ELSEVIER Brain Research 661 (1994) 137-146 BRAIN RESEARCH Research report Sustained tyrosine phosphorylation of p140 trkA in PC12h-R cells responding rapidly to NGF Masashi Yamada a, Toshihiko Ikeuchi a, Hiroko Tsukui a, Saburo Aimoto b, Hiroshi Hatanaka a,, a Dil~ision of Protein Biosynthesis, 3-2 Yamadaoka, Suita, Osaka 565, Japan b Research Center for Protein Engineering, Institute for Protein Research, Osaka Unil:ersity, 3-2 Yarnadaoka, Suita, Osaka 565. Japan Accepted 19 July 1994 Abstract The PC12h cell, a subclone of PC12 cells, has considerable activities of tyrosine hydroxylase (TH) and choline acetyltrans- ferase (CHAT) and shows an NGF-induced increase in both enzyme activities. The TH activity and its inducibility by NGF in PC12h cells were stably maintained in the passage of > 200 generations whereas the ChAT activity was not. We isolated a new cell line, PC12h-R (originally clone 8), from a long-term culture of PC12h cells. PC12h-R cells still showed the considerable TH activity, but not the ChAT activity, and maintained the inducibility of TH activity by NGF. Thus, the responses of PC12h-R cells to NGF were similar to those of chromaffin cells and sympathetic neurons. PC12h-R cells were found to extend neurites and differentiate into sympathetic neuron-like cells in response to NGF much more rapidly than PC12h cells. In addition, PCI2h-R cells showed sustained NGF-induced tyrosine phosphorylation of p140 ''kA and several cellular proteins, including 42-, 44- and 54-kDa proteins, in comparison with PC12h cells. We suggest that the NGF-induced sustained tyrosine phosphorylation signal in PCI2h-R cells may be correlated closely with their rapid NGF-induced differentiation into neuron-like cells. Keywords: PC12 cell; NGF; Neuronal differentiation; TrkA; Tyrosine phosphorylation 1. Introduction The rat pheochromocytoma PC12 cell line shows a sympathetic neuron-like phenotype in response to nerve growth factor (NGF) [15], a well-characterized neu- rotrophic factor [2,40] for sympathetic and sensory neurons in the PNS [18,66] and for basal forebrain cholinergic neurons in the CNS [22,28]. In PC12 cells treated with NGF, dramatic cellular events occur, in- cluding extension of neurites, cessation of mitosis, in- crease of biosynthesis of neurotransmitters and devel- opment of Na + and Ca 2+ channels [20]. The PC12 system is widely used as a model for investigating how NGF acts on neuronal precursor cells and neurons. In sympathetic neurons, NGF selectively induces the activity of tyrosine hydroxylase (TH) [65], a key enzyme * Corresponding author. Fax: (81) (6) 879-8626. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)00912-0 in catecholamine biosynthesis. In PC12 cells, however, NGF induces the activity of choline acethyltransferase (CHAT) [16,61], a key enzyme in acetylcholine biosyn- thesis, but fails to increase TH activity [11]. We have previously isolated the PC12h cell line [23], a subclone of the PC12 cell, which responds to NGF by increasing the TH activity. PC12h cells also show NGF responses similar to PC12 cells, including neurite outgrowth [24], increase of carbamylcholine-induced Na+-influx [46], induction of specific enkephalin binding [33] and neu- ronal differentiation of voltage-sensitive Ca :+ channels [64]. The action of NGF is initiated by binding to its receptors. Two types of NGF receptors, p75 NcwR and p140 trkA, have been identified [1,7,13,44]. The former, p75 NGFR, lacks a typical intracellular structure for sig- nal transduction, but the latter, p140 trkA, has an intra- cellular tyrosine kinase domain. The tyrosine kinase of p140 trkA is activated by the binding of NGF to its