This competing renewal focuses on the characterization of an intracellular receptor for the phorbol ester tumor promoters and the second messenger diacylglycerol (DAG), 2-chimaerin, and its relevance in the etiology of breast cancer. With the support of this RO1 we unambiguously established the concept of divergence in DAG signaling, which implies that DAG and phorbol ester tumor promoters can trigger responses via direct activation of effectors that do not belong to the protein kinase C (PKC) family, considered until not so long ago the only intracellular receptors for DAG and phorbol esters. 2-chimaerin possesses 3 well-defined regions: a N-terminal SH2 domain of unknown function (present only in a2- and 2-chimaerins), a C1 domain (responsible for DAG and phorbol ester binding), and a C-terminal GTPase Activating Protein (GAP) domain that has the ability to accelerate the hydrolysis of GTP from the small G-protein Rac, leading to Rac inactivation. We established that 2-chimaerin is a direct effector of the EGF receptor via the DAG pathway, and that activation of 2-chimaerin serves as a brake that limits the activation of Rac in response to tyrosine kinase receptor stimulation. Moreover, we determined that in breast cancer cells 2-chimaerin negatively modulates Rac1-mediated responses, including actin cytoskeleton reorganization, migration, invasion, proliferation, cell cycle progression, and cyclin D1 induction in response to ErbB receptor ligands, including EGF and heregulin. Remarkably, preliminary studies revealed that 2-chimaerin is down-regulated in human breast cancer cells and specimens, suggesting a potential tumor suppressor role for this DAG-regulated Rac-GAP. As an extension of our previous work we now propose 4 aims. In Aim 1 we will determine the relevance of -chimaerin in breast cancer progression using xenografts and also 1ith knock-out and knock-in mice for a hyperactive 2-chimaerin mutant (I130A allele). In Aim 2 we will analyze the expression of 2-chimaerin in a panel of human mammary tumors and their matched normal counterparts in order to validate our preliminary evidence that 2-chimaerin is down-regulated in breast cancer. We will also focus on promoter methylation as the potential cause for this down-regulation. In Aim 3 we will assess the regulation of 2-chimaerin activity and function by phosphorylation, as data revealed a PKC phosphorylation potentially modulates the Rac-GAP activity of 2-chimaerin. Lastly, in Aim 4 we will identify and validate potential proteins that interact with 2-chimaerin via the SH2 domain and determine the functional consequences of such interactons. The identification of a DAG/phorbol ester receptor unrelated to the PKC family that negatively regulates responses associated with the progression of breast cancer, and probably other neoplasias, not only has fundamental connotations in cancer signaling, but it may also reveal novel aspects of the etiology of cancer and have important therapeutic implications. PUBLIC HEALTH RELEVANCE: In this proposal we will characterize a receptor for the phorbol ester tumor promoters and the lipid second messenger diacylglycerol (DAG) named 2-chimaerin. We found that this protein negatively regulates important cellular functions involved in tumorigenesis and metastasis, and that it is down-regulated in breast cancer, suggesting a potential relationship with breast cancer etiology. This proposal will investigate the relevance of 2-chimaerin in breast cancer progression as well as key regulatory mechanisms that control its activation and function.