The role of Src protein in the process formation of PC12 cells induced by the proteasome inhibitor MG-132

The PC12 (rat pheochromocytoma) cell line is a popular model system to study neuronal differentiation. Upon prolonged nerve growth factor (NGF) exposure these tumor cells stop to divide, become polygonal, grow projections and start to look and behave like sympathetic neurons. Differentiation of PC12 cells can also be induced by peptidyl-aldehyde proteasome inhibitors, such as Z-Leu-Leu-Leu-al (also known as MG-132) or via infection of the cells with Rous sarcoma virus. The signal transduction pathways underlying process formation, however, are still not fully understood. The liganded NGF receptor initiates a protein kinase cascade a member of which is Extracellular Signal-Regulated Kinase (ERK). Active ERK1/2 enzymes phosphorylate various cytoplasmic proteins and can also be translocated into the nucleus, where they regulate gene expression by activating key transcription factors. Using immunological methods we detected phosphorylation of TrkA, prolongedactivation of Src, and ERK1/2 with nuclear translocation of the latter during MG-132-induced process formation of PC12 cells. Activated Src remained predominantly cytoplasmic. MG-132-induced sustained ERK1/2 activation, nuclear translocation and neuritogenesis required the intact function of Src since these phenomena were markedly reduced or failed upon chemical inhibition of Src tyrosine protein kinase activity.