Novel modulators and downstream targets of the cAMP receptors Epac and PKA

I: Obtain genomic DNA suitable for recombinant deletion of Epac1 in ES cells, produce Epac1 null mice. II: a) Use mapping data of more than 100 cAMP analogs for PKA type I,II and Epac to predict effect of cAMP modifications on binding. Design new discriminating analogs. b) Identify new ATP-site directed inhibitors of PKA. III. Study Epac1 k.o. mouse for adrenal medulla and sympathetic ganglion cell development. Use mouse to verify specificity of cAMP analogs for PKA or Epac. IV. (C. Krakstad) Search for mechanism explaining the synergistic upregulation of genes by Epac and PKA activators in PC-12 cells. Search for new proteins involved in Epac signaling by “signalomics” and the identification of Epac partners by proteomics. V. (C. Krakstad) Correlate cAMP-induced gene transcription in the absence and presence of Bcl-2 overexpression. Study whether Bcl-2 itself can modulate cell differentiation through gene transcription. VI. (F. Vandeput) Identify the proteins linking PKA act ivation to cdk5 activation and caspase activation in the cAMP-induced IPC-81 cell death model. Use IPC-81 model to test ATP directed cdk5 inhibitors for action in intact cells.