Tyrosines outside the Kinase Core and Dimerization Are Required for the Mitogenic Activity of RET/ptc2

doi:10.1074/jbc.270.42.24642

Tyrosines outside the Kinase Core and Dimerization Are Required for the Mitogenic Activity of RET/ptc2 (*)

From the (1)Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0654 and
(2) Divisione di Oncologia Sperimentale A, Istituto Nazionale Tumori, I20133 Milan, Italy

** To whom correspondence should be addressed.

Abstract

Defects in the c-ret proto-oncogene, a member of the protein tyrosine kinase receptor family, have recently been linked to two types of genetic syndromes, Hirschsprung's disease and the multiple endocrine neoplasia family of inherited cancers. RET/ptc2 is the product of a papillary thyroid carcinoma translocation event between the genes coding for c-ret and the type Iα regulatory subunit of protein kinase A (RIα) (Lanzi, C., Borrello, M., Bongarzone, I., Migliazza, A., Fusco, A., Grieco, M., Santoro, M., Gambetta, R., Zunino, F., Della Porta, G., and Pierotti, M.(1992) Oncogene 7, 2189-2194). The resulting 596-residue protein contains the first two-thirds of RIα and the entire tyrosine kinase domain of c-ret (RET). An in vivo assay of growth stimulatory effects was developed, which consisted of microinjecting a RET/ptc2 expression plasmid into the nuclei of 10T1/2 mouse fibroblasts and observing the incorporation of 5-bromodeoxyuridine. This assay was used to determine that only the dimerization domain of RIα fused to RET is required for RET/ptc2's mitogenic activity. In addition, all of the reported Hirschsprung's disease point mutations in the RET (S289P, R421Q, and R496G) inactivate RET/ptc2 in our assay, confirming that these are loss of function mutations. Two tyrosines outside the conserved kinase core were also identified that are essential for full mitogenic activity of RET/ptc2. These two tyrosines, Tyr-350 and Tyr-586, are potential sites for Src homology 2 and phosphotyrosine binding domain interactions.

Footnotes

↵^§ Supported by the Markey Charitable Trust as a Fellow; currently supported by National Institutes of Health Training Grant NCI T32 CA09523.
↵^¶ Supported by National Institutes of Health Research Service Award 5T32DK0704417.
↵* This research was supported in part by United States Army Grant AIBS1762 (to S. S. T.) and by the Italian Association for Cancer Research and Italian National Research Council Special Project ACRO (to M. A. P.). 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:

FMTC

familial medullary thyroid carcinoma

MEN

multiple endocrine neoplasia

RIα

type Iα regulatory subunit of cAMP-dependent protein kinase (protein kinase A)

RET

tyrosine kinase domain of the c-Ret receptor

SH2

Src homology 2 domain

DMEM

Dulbecco's modified Eagle's medium

FBS

fetal bovine serum

FITC

fluorescein isothiocyanate

BrdUrd

bromodeoxyuridine

EGF

epidermal growth factor.
- Received July 26, 1995.