Tyrosine Phosphorylation and Proteolysis

doi:10.1074/jbc.273.32.20589

Tyrosine Phosphorylation and Proteolysis

PERVANADATE-INDUCED, METALLOPROTEASE-DEPENDENT CLEAVAGE OF THE ErbB-4 RECEPTOR AND AMPHIREGULIN*

From the Departments of ^‡Biochemistry, ^¶Cell Biology, and ^‖Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146

Abstract

Enhancement of tyrosine phosphorylation in cells by the application of pervanadate, an extremely potent phosphotyrosine phosphatase inhibitor, provokes the rapid metalloprotease-dependent cleavage of ErbB-4, a transmembrane receptor tyrosine kinase. The pervanadate-induced proteolysis occurs in NIH 3T3 cells expressing transfected human ErbB-4 and in several cell lines that express endogenous ErbB-4. One product of this proteolytic event is a membrane-anchored molecule of approximately 80 kDa, which is heavily tyrosine phosphorylated and which possesses tyrosine kinase catalytic activity toward an exogenous substrate in vitro. This response to pervanadate is not dependent on protein kinase C activation, which has previously been demonstrated to also activate ErbB-4 cleavage. Hence, the pervanadate and 12-O-tetradecanoylphorbol-13-acetate-induced proteolytic cleavage of ErbB-4 seem to proceed by different mechanisms, although both require metalloprotease activity. Moreover, pervanadate activation of ErbB-4 cleavage, but not that of 12-O-tetradecanoylphorbol-13-acetate , is blocked by the oxygen radical scavenger pyrrolidine dithiocarbomate. A second phosphotyrosine phosphatase inhibitor, phenylarsine oxide, also stimulates a similar cleavage of ErbB-4 but, unlike pervanadate, is not sensitive to pyrrolidine dithiocarbomate. Last, pervanadate is shown to stimulate the proteolytic cell surface processing of a second and unrelated transmembrane molecule: the precursor for amphiregulin, an epidermal growth factor-related molecule. Amphiregulin cleavage by pervanadate occurred in the absence of a cytoplasmic domain and tyrosine phosphorylation of this substrate.

Footnotes

↵* This work was supported by National Institutes of Health Grants RO1 CA24071, RO1 CA46413, and T32 AR07491 and Department of Defense Grant DAMD17-94J-4024.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.
↵§ Current address: Dept. of Experimental Oncology, European Institute of Oncology, Via Ripamonti 435, 20141 Milan, Italy.
↵** To whom correspondence should be addressed: Dept. of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232-0146. Tel.: 615-322-6678; Fax: 615-322-2931.
Abbreviations:

EGF

epidermal growth factor

PDTC

pyrrolidine dithiocarbamate

DMEM

Dulbecco’s modified Eagle’s medium

PBS

phosphate-buffered saline

TGF

transforming growth factor

PLC

phospholipase C

PAO

phenylarsine oxide

PAGE

polyacrylamide gel electrophoresis

MDCK

Madin-Darby canine kidney.
- Received March 2, 1998.
- Revision received May 13, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.