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

Abstract

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.

Keywords: 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, an inhibitor of Src; Cy3; DMEM; DMSO; Dulbecco’s Modified Eagle’s Medium; EDTA; EGTA; ERK1/2; H-Ras; IκB; JNK; M-M17-26; MAP Kinase; MEK; MG-132; NGF; Neuronal differentiation; PBS; PC12; PC12 cell line; PD98059; PMSF; PP2; PVDF; Proteasome inhibition; Ras; SDS; Src; TBS; Tris-buffered saline; TrkA; a MAP kinase with the molecular weight of 38kDa; a TrkA-deficient PC12 variant clone; a chemical compound that inhibits MEK; a fluorophor cyanin dye; a monomeric G-protein; a non-receptor tyrosine kinase; a peptidyl-aldehyde proteasome inhibitor, also known as Z-Leu-Leu-Leu-al; a variant PC12 clone that stably expresses a dominant negative H-Ras mutant; an isoform of the small guanine nucleotide-binding protein Ras named after Harvey; c-Jun N-terminal kinase; dimethyl-sulfoxyde; ethylene glycol tetraacetic acid; ethylenediaminetetraacetic acid; extracellular signal-regulated kinase 1 and 2; inhibitor of kappa B; mitogen-activated protein kinase; mitogen-activated protein kinase/ERK kinase; nerve growth factor; nnr5; p38MAPK; phenylmethanesulfonyl fluoride; phosphate buffered saline; polyvinylidene difluoride; rat pheochromocytoma cell line; sodium dodecyl sulfate; tropomyosin-related kinase.