Sundberg Christina, Thodeti Charles Kumar, Kveiborg Marie, Larsson Christer, Parker Peter, Albrechtsen Reidar, Wewer Ulla M
Institute of Molecular Pathology, University of Copenhagen, Frederik V's Vej 11, Copenhagen, DK-2100, Denmark.
J Biol Chem. 2004 Dec 3;279(49):51601-11. doi: 10.1074/jbc.M403753200. Epub 2004 Sep 13.
The ADAM (a disintegrin and metalloprotease) family consists of multidomain cell-surface proteins that have a major impact on cell behavior. These transmembrane-anchored proteins are synthesized as proforms that have (from the N terminus): a prodomain; a metalloprotease-, disintegrin-like-, cysteine-rich, epidermal growth factor-like, and transmembrane domain; and a cytoplasmic tail. The 90-kDa mature form of human ADAM12 is generated in the trans-Golgi through cleavage of the prodomain by a furin-peptidase and is stored intracellularly until translocation to the cell surface as a constitutively active protein. However, little is known about the regulation of ADAM12 cell-surface translocation. Here, we used human RD rhabdomyosarcoma cells, which express ADAM12 at the cell surface, in a temporal pattern. We report that protein kinase C (PKC) epsilon induces ADAM12 translocation to the cell surface and that catalytic activity of PKCepsilon is required for this translocation. The following results support this conclusion: 1) treatment of cells with 0.1 microM phorbol 12-myristate 13-acetate (PMA) enhanced ADAM12 cell-surface immunostaining, 2) ADAM12 and PKCepsilon could be co-immunoprecipitated from membrane-enriched fractions of PMA-treated cells, 3) RD cells transfected with EGFP-tagged, myristoylated PKCepsilon expressed more ADAM12 at the cell surface than did non-transfected cells, and 4) RD cells transfected with a kinase-inactive PKCepsilon mutant did not exhibit ADAM12 cell-surface translocation upon PMA treatment. Finally, we demonstrate that the C1 and C2 domains of PKCepsilon both contain a binding site for ADAM12. These studies show that PKCepsilon plays a critical role in the regulation of ADAM12 cell-surface expression.
ADAM（一种解聚素和金属蛋白酶）家族由多结构域细胞表面蛋白组成，这些蛋白对细胞行为有重大影响。这些跨膜锚定蛋白以前体形式合成，从前体的N端开始依次为：前结构域；金属蛋白酶、解聚素样、富含半胱氨酸、表皮生长因子样和跨膜结构域；以及细胞质尾。人ADAM12的90 kDa成熟形式是在反式高尔基体中通过弗林蛋白酶切割前结构域而产生的，并在细胞内储存，直到作为组成型活性蛋白转运到细胞表面。然而，关于ADAM12细胞表面转运的调控知之甚少。在这里，我们使用人RD横纹肌肉瘤细胞，其以时间模式在细胞表面表达ADAM12。我们报告蛋白激酶C（PKC）ε诱导ADAM12转运到细胞表面，并且这种转运需要PKCε的催化活性。以下结果支持这一结论：1）用0.1 microM佛波酯12 -肉豆蔻酸酯13 -乙酸酯（PMA）处理细胞增强了ADAM12细胞表面免疫染色；2）ADAM12和PKCε可以从PMA处理细胞的富含膜的组分中进行共免疫沉淀；3）用EGFP标记的、肉豆蔻酰化的PKCε转染的RD细胞在细胞表面表达的ADAM12比未转染细胞更多；4）用激酶失活的PKCε突变体转染的RD细胞在PMA处理后未表现出ADAM12细胞表面转运。最后，我们证明PKCε的C1和C₂结构域都含有ADAM12的结合位点。这些研究表明PKCε在ADAM12细胞表面表达的调控中起关键作用。
