Regulation of ADAM12 cell-surface expression by protein kinase C ∈

Abstract

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-ricli, 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) ∈ induces ADAM12 translocation to the cell surface and that catalytic activity of PKC∈ is required for this translocation. The following results support this conclusion: 1) treatment of cells with 0.1 μM phorbol 12-myristate 13-acetate (PMA) enhanced ADAM12 cell-surface immunostaining, 2) ADAM12 and PKCε could be co-immunoprecipitated from membrane-enriched fractions of PMA-treated cells, 3) RD cells transfected with EGFP-tagged, myristoylated PKC∈ expressed more ADAM12 at the cell surface than did non-transfected cells, and 4) RD cells transfected with a kinase-inactive PKC∈ mutant did not exhibit ADAM12 cell-surface translocation upon PMA treatment. Finally, we demonstrate that the C1 and C2 domains of PKC∈ both contain a binding site for ADAM12. These studies show that PKC∈ plays a critical role in the regulation of ADAM12 cell-surface expression.