The Shedding of Membrane-anchored Heparin-binding Epidermal-like Growth Factor Is Regulated by the Raf/Mitogen-activated Protein Kinase Cascade and by Cell Adhesion and Spreading*
- From the Departments of Surgical Research and Pathology, Children’s Hospital and Harvard Medical School, Boston, Massachusetts 02115
Abstract
Heparin-binding epidermal-like growth factor (HB-EGF) is synthesized as a transmembrane precursor (HB-EGFTM). The addition of phorbol ester (PMA, phorbol 12-myristate 13-acetate) to cells expressing HB-EGFTM results in the metalloproteinase-dependent release (shedding) of soluble HB-EGF. To analyze mechanisms that regulate HB-EGF shedding, a stable cell line was established expressing HB-EGFTM in which the ectodomain and the cytoplasmic tail were tagged with hemagglutinin (HA) and Myc epitopes, respectively (HB-EGFTMHA/Myc). HB-EGFTMHA/Myc cleavage was followed by the appearance of soluble HB-EGFHA in conditioned medium, the loss of biotinylated cell-surface HB-EGFTMHA/Myc, and the appearance of a Myc-tagged cytoplasmic tail fragment in cell lysates. By using this approach, several novel metalloproteinase-dependent regulators of HB-EGFTM shedding were identified as follows. (i) HB-EGFTMHA/Myc shedding induced by PMA was blocked by the mitogen-activated protein (MAP) kinase kinase inhibitor, PD98059. PMA activated MAP kinase within 5 min, but HB-EGFTMHA/Myc shedding did not occur until 20 min, suggesting that MAP kinase activation was a necessary step in the pathway of PMA-induced HB-EGFTM cleavage. (ii) Activation of an inducible Raf-1 kinase, ΔRaf-1:estrogen receptor, resulted in a rapid MAP kinase activation within 10 min and shedding of HB-EGFTMHA/Myc within 20–40 min. (iii) Serum induced MAP kinase activation and HB-EGFTMHA/Myc shedding that were inhibited by PD98059. (iv) Whereas PMA induced HB-EGFTMHA/Myc shedding in attached cells, no shedding occurred when the cells were placed in suspension. Shedding was fully restored shortly after cells were allowed to spread on fibronectin, and the extent of PMA-induced shedding increased with the extent of cell spreading. PMA induced the same level of MAP kinase activation whether the cells were attached or in suspension suggesting that although MAP kinase activation might be necessary for shedding, it was not sufficient. Taken together, these results suggest that there are two components of cell regulation that contribute to the shedding process, not previously recognized, the Raf-1/MAP kinase signal transduction pathway and cell adhesion and spreading.
Footnotes
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↵* This work was supported by National Institutes of Health Grants GM RO1.47397 (to M. K.), CA 45548, and HL 56398 (to D. I.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed: Children’s Hospital, 300 Longwood Ave., Boston, MA 02115. Tel.: 617-355-7503; Fax: 617-355-7291; E-mail: klagsbrun@a1.tch.harvard.edu.
- Abbreviations:
- TGF-α
-
transforming growth factor-α
- TNF-α
-
tumor necrosis factor-α
- HB-EGF
-
heparin-binding EGF-like growth factor
- EGF
-
epidermal growth factor
- PMA
-
phorbol 12-myristate 13-acetate
- MAP kinase
-
mitogen-activated protein kinase
- ERK
-
extracellular signal-regulated kinase
- MDC9
-
metalloproteinase/disintegrin/cysteine-rich protein 9
- ADAM
-
a disintegrinand metalloproteinase
- β-APP
-
β-amyloid precursor protein
- α-MEM
-
α-minimal essential medium
- CHO
-
Chinese hamster ovary
- HA
-
hemagglutinin
- CM
-
conditioned medium
- MEK
-
MAP kinase/ERK
- PBS
-
phosphate-buffered saline
- BSA
-
bovine serum albumin
- ER
-
estrogen receptor
- CAPS
-
3-(cyclohexylamino)propanesulfonic acid
- CHAPS
-
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
-
- Received April 27, 1999.
- Revision received July 15, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
