Role of Src Kinases in the ADAM-mediated Release of L1 Adhesion Molecule from Human Tumor Cells

doi:10.1074/jbc.275.20.15490

Role of Src Kinases in the ADAM-mediated Release of L1 Adhesion Molecule from Human Tumor Cells*

From the Tumor Immunology Programme, 0710, German Cancer Research Center, D-69120 Heidelberg, Germany

Abstract

The ectodomain of certain transmembrane molecules can be released by proteolysis, and the solubilized antigens often exert important biological functions. We demonstrated before that the L1 adhesion molecule is shed from the cell surface. Here we show that L1 release in AR breast carcinoma cells is mediated by a member of the disintegrin metalloproteinase (ADAM) family of proteinases. Up-regulation of L1 shedding by phorbol ester or pervanadate involved distinct mechanisms. Pervanadate induced shedding and rounding-up of cells from the substrate, which was blocked by the Src kinase inhibitor PP2. Tyr phosphorylation of the L1 cytoplasmic tail and the Src kinase Fyn was observed following pervanadate treatment. Up-regulation of L1 release and activation of Fyn occurred also when cells were detached by EDTA suggesting that the regulation of L1 shedding by this pathway was linked to cell morphology and adhesion. The phorbol 12-myristate 13-acetate-induced shedding was inhibited by the protein kinase C inhibitor bisindolylmaleimide I and by PD98059, a specific inhibitor of the mitogen-activated protein kinase pathway. Soluble L1 binds to the proteoglycan neurocan and in bound form could support integrin-mediated cell adhesion and migration. We propose that the release of cell-associated adhesion molecules such as L1 may be relevant to promote cell migration.

Footnotes

↵* This work was supported by a grant from the German-Israeli Cooperation in Cancer Research (to P. G. and P. A.).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.
↵‡ Supported by a grant from MINERVA-Stiftung.
↵§ To whom correspondence should be addressed: Tumor Immunology Programme, G0100, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Tel.: 06221-423714; Fax: 06221-423702; E-mail: P.Altevogt@dkfz-heidelberg.de.
↵2 S. Mechtersheimer and S. Beer, unpublished results.
Abbreviations:

TNF

tumor necrosis factor

TGF

transforming growth factor

ADAM

a disintegrin and metalloproteinase

TACE

tumor necrosis factor-α converting enzyme

EGF

epidermal growth factor

HB

heparin binding

PMA

phorbol 12-myristate 13-acetate

CHO

Chinese hamster ovary

FACS

fluorescence-activated cell sorter

mAb

monoclonal antibody

MAP

mitogen-activated protein

PBS

phosphate-buffered saline (lacking Ca²⁺ and Mg²⁺)

BSA

bovine serum albumin
- Received January 24, 2000.
- Revision received February 25, 2000.
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