606480:ADAM12 protein, human; D019204:GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate