Signalling of the Ret receptor tyrosine kinase through the c-Jun NH 2 - terminal protein kinases (JNKs): evidence for a divergence of the ERKs and JNKs pathways induced by Ret Mario Chiariello 1 *, Roberta Visconti 1 *, Francesca Carlomagno 1 , Rosa Marina Melillo 1 , Cecilia Bucci 1 , Vittorio de Franciscis 1 , Gary M Fox 2 , Shuqian Jing 2 , Omar A Coso 3 , J Silvio Gutkind 3 , Alfredo Fusco 4 and Massimo Santoro 1 1 Centro di Endocrinologia ed Oncologia Sperimentale del CNR, c/o Dipartimento di Biologia e Patologia Cellulare e Molecolare, Facolta Á di Medicina e Chirurgia, Universita Á di Napoli `Federico II', via S Pansini 5, 80131 Naples, Italy; 2 Amgen, 1840 DeHavilland Drive, Thousand Oaks, California 91320-1789, USA; 3 Oral and Pharyngeal Cancer Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892, USA; 4 Dipartimento di Medicina Sperimentale e Clinica, Facolta Á di Medicina e Chirurgia di Catanzaro, Universita Á di Reggio Calabria, via T Campanella 5, 88100 Catanzaro, Italy The RET proto-oncogene encodes a functional receptor tyrosine kinase (Ret) for the Glial cell line Derived Neurotrophic Factor (GDNF). RET is involved in several neoplastic and non-neoplastic human diseases. Oncogenic activation of RET is detected in human papillary thyroid tumours and in multiple endocrine neoplasia type 2 syndromes. Inactivating mutations of RET have been associated to the congenital megacolon, i.e. Hirsch- prung's disease. In order to identify pathways that are relevant for Ret signalling to the nucleus, we have investigated its ability to induce the c-Jun NH 2 -terminal protein kinases (JNK). Here we show that triggering the endogenous Ret, expressed in PC12 cells, induces JNK activity; moreover, Ret is able to activate JNK either when transiently transfected in COS-1 cells or when stably expressed in NIH3T3 ®broblasts or in PC Cl 3 epithelial thyroid cells. JNK activation is dependent on the Ret kinase function, as a kinase-de®cient RET mutant, associated with Hirschsprung's disease, fails to activate JNK. The pathway leading to the activation of JNK by RET is clearly divergent from that leading to the activation of ERK: substitution of the tyrosine 1062 of Ret, the Shc binding site, for phenylalanine abrogates ERK but not JNK activation. Experiments conducted with dominant negative mutants or with negative regulators demonstrate that JNK activation by Ret is mediated by Rho/Rac related small GTPases and, particularly, by Cdc42. Keywords: tyrosine kinase; transformation; jun; MEN2; thyroid Introduction RET encodes a tyrosine kinase receptor (Ret) (Takahashi et al., 1988), which is expressed, during embryogenesis, in kidney and nervous system, includ- ing neurons of the enteric tract. Consistently, RET- de®cient mice show lack of enteric neurons and renal dysgenesis (Schuchardt et al., 1994), and `loss of function' RET mutations are associated with Hirsch- sprung's disease (Romeo et al., 1994; Edery et al., 1994; Pasini et al., 1995; Carlomagno et al., 1996), which is characterized by the congenital defect of intestinal innervation. Recently, GDNF (glial cell line derived neurotrophic factor) has been identi®ed as one functional ligand for Ret. GDNF binds to GDNFR-a, a glycosyl phosphatidylinositol-linked cell surface receptor, which, in turn, mediates Ret activation (Jing et al., 1996; Treanor et al., 1996). Consistent with that, mice with targeted disruption of the GDNF gene show a phenotype similar to that of RET knock-out mice (Sanchez et al., 1996). Deregulation of the RET function has been demonstrated to be oncogenic. RET activation by gene rearrangement is found in nearly 40% of sporadic thyroid papillary carcinomas. These rearrangements lead to the fusion of its tyrosine kinase encoding domain to heterologous genes, generating the chimeric RET/PTC oncogenes (Grieco et al., 1990). RET/PTC1, the one more frequently isolated, is generated by the fusion of RET to the 5'-terminal region of a gene designated H4 (Grieco et al., 1990). In addition, germline RET point mutations, responsible for the inheritance of the MEN2A (multiple endocrine neoplasia type 2A), MEN2B, and FTMC (familial medullary thyroid carcinoma) syndromes (Donis-Keller et al., 1993; Mulligan et al., 1993; Carlson et al., 1994; Hofstra et al., 1994), also lead to an activation of the transforming potential of RET (Santoro et al., 1995; Asai et al., 1995). In most of MEN2A and FMTC cases substitution of extracellular cysteines leads to a constitutive dimerization of the receptor (Santoro et al., 1995; Asai et al., 1995); in contrast, in the majority of MEN2B cases, a M918T mutation causes a change of Ret substrate speci®city (Santoro et al., 1995; Songyang et al., 1995). Receptor tyrosine kinases' signals are, at least in part, transduced by intracellular serine/threonine kinases designated MAPK (mitogen activated protein kinases) (reviewed by Marshall, 1995). MAPK include extracellular signal-regulated protein kinase (p44 and p42 ERK1 and ERK2, respectively), c-Jun amino- terminal kinases (JNKs), also designated stress- activated protein kinase (SAPKs), and p38 kinase. MAPK are activated by upstream dual-speci®c kinases. Correspondence: M Santoro *The ®rst two authors contributed equally to the work Received 9 June 1997; revised 8 December 1997; accepted 9 December 1997 Oncogene (1998) 16, 2435 ± 2445  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc