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 this response in differentiating SH-SY5Y/TrkA cells. NGF stimulated increased accumulation of Ras-GTP and a threefold activation of Raf-1. In contrast, 12-O-tetradecanoylphorbol-13-acetate (TPA) had no effect on the amount of Ras-GTP but led to a smaller activation of Raf-1. NGF caused a limited increase in phosphorylation of Raf-1 compared with TPA, and NGF-induced Raf activity was independent of PKC. Analysis of phosphorylation of the endogenous PKC 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 results 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.
Söderholm
H
H
Tumour Biology, Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University, SE-751 85, Uppsala, Sweden.
Olsson
A
A
Lavenius
E
E
Rönnstrand
L
L
Nånberg
E
E
D015153:Blotting, Western; D002273:Carcinogens; D002454:Cell Differentiation; D004789:Enzyme Activation; D006801:Humans; D008957:Models, Genetic; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism; D009227:Myristic Acids / Q000378:metabolism; D020932:Nerve Growth Factor / Q000494:pharmacology*; D009447:Neuroblastoma / Q000201:enzymology*; D010641:Phenotype; D010703:Phorbol Esters; D010766:Phosphorylation; D011233:Precipitin Tests; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate; D013997:Time Factors; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured; D015854:Up-Regulation; D018631:ras Proteins / Q000378:metabolism*
D002273:Carcinogens; D009227:Myristic Acids; D010703:Phorbol Esters; D020033:Protein Isoforms; D020932:Nerve Growth Factor; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D020559:Monomeric GTP-Binding Proteins; D018631:ras Proteins; D013755:Tetradecanoylphorbol Acetate