Phorbol ester-induced activation of a membrane-bound candidate pro- transforming growth factor-α processing enzyme

T. Harano, K. Mizuno

Research output: Contribution to journalArticlepeer-review

21 Citations (Scopus)


Transforming growth factor-α (TGF-α) is synthesized and transported to the cell surface as a membrane-anchored precursor (proTGF-α) that is converted to the soluble form by proteolytic cleavage. ProTGF-α cleavage is activated in response to tumor-promoting phorbol ester or to calcium ionophore. Mechanism(s) controlling conversion of the membrane-anchored precursor to the soluble TGF-α are unknown, and the responsible protease has not been identified. Using the fluorogenic substrate succinyl-Ala-Ala-Ala-4- methylcoumaryl-7-amide (Suc-Ala-Ala-Ala-MCA), containing the sequence similar to the cleavage sites for proTGF-α processing, we identified a putative candidate proTGF-α-converting enzyme in the membrane fractions of Chinese hamster ovary (CHO) cells. The enzyme activity was about 5-6-fold increased by exposure of the cells with phorbol ester or with calcium ionophore. The enzyme activity was released from the cells by trypsin added exogenously; hence, the activity probably locates on the outer surface of the cell membrane. The enzyme was partially purified from membranes of phorbol ester- treated CHO cells. The molecular mass of the enzyme was estimated to be about 84 kDa by gel filtration analysis. Inhibitor profile of the enzyme, including inhibition by serine protease inhibitors and no inhibition by elastatinal, coincides with that previously reported for proTGF-α cleavage activity on CHO cells. These findings suggest that the enzyme is a processing protease involved in the cleavage of proTGF-α and that induction of proTGF-α conversion by phorbol ester or by calcium ionophore can be attributed to activation of the processing protease. In the cell-free reconstitution system, the membrane-bound Suc-Ala-Ala-Ala-MCA-hydrolyzing enzyme from CHO cells was also activated by phorbol ester in the presence of the soluble fraction, ATP, and vanadate. Requirement of vanadate, an inhibitor for protein phosphatases, for activation of the enzyme in this system suggests that phosphorylation-dephosphorylation probably plays a key role in the regulated cleavage of proTGF-α.

Original languageEnglish
Pages (from-to)20305-20311
Number of pages7
JournalJournal of Biological Chemistry
Issue number32
Publication statusPublished - 1994
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


Dive into the research topics of 'Phorbol ester-induced activation of a membrane-bound candidate pro- transforming growth factor-α processing enzyme'. Together they form a unique fingerprint.

Cite this