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FRIBIO-Biologi og biomedisin

Elucidation of downstream signaling events of cAMP using new tools distinguishing the roles of EPAC and PKA

Tildelt: kr 3,1 mill.

The present project will receive Epac and PKA modulating cAMP analogs. I. We will test selected new analogs in a newly developed and unique assay for binding to PKA holoenzyme II. The best activators (current or novel) will be used, separately and jointl y, to define effect of Epac and PKA activation on the surface morphology (by SEM) and internal fine morphology (by TEM) of PC-12 and neuroblastoma cells, especially early neurite extensions. The analog studies will be supplemented by transfection using do minant negative Epac1 and RI subunit of PKA. The effect of Epac activation on protein and phosphoprotein composition of such cells will be studied by SILAC labeling and proteomics (and phosphoepitope-specific antibodies). A cAMP analog affinity resin (cu stom synthesized by BioLog) will be used to pull-down Epac and eventual partners. Importance for neuritogenesis of induced, downregulated or partner proteins will be studied after appropriate transfection. III. 3T3-L1 fibroblasts and primary fibroblasts from Epac-/- animals will be compared for adipocyte conversion by Epac and PKA activators. In vivo studies on white/brown adipose tissue in k.o. mice will be performed to validate findings. IV. We will study effects of Epac activators on gene expressi on in primary hepatocytes, and compare these findings with hepatoma cells with genetically modfied Epac and transcription factors.

Budsjettformål:

FRIBIO-Biologi og biomedisin

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