Serum response factor (SRF), as the master regulatory platform, may play a central role in the commitment of cardiac progenitors by virtue of its obligatory requirement for mesoderm formation and by its ability for making specific protein-protein associations with other early cardiac enriched transcription cofactors. Analysis of SRF null mutants supports the idea that SRF is an essential regulator of mammalian cardiac mesoderm formation and places SRF at a very high point in the regulatory hierarchy for cardiac cell commitment and differentiation. We discovered that the regulated phosphorylation of two conserved SRF MADS box residues Thr159 and Ser162 constitutes a novel gene switch for the activation and repression of SRF dependent cardiogenic gene programs. Protein kinase C (PKC) has a profound inhibitory activity on SRF, directing phosphorylation primarily to Ser162 and secondarily to Thr159. It is likely that VEGF by signaling through the Flkl receptor and driving PKC activity may inactivate SRF myogenic activity in part by blocking the expression of cardiogenic contractile protein gene activity. In addition, VEGF may exert inhibitory activity that forms the posterior border of the heart forming region and may signal later during the transition from forming the cardiac cushion to the appearance of the noncontractile endocardium. The phosphorylated SRF gene switch may also allow for the activation of the endothelial-hemangiogenic program at the expense of cardiogenic gene activity. Thus, the central theme of this proposal will be to determine how the phosphorylation of SRF, at two sites that are only three amino acids apart in the alpha 1 coil of the MADS box, is networked to regulate downstream gene targets during the appearance of cardiac mesoderm and the elaboration and formation of the embryonic mouse heart. By the following four aims: Aim I: Does phosphorylation of SRF MADS box occur during cardiogenesis and the elaboration of the murine heart? Aim II Does phosphorylation of the SRF MADS box act as a gene regulatory switch? Aim III Does phosphorylation of SRF MADS box have a direct role in specifying early cardiac and or endothelial/hemangiogenic programs? Aim IV Does the VEGF signaling pathway repress SRF dependent cardiogenic gene activity?

R01HL079628

2005-01-15

2008-12-31