Mitogenic Signaling by Ret/ptc2 Requires Association with Enigma via a LIM Domain

doi:10.1074/jbc.271.22.12691

Mitogenic Signaling by Ret/ptc2 Requires Association with Enigma via a LIM Domain*

From the Departments of ^‡ Chemistry and Biochemistry
^¶ Biology, and
^″ Medicine, University of California, San Diego, La Jolla, California 92093-0654

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Abstract

The ret/ptc2 papillary thyroid cancer oncogene, an oncogenic form of the c-Ret receptor tyrosine kinase, is the product of a somatic crossover event fusing the dimerization domain of the type Iα regulatory subunit of cyclic AMP-dependent protein kinase (RI) with the tyrosine kinase domain of c-Ret. Mitogenic activity of Ret/ptc2 required dimerization via the N terminus of RI and a tyrosine residue located C-terminal to the kinase core of Ret, Tyr-586 (Durick, K., Yao, V. J., Borrello, M. G., Bongarzone, I., Pierotti, M. A. and Taylor, S. S. (1995) J. Biol. Chem. 270, 24642-24645). Using the yeast two-hybrid system, Ret/ptc2 binding proteins were identified, and the sites of interaction with Ret/ptc2 were mapped. The SH2 domains of phospholipase Cγ and Grb10 were both identified, and binding depended on phosphorylation of Tyr-539 and Tyr-429, respectively. These interactions, however, were not required for mitogenic signaling. The second of the three LIM domains in Enigma (Wu, R. Y., and Gill, G. N. (1994) J. Biol. Chem. 269, 25085-25090) was also identified as a Ret/ptc2 binding domain. Enigma, a 455-residue protein, was discovered based on its interaction with the insulin receptor through the C-terminal LIM domain. Although the association with Enigma required Tyr-586 of Ret/ptc2, the interaction was phosphorylation-independent. In contrast to the SH2 interactions, disruption of the interaction with Enigma abolished Ret/ptc2 mitogenic signaling, suggesting that LIM domain recognition of an unphosphorylated tyrosine-based motif is required for Ret signal transduction.

Footnotes

↵^§ Supported by the Markey Charitable Trust as a Fellow and currently supported by National Institutes of Health Training Grant NCI T32 CA09523.
↵^∥ Supported by United States Army Breast Cancer Research Fellowship DAMD 17-94-J-4124.
↵* This research was supported in part by United States Army Grant AIBS1762 (to S. S. T.) and National Institutes of Health Grant DK13149 (to G. N. G.). 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:

ptc

papillary thyroid carcinoma

RI

type Iα regulatory subunit of cyclic AMP-dependent protein kinase

PLCγ

phospholipase C-γ

GST

glutathione S-transferase

EGF

epidermal growth factor

EGFR

epidermal growth factor receptor

SH2

Src homology 2.
↵² Wu, R.-Y., Durick, R., Songyang, Z., Cantley, L. C., Taylor, S. S., and Gill, G. N. (1996) J. Biol. Chem., in press.
- Received March 8, 1996.
- Revision received March 29, 1996.