How the extracellular environment influences intracellular signaling pathways that regulate cell growth and differentiation has been a major challenge over the past decade. Mutations in genes controlling these processes result in cellular transformation and cancer development. A family of proteins containing LIM domains has been recognized as playing important roles in the control of gene expression, cell fate determination, remodeling of the cytoskeleton, and as potential mediators of communication between the cytosol and the nucleus in response to extracellular signals. LIM domains function as versatile protein modules, capable of acting within diverse cellular contexts and in multiple subcellular compartments. Many have been shown to participate in direct protein-protein interactions. LIM domain-containing proteins have been classified according to sequence homologies among the LIM domains and overall protein structure. Group 3 proteins are cytosolic and contain 3 to 4 tandem LIM domains at the C-terminus in association with distinct N-terminal domains. Some are components of fusion proteins derived from chromosomal translocation present in some cancers. A major challenge is to understand how group 3 LIM proteins couple extracellular stimuli to specific cellular responses. The PI has identified and characterized a novel group 3 LIM protein, Ajuba (""""""""curiosity"""""""" in Urdu, an Indian dialect). Ajuba shuttles between the cytoplasm and the nucleus of cells. Deletion of a leucine-rich nuclear export signal in Ajuba results in an accumulation of Ajuba in the cell nucleus. In embryonal carcinoma cells nuclear accumulation of Ajuba results in a cell growth arrest and spontaneous differentiation. Ajuba associates with the cytosolic adapter protein Grb2 leading to a Ras-dependent activation of mitogen-activated protein kinase activity. When expressed in Xenopus oocytes, Ajuba promotes meiotic maturation. Thus Ajuba is a cytosolic protein that transduces signals to the nucleus in response to extracellular stimuli. In this proposal the PI has designed experiments to 1) determine how Ajuba alters MAPK signaling pathways; 2) determine how Ajuba regulates multipotent embryonal carcinoma cell proliferation and differentiation; 3) determine the signals regulating cytosolic-nuclear cytosolic translocation of Ajuba; 4) identify cytosolic and nuclear proteins that interact with Ajuba and determine how these interactions affect Ajuba function in specific subcellular compartments.
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