The crosstalk between the ERK and the cAMP signalling pathways in PC12 Cells

QR Microbiology;QP Physiology

Gormand, Amelie ; Kolch, Walter

University:University of Glasgow

Department:Institute of Molecular Cell and Systems Biology

关键词: Cell signalling, ERK pathway, cAMP, PC12 cells, Raf kinases, cancer;   

Others  :  http://theses.gla.ac.uk/378/1/2008Gormandphd.pdf

来源: University of Glasgow

【 摘 要 】

The extracellular-regulated kinase (ERK) signalling pathway is involved in the control of different biological processes such as survival, proliferation and differentiation. In PC12 cells, the ERK signalling pathway integrates different external stimuli: epidermal growth factor (EGF) stimulates the ERK pathway transiently and induces cell proliferation, whereas nerve growth factor (NGF) induces sustained activation of ERK and promotes cell differentiation into sympathetic-like neurons. The second messenger 3’,5’-cyclic adenosine monophosphate (cAMP) controls a plethora of cellular events from metabolic to cellular signalling pathways. Over the years, lines of evidence have shown that cAMP is also involved in the regulation of cell growth and cell differentiation, suggesting a possible crosstalk between these two pathways.Selective phosphodiesterase (PDE) inhibitors were used to block the degradation of cAMP. These inhibitors increased the level of cAMP, but had different effects on the activation of ERK. Upon both NGF and EGF stimulations, cilostamide had the strongest effect and doubled the intensity of the phosphorylation of ERK, identifying PDE3 to control the level of cAMP relevant for the regulation of the ERK pathway. The treatment with cilostamide enhanced the differentiation of PC12 cells and the combination of both cilostamide and rolipram (a PDE4 inhibitor) turned the proliferative effect of EGF into a differentiation effect.The route for cAMP in the regulation of the ERK pathway was decomposed by using the cAMP analogues 8-pCPT-2’-O-Me-cAMP and 6-Bnz-cAMP. They specifically activate the Exchange protein activated by cyclic AMP (Epac) and the cAMP regulated protein kinase (PKA) respectively, which were hypothesised to be the effectors of cAMP in the regulation of ERK. The Epac agonist mimicked the effects of cilostamide on the activation of ERK, but failed to enhance cell differentiation. The PKA agonist reduced the phosphorylation ERK upon EGF. It was suggested that the activation of ERK in response to cAMP was mainly mediated through Epac rather than PKA, and that the activation of both PKA and Epac are required to induce cellular differentiation.To elucidate the differential regulation of the activation of ERK upon NGF and EGF stimulation and in response to cAMP, the activity of Ras and Rap1 were measured by affinity pulldown assays. Upon EGF the signal was transduced through Ras only, whereas upon NGF the signal was mediated through both Ras and Rap1. cAMP sensitised Rap1 that became activated upon EGF stimulation indicating that cAMP can switch on the Rap1/B-Raf pathway. This correlated with the increase in the phosphorylation of ERK in response to high levels of cAMP upon EGF stimulation. Then, the role of Raf-1 and C3G, a guanine exchange factor for Rap1, were investigated using small interfering RNA. The depletion of Raf-1 showed that Raf-1 is not essential for transducing the mitogen signal upon NGF stimulation and suggested that Ras mediates the signal through B-Raf upon EGF stimulation to compensate for the loss of Raf-1. The depletion of C3G also confirmed that the activation of ERK in response to cAMP is mediated through the Rap1/B-Raf pathway.Finally, the interaction between Raf-1 and AKAP79 was demonstrated for the first time suggesting the existence of a complex between Raf-1, AKAP and PKA and therefore a possible molecular mechanism for the inhibition of Raf-1 by cAMP through PKA. The data presented in this thesis demonstrates that cAMP participates to finely tune the regulation of the ERK signalling pathway and can be use as a tool to elucidate the network comprising the ERK cascade.