Phorbol 12-myristate 13-acetate-induced ectodomain shedding and phosphorylation of the human meprinbeta metalloprotease.

Abstract

Shedding of proteins localized at the cell surface is an important regulatory step in the function of many of these proteins. Human meprin (N-benzoyl-l-tyrosyl-p-aminobenzoic acid hydrolase, PPH, EC 3.4.24.18) a zinc-metalloendopeptidase of the astacin family is an oligomeric protein complex of alpha- and beta-subunits and is expressed abundantly in the intestine and kidney as well as in leukocytes of the lamina propria and in cancer cells. In transfected cells intracellular proteolytic removal of the membrane anchor results in the secretion of the meprin alpha-subunit. In rats and mice, the beta-subunit exists in a membrane-anchored form. In contrast, human meprinbeta is constitutively converted into a secretable form. We now show that phorbol 12-myristate 13-acetate (PMA) stimulates an increased release of hmeprinbeta from transfected COS-1 cells, whereas hmeprinalpha secretion is not influenced. This stimulatory effect is inhibited by the protein kinase C (PKC) inhibitor staurosporine, suggesting that activation of PKC mediates PMA-induced hmeprinbeta shedding. The use of different protease inhibitors shows that two different metalloprotease activities are responsible for the constitutive and the PMA-stimulated hmeprinbeta shedding. We identified tumor necrosis factor alpha-converting enzyme (TACE or ADAM17) as the protease that mediates the PMA-induced release. We also demonstrate that hmeprinbeta is phosphorylated by PMA treatment on Ser687 within a PKC consensus sequence in the cytosolic domain of the protein. This phosphorylation of hmeprinbeta is not, however, implicated in the enhanced secretion by PMA treatment.

• Authors: Dagmar Hahn, Anastassios Pischitzis, Sandra Roesmann, Marianne K Hansen, Boris Leuenberger, Ursula Luginbuehl, Erwin E Sterchi
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 278 Issue 44 Pg. 42829-39 (Oct 31 2003) ISSN: 0021-9258 [Print] United States
• PMID: 12941954 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Enzyme Inhibitors
• Protease Inhibitors
• Protein Kinase C
• Metalloendopeptidases
• meprin B
• Staurosporine
• Tetradecanoylphorbol Acetate

Topics

• Amino Acid Sequence
• Animals
• Blotting, Western
• COS Cells
• Cytosol (metabolism)
• Electrophoresis, Polyacrylamide Gel
• Enzyme Inhibitors (pharmacology)
• Humans
• Metalloendopeptidases (chemistry, metabolism)
• Mice
• Models, Biological
• Molecular Sequence Data
• Phosphorylation
• Precipitin Tests
• Protease Inhibitors (pharmacology)
• Protein Kinase C (antagonists & inhibitors)
• Protein Structure, Tertiary
• Sequence Homology, Amino Acid
• Staurosporine (pharmacology)
• Tetradecanoylphorbol Acetate
• Time Factors
• Transfection