Altered Expression of Protein-tyrosine Phosphatase 2C in 293 Cells Affects Protein Tyrosine Phosphorylation and Mitogen-activated Protein Kinase Activation

doi:10.1074/jbc.270.20.11765

Altered Expression of Protein-tyrosine Phosphatase 2C in 293 Cells Affects Protein Tyrosine Phosphorylation and Mitogen-activated Protein Kinase Activation (*)

From the (1) Departments of Biochemistry and
(2) Pharmacology, University of Washington, Seattle, Washington 98195 and the
(3) Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec, Canada H4P 2R2

^§Recipient of a research fellowship from SUGEN, Inc. To whom correspondence and reprint requests should be addressed. Tel.: 206-543-3553; Fax: 206-685-1792.

Abstract

PTP2C, an SH2 domain-containing protein-tyrosine phosphatase, is recruited to the growth factor receptors upon stimulation of cells. To investigate its role in growth factor signaling, we have overexpressed by approximately 6-fold the native PTP2C and a catalytically inactive mutant of the enzyme in 293 human embryonic kidney cells. The native PTP2C was located entirely in the cytosol, while the inactive mutant was nearly equally distributed in cytosolic and membrane fractions. Expression of the latter caused hyperphosphorylation on tyrosine of a 43-kDa protein, which was co-immunoprecipitated and co-partitioned in the plasma membrane fraction with the inactive PTP2C mutant. This protein may represent a physiological substrate of PTP2C. Overexpression of the native PTP2C enhanced epidermal growth factor (EGF)-stimulated mitogen-activated protein (MAP) kinase kinase activity by 30%, whereas expression of the inactive mutant reduced the stimulated activity by 50%. Similar effects were observed for the activation of MAP kinase as determined by activity assay, gel mobility shift, and tyrosine phosphorylation. The data suggest that the phosphatase activity of PTP2C is partly required for MAP kinase activation by EGF and that PTP2C may function by dephosphorylating the 43-kDa membrane protein.

Footnotes

↵* This work was supported in part by Grants DK07902 and GM42508 from the National Institutes of Health and grants from the Muscular Dystrophy Association of America and the International Human Frontier Sciences Programs. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

SH2

src homology 2

PTP

protein- tyrosine phosphatase

MAP kinase

mitogen-activated protein kinase

EGF

epidermal growth factor

PDGF

platelet-derived growth factor

MBP

myelin basic protein

PAGE

polyacrylamide gel electrophoresis.