Regulation of ADAM12 cell-surface expression by protein kinase C epsilon.
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.
Sundberg
Christina
C
Institute of Molecular Pathology, University of Copenhagen, Frederik V's Vej 11, Copenhagen, DK-2100, Denmark.
Thodeti
Charles Kumar
CK
Kveiborg
Marie
M
Larsson
Christer
C
Parker
Peter
P
Albrechtsen
Reidar
R
Wewer
Ulla M
UM
D051722:ADAM Proteins; D000072199:ADAM12 Protein; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D016466:CHO Cells; D002384:Catalysis; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000737:chemistry; D008856:Microscopy, Fluorescence; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000378:metabolism / Q000502:physiology*; D051744:Protein Kinase C-epsilon; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D013755:Tetradecanoylphorbol Acetate; D013997:Time Factors; D014162:Transfection
D018076:DNA, Complementary; D008565:Membrane Proteins; D020033:Protein Isoforms; D049452:Green Fluorescent Proteins; C497255:PRKCE protein, human; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon; D051722:ADAM Proteins; D000072199:ADAM12 Protein; C000606480:ADAM12 protein, human; D008666:Metalloendopeptidases; D013755:Tetradecanoylphorbol Acetate