[CANCER RESEARCH 62, 2414 –2422, April 15, 2002] Repair by Src Kinase of Function-impaired RET with Multiple Endocrine Neoplasia Type 2A Mutation with Substitutions of Tyrosines in the COOH-Terminal Kinase Domain for Phenylalanine 1 Masashi Kato, Kozue Takeda, Yoshiyuki Kawamoto, Toshihide Iwashita, Anwarul A. Akhand, Takeshi Senga, Masahiko Yamamoto, Gen Sobue, Michinari Hamaguchi, Masahide Takahashi, and Izumi Nakashima 2 Departments of Immunology [M. K., K. T., Y. K., A. A. A., I. N.], Pathology [T. I., M. T.], and Neurology [M. Y., G. S.] and Laboratory of Molecular Pathogenesis [T. S., M. H.], Nagoya University Graduate School of Medicine, Nagoya, Aichi 466-8550, Japan ABSTRACT An oncogenic mutant of c-RET as a receptor-type tyrosine kinase, termed RET-MEN2A, displays both cell-transforming activity in vivo and strong catalytic activity in vitro. In this study, we compared the activities of mutant RET-MEN2A with substitutions of each of nine tyrosines for phenylalanine (Y1062F, Y1015F, Y981F, Y952F, Y928F, Y905F, Y900F, Y864F, and Y826F), which had been transfected into NIH 3T3 cells. In RET-MEN2A with the Y905F mutation, the cell-transforming activity was drastically reduced with a great reduction in the in vitro catalytic activity. Unexpectedly, we found that in vitro kinase activity was severely impaired in RET-MEN2A with Y981F, Y952F, or Y928F mutation, which displayed near-normal cell-transforming activity and only a partially impaired ty- rosine phosphorylation level in vivo. Phosphoamino acid analysis actually demonstrated some increase in phosphotyrosine in the Y905F mutant but no or barely detectable increase in the Y981F, Y952F, or Y928F mutant after incubation for in vitro kinase assay. This suggested a crucial role of the Y981/Y952/Y928-linked structural integrity of the COOH end of the catalytic domain of RET in starting Y905 autophosphorylation. Interest- ingly, the apparent defect in intrinsic kinase activity in vitro in the Y981F, Y952F, or Y928F mutant, but not the reduction in activity in the Y905F mutant, could be partially repaired or restored by c-Src or, more exten- sively, by v-Src, which promoted Y905 phosphorylation in trans. A com- plex was shown to be formed between v-Src and RET-MEN2A through association of both with a cholesterol-rich membrane microdomain known as “a raft,” possibly for efficient contact of submembranous domains of Src and RET to promote phosphorylation of Y905 of the latter. Finally, endogenous c-Src was shown to promote Y905 phosphorylation of the Y981F mutant in vivo. These results reveal a novel Src kinase-mediated repair mechanism of otherwise function-impaired mutant RET kinases. INTRODUCTION The c-RET proto-oncogene encodes a receptor-PTK, 3 that is an essential component of a signaling pathway required for renal orga- nogenesis and enteric neurogenesis (1, 2). Germ-line single-point mutations of the c-RET proto-oncogene are associated with MEN2A and MEN2B, familial medullary thyroid carcinoma, and Hirschsprung disease (3– 8). The catalytic activities of many PTKs are regulated through control of the phosphorylation/dephosphorylation of specified tyrosine residues (Y) in kinase molecules. The RET protein has 16 tyrosines in the intracellular domain, including six tyrosines (Y864, Y900, Y905, Y928, Y952, and Y981) in its kinase domain 2, and Y905 has been suggested to play a crucial role in its catalytic and cell-transforming activity (9). Y1062 on the COOH-terminal tail of the RET protein has been reported to be the binding site of Shc and to thereby be involved also in cell-transforming activity (9, 10). Y1062 and Y1015, which was identified as the binding site for phospholipase C (11), on the COOH-terminal tail and Y826 on the kinase insert in the RET protein have also been reported to be autophosphorylation sites of RET (12). It is unknown, however, whether any tyrosines downstream of Y905 in the kinase domain are indispensably involved in the structural integrity needed for the catalytic activity of RET kinase. From a comparison of the amino acid sequence of c-RET (13, 14) with those of c-Src and other PTKs such as Lck, Hck, IRK, FgfRK, and cAPK (15), it is evident that all of these PTKs have a kinase domain with a conserved basic structure scattered with a number of highly conserved amino acids, including Y905 of c-RET (correspond- ing to Y416 of c-Src) in the activation segment (13–15) and two specified cysteines (C) with an interval of 10 amino acids (C976 of c-RET, corresponding to C487 of c-Src; C987 of c-RET, correspond- ing to C498 of c-Src) near the COOH end of the catalytic domain (15–18). Y928, Y952, and Y981 of c-RET, which are located down- stream of the Y905-containing activation segment-like sequence of the kinase domain, are also relatively conserved in a number of PTKs (Y981 in Lck, Hck, and FgfRK; Y952 in Src, Lck, Hck, IRK, FgfRK, and cAPR; and Y928 in FgfRK and cAPR). Xu et al. (15), who determined the three-dimensional structure of c-Src, suggested that both the “C” helix of the kinase NH 2 lobe and activation segment with a major autophosphorylation site of the kinase COOH lobe form a local switch for activation of the kinase, which is under global regulation linked to the change in conformation of the whole kinase molecule. A well-known global regulation of c-Src kinase is linked to change in molecular conformation attributable to association or dis- sociation of the regulatory tyrosine (Y527)-containing COOH-termi- nal tail of the kinase domain with the SH2 domain of the kinase (15, 19). The potential role of the COOH-terminal kinase domain upstream of the tail and downstream of the activation segment in the global regulation of the catalytic activity has not been investigated. In this study, we established mutants of RET-MEN2A in which each of nine tyrosines was replaced by phenylalanine (F), and we compared their kinase activities. The results show that whereas Y905 is needed for both cell-transforming activity and full in vitro kinase activity, Y981-, Y952-, and Y928-linked structural integrity of the COOH-terminal catalytic domain, downstream of the Y905-bearing activation segment, is essential for the tyrosine 905-dependent cata- lytic activity of RET-MEN2A to start in vitro, suggesting an involve- ment of this part of the molecule in the global regulation of the Y905-dependent local switch for kinase activation. Interestingly, the impaired in vitro catalytic activity of RET-MEN2A with the Y981F, Y952F, or Y928F mutation was partially restored by Src kinase, Received 11/21/00; accepted 2/15/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by Grants-in-Aid for Center of Excellence (COE) Research, Scientific Research on Priority Areas and for Scientific Research (C) from the Ministry of Education, Science, Sports and Culture of Japan, the Fund for Comprehensive Research on Aging and Health from the Ministry of Health and Welfare, the Lydia O’Leary Memorial Foundation, and the Aichi Cancer Research Foundation. 2 To whom requests for reprints should be addressed, at Department of Immunology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi 466, Japan. Phone: 81-52-744-2134; Fax: 81-52-744-2972; E-mail: inakashi@ med.nagoya-u.ac.jp. 3 The abbreviations used are: PTK, protein tyrosine kinase; MBP, myelin basic protein; RET-MEN2A, RET with multiple endocrine neoplasia type 2A mutation; GDNF, glial cell-derived neutrophic factor; PP1, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo- [3,4-d]pyrimidine; EGFR, epidermal growth factor receptor. 2414 Research. on December 13, 2021. © 2002 American Association for Cancer cancerres.aacrjournals.org Downloaded from