Pro-Tumor Necrosis Factor-α Processing Activity Is Tightly Controlled by a Component That Does Not Affect Notch Processing*
- From the Laboratori de Recerca Oncològica, Servei d’Oncologia Mèdica, Hospital General Universitari Vall d’Hebron, Psg. Vall d’Hebron 119–129, Barcelona 08035, Spain and‡Immunex Corporation, Seattle, Washington 98101
Abstract
The extracellular domain of a heterogeneous group of transmembrane proteins can be proteolytically released from the cell surface, a process known as protein ectodomain shedding. Despite the biomedical importance of several substrates of the shedding system, such as the β-amyloid precursor protein (βAPP), little is known about the regulation of protein ectodomain shedding, and the only protease known to be involved is the metalloprotease disintegrin, tumor necrosis factor-α converting enzyme (TACE). Here, we show that previously described pro-transforming growth factor-α shedding-defective cell mutants (M2 cells), known to be defective in ectodomain shedding of several molecules, that include βAPP, fail to shed the ectodomain of pro-TNF-α. The target of the mutation is a component required for TACE activity, since transfection of TACE into M2 cells has no effect on the shedding of pro-TNF-α and somatic cell fusions between M2 cells and TACE null cells recover the ability to shed pro-TNF-α, pro-transforming growth factor-α, and βAPP. Furthermore, we show that TACE is also necessary for the shedding of βAPP since TACE null cells show defective βAPP shedding. Biochemical evidence shows that the component that controls TACE is different from protein kinase C, the only known activator of protein ectodomain shedding, and that this component does not affect biosynthesis or processing of TACE or other metalloprotease disintegrins. The component mutated in M2 cells is likely to control only a subset of metalloprotease disintegrins involved in regulated ectodomain shedding, since Notch processing, a process known to be dependent on the activity of another metalloprotease disintegrin, Kuzbanian, is normal in M2 cells.
Footnotes
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↵* This work was supported by Spanish Comisión Interministerial de Ciencia y Tecnologı́a Grant SAF97-0229, Fundació La Marató de TV3 Grant 036/97, a predoctoral fellowship from the Spanish ministry of education (to A. M.-S.), and a postdoctoral fellowship from the Fundació per a la Recerca i Docència dels Hospitals Vall d’Hebron (to J. F.-L.).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: Laboratori de Recerca Oncològica, Hospital General, Psg. Vall d’Hebron 119–129, Barcelona 08035, Spain. Tel.: 34-93-274-6077; Fax: 34-93-274-6059; E-mail: jarribas{at}hg.vhebron.es.
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↵2 J. Peschon, J. Slack, P. Reddy, K. Stocking, S. Sunnarborg, D. Lee, W. Russell, B. Castner, R. Johnson, J. Fitzner, R. Boyce, N. Nelson, C. Kozlosky, M. Wolfson, C. Rauch, D. Cerretti, R. Paxton, C. March, and R. Black, submitted for publication.
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↵3 J. D. Buxbaum, K.-N. Liu, Y. Luo, J. L. Slack, K. L. Stocking, J. L. Peschon, R. Johnson, D. P. Cerretti, and R. A. Black, submitted for publication.
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↵4 J. Ureña, J. Baselga, and J. Arribas, submitted for publication.
- Abbreviations:
- PKC
-
protein kinase C
- TNF-α
-
tumor necrosis factor-α
- TGF-α
-
transforming growth factor-α
- IL
-
interleukin
- TACE
-
tumor necrosis factor-α converting enzyme
- βAPP
-
β-amyloid precursor protein
- MMP
-
matrix metalloprotease
- DMEM
-
Dulbecco’s modified Eagle’s medium
- CHO
-
Chinese hamster ovary
- PMA
-
phorbol 12-myristate 13-acetate
- PBS
-
phosphate-buffered saline
- PAGE
-
polyacrylamide gel electrophoresis.
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- Received May 13, 1998.
- Revision received June 22, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
