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Scaffold proteins and T cell signaling

The role of scaffold proteins in T cell signaling: T cell antigen recognition is associated with the formation of a structured interface between antigen-presenting and responding cells that is believed to facilitate the specific transmission of activating and desensitizing stimuli. Engagement of the T cell antigen receptor (TCR) during antigen presentation initiates a sensitive, highly regulated response that relies on the coordinated action of a large number of signaling proteins. Recent evidence has shown that extensive rearrangements of membrane and cytoskeletal elements attend the activation response, and agents that disrupt the organization or localization of these elements interfere with antigen recognition. These observations led to our interest in identifying molecules capable of coordinating a large numbers of dissimilar signaling molecules and coupling membrane events to the underlying cytoskeleton.

Scaffold proteins with PDZ domains are known to coordinate multiple signaling molecules and to couple membrane events to the underlying cytoskeleton. Using genetic and molecular approaches we are investigating the role of PDZ domain containing scaffold proteins in T cell receptor signaling. We are particularly interested in understanding how scaffold proteins modulate positive and negative signaling outputs following T cell receptor activation. We have generated knockout ES cells and mice of a number of novel PDZ domain proteins 1) To define the biochemical functions of these proteins 2) To track signaling in vivo using GFP/RFP tagged proteins. 3) To elucidate the roles of these proteins in mouse models of inflammation.

Application of high throughput methods of modern genomic biology to discover novel activators of Serum response element: Inappropriate activation of these signaling pathways has important consequences that result in neoplasia. To discover novel molecules that activate diverse cell signaling pathways, we utilize a simple automated method based on sib-selection screening of arrayed collections of robotically picked bacterial colonies harboring mammalian cDNAs.. Pools of cDNA are transfected into reporter cell lines that express a basal promoter that is very strongly induced when response elements such as Serum response element (SRE) , p53 and HIF-1_ are placed upstream. Luciferase or GFP is placed downstream of the synthetic promoter. To date we have been successful at isolating new and known genes that induce the serum response element from cDNA libraries prepared from normal tissues. Genes discovered as activators of these pathways will then be confirmed using RNA interference and dominant negative epistasis studies. Ultimately, we would like to use this information to identify molecular targets that would interfere with inflammation and neoplasia

References:

1. Xavier R, Rabizadeh S, Ishiguro K, Andre N, Bernabe Ortiz J, Wachtel W, Shaw A, Morris M, Lopez-Ilasaca M, Swat W, Seed B. Journal of Cell Biology 2004 ( in press)

2. Rabizadeh S*, Xavier R*, Ishiguro K, Benabeortiz J. Lopez-Ilasaca M, Khokhlatchev A, Mollahan P. Pfeifer GP. Avruch J, Seed B. A common scaffold for the proliferative and anti-proliferative programs of Ras. JBC 2004 (in press)

3. Ishiguro K, Xavier R. Homer-3 regulates activation of serum response element in T cells via its EVH1 domain. Blood. 2004. 15;103(6):2248-56

4. Xavier R, Brennan T, Li Q, McCormack C, Seed B. Membrane compartmentation is required for efficient T cell activation. Immunity 1998:8;1-20.

5. Fukumura D*, Xavier R*, Sugiura T, Chen Y, Park EC et al. Tumor Induction of VEGF promoter activity in stromal cells. Cell 1998:94:715-725.


	Ramnik Xavier, M.D., Ph.D Scaffold proteins and T cell signaling The role of scaffold proteins in T cell signaling: T cell antigen recognition is associated with the formation of a structured interface between antigen-presenting and responding cells that is believed to facilitate the specific transmission of activating and desensitizing stimuli. Engagement of the T cell antigen receptor (TCR) during antigen presentation initiates a sensitive, highly regulated response that relies on the coordinated action of a large number of signaling proteins. Recent evidence has shown that extensive rearrangements of membrane and cytoskeletal elements attend the activation response, and agents that disrupt the organization or localization of these elements interfere with antigen recognition. These observations led to our interest in identifying molecules capable of coordinating a large numbers of dissimilar signaling molecules and coupling membrane events to the underlying cytoskeleton. Scaffold proteins with PDZ domains are known to coordinate multiple signaling molecules and to couple membrane events to the underlying cytoskeleton. Using genetic and molecular approaches we are investigating the role of PDZ domain containing scaffold proteins in T cell receptor signaling. We are particularly interested in understanding how scaffold proteins modulate positive and negative signaling outputs following T cell receptor activation. We have generated knockout ES cells and mice of a number of novel PDZ domain proteins 1) To define the biochemical functions of these proteins 2) To track signaling in vivo using GFP/RFP tagged proteins. 3) To elucidate the roles of these proteins in mouse models of inflammation. Application of high throughput methods of modern genomic biology to discover novel activators of Serum response element: Inappropriate activation of these signaling pathways has important consequences that result in neoplasia. To discover novel molecules that activate diverse cell signaling pathways, we utilize a simple automated method based on sib-selection screening of arrayed collections of robotically picked bacterial colonies harboring mammalian cDNAs.. Pools of cDNA are transfected into reporter cell lines that express a basal promoter that is very strongly induced when response elements such as Serum response element (SRE) , p53 and HIF-1_ are placed upstream. Luciferase or GFP is placed downstream of the synthetic promoter. To date we have been successful at isolating new and known genes that induce the serum response element from cDNA libraries prepared from normal tissues. Genes discovered as activators of these pathways will then be confirmed using RNA interference and dominant negative epistasis studies. Ultimately, we would like to use this information to identify molecular targets that would interfere with inflammation and neoplasia References: 1. Xavier R, Rabizadeh S, Ishiguro K, Andre N, Bernabe Ortiz J, Wachtel W, Shaw A, Morris M, Lopez-Ilasaca M, Swat W, Seed B. Journal of Cell Biology 2004 ( in press) 2. Rabizadeh S, Xavier R, Ishiguro K, Benabeortiz J. Lopez-Ilasaca M, Khokhlatchev A, Mollahan P. Pfeifer GP. Avruch J, Seed B. A common scaffold for the proliferative and anti-proliferative programs of Ras. JBC 2004 (in press) 3. Ishiguro K, Xavier R. Homer-3 regulates activation of serum response element in T cells via its EVH1 domain. Blood. 2004. 15;103(6):2248-56 4. Xavier R, Brennan T, Li Q, McCormack C, Seed B. Membrane compartmentation is required for efficient T cell activation. Immunity 1998:8;1-20. 5. Fukumura D, Xavier R, Sugiura T, Chen Y, Park EC et al. Tumor Induction of VEGF promoter activity in stromal cells. Cell 1998:94:715-725.