Acidic substitution of the activation loop tyrosines in TrkA supports nerve growth factor-dependent, but not nerve growth factor-independent, differentiation and cell cycle arrest in the human neuroblastoma cell line, SY5Y Ela A Gryz 1,2,4 and Susan O Meakin* ,1,2,3 1 Laboratory of Neural Signalling, Cell Biology Group, The Robarts Research Institute, 100 Perth Drive, London, Ontario, Canada N6A 5K8; 2 Graduate Program in Neuroscience, The University of Western Ontario, London, Ontario, Canada N6A 5C1; 3 Department of Biochemistry, The University of Western Ontario, London, Ontario, Canada N6A 5C1 TrkA is the receptor tyrosine kinase (RTK) for nerve growth factor (NGF) and stimulates NGF-dependent cell survival and differentiation in primary neurons and also differentiation of neuroblastomas and apoptosis of me- dulloblastomas. We have previously shown that aspartic acid and glutamic acid substitution (AspGlu and GluAsp) of the activation loop tyrosines in TrkA (Tyr 683 and Tyr 684 ) supports NGF-independent neuritogenesis and cell survi- val in PC12 cell-derived nnr5 cells. In this study, the AspGlu and GluAsp mutant Trks have been analysed for their ability to support NGF-independent and NGF- dependent neuritogenesis, proliferation and cell signalling in the human neuroblastoma cell line, SY5Y. We find that the AspGlu and GluAsp mutant Trks support NGF- dependent, but not NGF-independent, autophosphoryla- tion, neuritogenic responses and/or inhibit cell cycle progression. The NGF-dependent neuritogenic responses are lower for the mutant Trks (approximately 30–60% for AspGlu and 50–60% for GluAsp), relative to wild-type TrkA. While both the AspGlu and GluAsp mutant Trks support NGF-dependent transient phosphorylation of Shc, PLCc-1, AKT, FRS2, SH2B as well as prolonged MAP kinase activation, the GluAsp mutant induces stronger NGF-dependent tyrosine phosphorylation of FRS2 and SH2B, as well as a stronger reduction in bromodeoxyur- idine (BrdU) incorporation. Collectively, these data suggest that neither absolute levels of receptor autopho- sphorylation, high levels of TrkA expression nor the activation of a specific signalling pathway is dominant and absolutely essential for neuritogenesis and cell cycle arrest of SY5Y cells. Oncogene (2003) 22, 8774–8785. doi:10.1038/sj.onc.1206890 Keywords: nerve growth factor; TrkA; tyrosine kinase receptor; kinase activation; activation loop; neurotrophins; mutagenesis; neuroblastomas; neuronal tumors; signalling Introduction New growth factor (NGF) and the related neurotro- phins (brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3)), affect the biological behavior of neuronal cells during the development, survival and differentiation of the central and peripheral nervous systems. The biological effects of the neurotrophins are also detected in the progression and development of glial tumors and neuronal tumors such as medulloblastomas and neuroblastomas (NBs). NB is a malignant childhood tumor of neural crest origin, which can arise anywhere along the sympathetic ganglia chain or within the adrenal medulla. While some tumors may spontaneously stop proliferating and regress, others continue to proliferate and can metasta- size to the CNS. Neurotrophins and their tyrosine kinase receptors (TrkA, TrkB and TrkC) play an important role in the biology and clinical outcome of NBs. Observations from several studies indicate, first of all, that expression of TrkA and TrkC correlate with good prognosis of the disease (Kogner et al., 1993; Nakagawara et al., 1993; Ryden et al., 1996; Yamashiro et al., 1996). Secondly, correlations between prognostic markers and the progression of tumor development have been established. Specifically, N-myc amplification is associated with poor prognosis (Brodeur et al., 1984; Seeger et al., 1985) and, most importantly, the examination of TrkA expression and N-myc amplifica- tion has shown that TrkA expression is associated with an absence of N-myc amplification, lower disease stage, lower patient age and a favorable outcome (Nakagawara et al., 1992). Finally, tumors expressing full-length TrkB and BDNF are usually associated with poor prognosis and N-myc amplification (Nakagawara et al., 1994). Collectively, these findings suggest that differential activation of the Trks in NBs may have Received 29 May 2003; revised 3 June 2003; accepted 19 June 2003 *Correspondence: SO Meakin, Laboratory of Neural Signalling, Cell Biology Group, The Robarts Research Institute, 100 Perth Drive, London, Ontario, Canada N6A 5K8; E-mail: smeakin@robarts.ca 4 Current address: The Hospital for Sick Children, 555 University Ave., Toronto, Ontario, Canada M5G 1X8 Oncogene (2003) 22, 8774–8785 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc