A novel point of crosstalk between the cAMP and Raf-1 signalling pathways

Crosstalk between the cAMP and ERK signalling pathway is crucially involved in the regulation of cell proliferation, apoptosis, transformation and survival. The relationship between these pathways is of vital importance because irregularities in the Ras/Raf/MEK/ERK pathway, by way of mutated or aberrantly expressed proteins, play a major role in cancer pathogenesis. The work presented in this thesis focuses on a new point of crosstalk between these two key pathways: the direct interaction between Raf-1 and PDE8A1. One of the main cAMP effectors, protein kinase A (PKA), can phosphorylate and inhibit Raf-1 at Ser259. The only method of degrading the cAMP signal, and thereby preventing PKA inhibition of Raf-1, is through its hydrolysis by phosphodiesterases (PDEs). To date, no PDE has been implicated in the regulation of Raf-1 and these results represent the first time a PDE has been shown to modulate Raf-1 activity. Proteomic screenings of Raf-1 immunopurifications found PDE8A to be associated, and the first part of this thesis investigates the relationship between Raf-1 and PDE8A1. First of all, the PDE8A/Raf-1 complex was verified with immunoprecipitations, in vitro binding assays and BiaCore analyses. PDE8A1 was shown to regulate the phosphorylation state of S259, and therefore activation state, of Raf-1. The interaction sites between PDE8A and Raf-1 were mapped using the novel peptide array technology that has been used previously in our laboratory. Based on the peptide array results, a cell-permeable PDE8A peptide was created to disrupt the PDE8A/Raf-1 interaction. This cell permeable peptide significantly increased Ca2+ signalling within mouse cardiomyocytes when they were stimulated with isoproterenol. In addition, the cell-permeable peptide significantly reduced the basal levels of phospho-ERK1/2 by approximately 50% in HEK293 cells. Immunocytochemistry showed PDE8A to be uniquely targetted to the mitochondria, where it colocalised with a distinct pool of Raf-1. After exposing cells to epidermal growth factor (EGF), the remaining Raf-1 relocalised from the plasma membrane and cytoplasm to the PDE8A-rich mitochondrial compartments. Over-expresssed dominant-negative PDE8A1 (D/N PDE8A), where the PDE8A1 sequence was mutated to render the protein catalytically inactive, caused a large increase in phosphorylated Raf-1 at the inhibitory S259 site. HEK293 cells overexpressing D/N PDE8A were found to be hypersensitive to the cytotoxic agents, doxorubicin, staurosporine, and hydrogen peroxide. Based on this evidence showing PDE8A1 to regulate Raf-1 phosphorylation and activity, these results suggest that the PDE8A/Raf-1 signalling junction could be a selective novel target for cancer treatment. In the second part of the thesis, the post-translational modification of PDE8A1, in particular the phosphorylation of PDE8A1 was investigated. A novel phosphorylation site, S359, was identified and specially developed phospho-specific PDE8A antibodies showed a major increase in PKA-mediated phosphorylation of PDE8A when cAMP levels were raised using forskolin in HEK293 cells. Experiments involving S359 mutants suggest that phosphorylation of PDE8A1 increases the cAMP hydrolysing activity of the enzyme. A putative sumoylation site of PDE8A1, K435, was also identified. In addition to the work outline above, the interaction between AKAP79 and Raf-1 was investigated. Despite promising proteomics results, the AKAP79/Raf-1 interaction could not be verified and requires further investigation. In summary, the work in this thesis contributes further to our understanding of the crosstalk between the cAMP and Raf/MEK/ERK signalling pathways in cells, and the regulation of one of the lesser-studied PDEs, PDE8A