Activation of Ras, Raf-1 and protein kinase C in differentiating human neuroblastoma cells after treatment with phorbolester and NGF

The human neuroblastoma cell line SH-SY5Y/TrkA differentiates in vitro and acquires a sympathetic phenotype in response to phorbolester (activator of protein kinase C, PKC) in the presence of serum or growth factors, or nerve growth factor (NGF). We have now investigated to what extent phorbolester and NGF cause activation of Ras and Raf-1 and the involvement of PKC in thi s response in differentiating SH-SY5Y/TrkA cells. NGF stimulated increased accumulation of Ras-GTP and a threefold activation of Raf-1. In contrast, 1 2-O-tetradecanoylphorbol-13-acetate (TPA) had no effect on the amount of Ra s-GTP but led to a smaller activation of Raf-1. NGF caused a limited increa se in phosphorylation of Raf-1 compared with TPA, and NGF-induced Raf activ ity was independent of PKC. Analysis of phosphorylation of the endogenous P KC substrate myristoylated alanine-rich C-kinase substrate (MARCKS), and of subcellular distribution of PKC-alpha, -delta, and -epsilon revealed that NGF only caused a very small activation of PKC in SH-SY5Y/TrkA cells. The r esults identify Raf-1 as a target for both TPA- and NGF-induced signals in differentiating SH-SY5Y/TrkA cells and demonstrate that signalling to Raf-1 was mediated via distinct mechanisms. (C) 2001 Elsevier Science Inc. All r ights reserved.