Cripto Enhances the Tyrosine Phosphorylation of Shc and Activates Mitogen-activated Protein Kinase (MAPK) in Mammary Epithelial Cells

doi:10.1074/jbc.272.6.3330

Cripto Enhances the Tyrosine Phosphorylation of Shc and Activates Mitogen-activated Protein Kinase (MAPK) in Mammary Epithelial Cells*

From the ^a Tumor Growth Factor Section, Laboratory of Tumor Immunology and Biology, NIC, National Institutes of Health, Bethesda, Maryland 20892 the
^b ICRF Oncology Unit, Hammersmith Hospital, London, United Kingdom the
^c Department of Pathology, BML-350, Yale University School of Medicine, New Haven, Connecticut 06520-8023 the
^e Friedrich Meischer-Institut, CH-4002 Basel, Switzerland the
^f Istituto Internazionale di Genetica e Biofisca, Naples, Italy the
^g Vincent T. Lombardi Cancer Research Center, Georgetown University, Washington, D. C. 20007 the
^h Oncologia Sperimentale d Istituto Nazionale Per lo Studio e La Cura, Dei Tumori-Fondazione Pascale, Naples, Italy, and the
^j Cattedra di Oncologia Medica, II Facoltá di Medicina e Chirurgia, Universitá degli Studi di Napoli Federico II, 80131 Naples, Italy

^k To whom all correspondence and reprint requests should be addressed:
Bldg. 10, Rm. 5B39, Tumor Growth Factor Section, Laboratory of Tumor Immunology and Biology, NCI, National Institutes of Health, Bethesda, MD 20892.
Tel.: 301-496-9536; Fax: 301-402-8656; E-mail: davetgfa{at}helix.nih.gov

Abstract

Cripto-1 (CR-1), a recently discovered protein of the epidermal growth factor (EGF) family, was found to interact with a high affinity, saturable binding site(s) on HC-11 mouse mammary epithelial cells and on several different human breast cancer cell lines. This receptor exhibits specificity for CR-1, since other EGF-related peptides including EGF, transforming growth factor α, heparin-binding EGF-like growth factor, amphiregulin, epiregulin, betacellulin, or heregulin β1 that bind to either the EGF receptor or to other type 1 receptor tyrosine kinases such as erb B-3 or erb B-4 fail to compete for binding. Conversely, CR-1 was found not to directly bind to or to activate the tyrosine kinases associated with the EGFR, erb B-2, erb B-3, or erb B-4 either alone or in various pairwise combinations which have been ectopically expressed in Ba/F3 mouse pro-B lymphocyte cells. However, exogenous CR-1 could induce an increase in the tyrosine phosphorylation of 185- and 120-kDa proteins and a rapid (within 3-5 min) increase in the tyrosine phosphorylation of the SH2-containing adaptor proteins p66, p52, and p46 Shc in mouse mammary HC-11 epithelial cells and in human MDA-MB-453 and SKBr-3 breast cancer cells. CR-1 was also found to promote an increase in the association of the adaptor Grb2-guanine nucleotide exchange factor-mouse son of sevenless (mSOS) signaling complex with tyrosine-phosphorylated Shc in HC-11 cells. Finally, CR-1 was able to increase p42^erk-2 mitogen-activated protein kinase (MAPK) activity in HC-11 cells within 5-10 min of treatment. These data demonstrate that CR-1 can function through a receptor which activates intracellular components in the ras/raf/MEK/MAPK pathway.

Footnotes

↵^d Supported by the National Cancer Institute, United States Public Health Service Grant HD-07149, and United States Army Medical Research and Material Command Grant DAMD-17-94-J-4036.
↵ⁱ Supported by the United States Army Medical Research and Material Command Grant DAMD-17-94-J-4498.
↵* This work was supported in part by the National Cancer Institute, United States Public Health Service Grant CA-45708, and United States Army Medical Research and Material Command Grant DAMD-17-94-J-4476 (to D. F. S.). 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.
↵¹ The abbreviations used are:

CR-1

cripto-1

EGF

epidermal growth factor

TGFα

transforming growth factor α

AR

amphiregulin

HRG

heregulin

BTC

betacellulin

EGFR

EGF receptor

mSOS

mouse Son of Sevenless, gunanine nucleotide exchange factor

MAPK

mitogen-activated protein kinase

erk

extracellular signal-regulated protein kinase

MEK

mitogen-activated erk-activating kinase

MBP

myelin basic protein

Shc

Src homologous and collagen protein

Grb2

growth factor receptor-bound protein

PAGE

polyacrylamide gel electrophoresis

Tricine

N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
↵² S. Kannan, manuscript in preparation.
↵³ M. Seno, manuscript in preparation.
- Received June 19, 1996.
- Revision received November 7, 1996.