The aim of this proposal is to characterize the signaling pathways utilized by growth factor receptors to activate immediate early genes. The proto-type immediate early gene c-fos is regulated by a Serum Response Element (SRE), which binds Serum Response Factor (SRF). SRF is activated by two pathways. One is MARK phosphorylation of the SRF cofactors, the ternary complex factors (TCFs), and the second is signaling through the small GTPase RhoA. In this proposal the pathways used by RhoA to activate the c-fos promoter will be studied. The SRF coactivators MKL1/2 will be studied as they have been shown to be required for serum and RhoA activation of SRF target genes. MKL1is rapidly phosphorylated in response to serum treatment of cells. The sites of inducible phosphorylation have been mapped and will be tested for their requirement for serum induction of target genes. The responsible protein kinase will then be identified. The role of domains of MKL1 in its regulation will also be determined and complexing proteins to critical domains will be isolated. RhoA signaling to SRF has been proposed to involve changes in actin filaments. However, mutations in RhoA argue against this model. This suggests that a novel RhoA effector is used to signal to SRF. Targets of RhoA involved in SRF activation will be purified using lack of binding to RhoA mutants as selection criteria. We have identified many serum inducible genes by microarray analysis and found that a subset is dependent upon MKL1 for its induction. It is unclear how many of the other genes are activated by TCF or even by SRF. We will develop cell lines defective in these factors and use microarrays to determine which mechanisms regulate the entire class of immediate early genes. This will likely identify genes activated independently of these factors. These genes will be mapped for novel regulatory factors to identify new signaling pathways. We also found in microarrays that there are many serum repressible genes. The mechanisms for this repression have not been extensively studied and many of the repressed genes have anti-proliferative effects. We will characterize regulation of these genes to identify new signaling pathways in the immediate early response.
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