Regulation of ADAM12 Cell-surface Expression by Protein Kinase C ϵ (original) (raw)

2004, Journal of Biological Chemistry

The ADAM (a disintegrin and metalloprotease) family consists of multidomain cell-surface proteins that have a major impact on cell behavior. These transmembraneanchored 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) ⑀ 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 M phorbol 12-myristate 13-acetate (PMA) enhanced ADAM12 cell-surface immunostaining, 2) ADAM12 and PKC⑀ 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. Cells possess a diverse array of surface proteins, lipids, and carbohydrates that provide active gateways for the selective intake and release of molecular information, which is important in regulating cell behavior. In fact, many disease processes relate to disorganized cell-surface communication systems. ADAMs 1 belong to a large family of cell-surface proteins with over 30 members. The prototypical ADAM molecule is a transmembrane glycoprotein composed of several distinct domains, including a prodomain and a metalloprotease, disintegrin-like, cysteine-rich, epidermal growth factor-like, transmembrane, and cytoplasmic domain. ADAMs play important roles in cell adhesion, interacting with integrins and syndecans, and in the proteolysis of the ectodomains of cell-surface proteins, such as growth factors, growth factor receptors, and cytokines (1-5). For example, ADAM17 (TACE) mediates release of tumor necrosis factor-␣, transforming growth factor-␣, ␤-amyloid, L-selectin, TRANCE, and amphiregulin precursor proteins (6-11). ADAMs 9, 10, and 12 have been shown to cleave membraneanchored, heparin-binding epidermal growth factor (12-15). Important in vivo functions have been reported for several ADAMs (3). For example, the finding that ADAM9,-10, and-17 have, or mediate, ␣-secretase activity could be used to design new treatment strategies for Alzheimer's disease (7, 14, 16, 17). Overexpression of ADAMs has been observed in many human cancers (18, 19), suggesting that ADAMs could promote tumor growth and metastasis by modulating growth factor shedding and cell adhesion. A recent genome-wide scan and polymorphism analysis of a large group of patients identified ADAM33 on chromosome 20 as a putative asthma susceptibility gene (20). We have demonstrated that ADAM12-S, which is present in the serum of pregnant women but not in that of women who are not pregnant (21), can be used as a first-trimester maternal serum marker for Down syndrome (22). Gene-ablation experiments in mice revealed that ADAM17 (TACE)-deficient mice have severe perinatal and postnatal defects primarily related to eye, hair, and skin anomalies, including failure of eyelid fusion (10). In contrast, ADAM9-deficient mice have an apparently normal phenotype (10, 14). ADAM12 deficiency confers increased perinatal mortality, although the reason for this is not yet well understood (23). Surviving ADAM12-null mice have defects in adipose tissue (23), and mice overexpressing ADAM12 under the muscle creatine kinase promoter exhibit increased adipogenesis (24), supporting the idea that ADAM12 is involved in mesenchymal cell differentiation. ADAM12, originally named meltrin-␣ (25), has been implicated in muscle cell function in vivo and in vitro (25-30). In the original study, expression of a truncated version of