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The Koretzky Laboratory | Email Us
Abramson Family Cancer Research Institute
421 Curie Boulevard
427BRB II/III
Philadelphia, PA 19104-6160
Ph: (215) 746-5541/5542
Fax: (215) 746-5525
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Research
The immune system contains a variety of different types of cells which must work together to fend off invasions. The Signal Transduction Program is pursuing a better understanding of the molecular biology and biochemistry involved in the communication required.
Much has been learned in recent years about the most proximal (local) events which occur inside T lymphocyte cells after the receptors on the cells' surface engage with peptide fragments from foreign pathogens. A major challenge facing investigators today is to understand how these signals are integrated and regulated so that the cells appropriately become activated to fight the invasion.
Our laboratory makes use of cell-free biochemical systems, model cell lines, and whole animals which have been genetically manipulated to probe various signal transduction pathways. Over the past several years we have become particularly interested in the regulation and integration of second messenger cascades by adapter molecules, those proteins which possess no intrinsic enzymatic properties, but which function by bridging protein-protein interactions.
In an effort to understand better the regulation of signal transduction via the T cell antigen receptor (TCR), we have focused recently on identifying and characterizing novel substrates of the protein tyrosine kinases which are activated following engagement of the TCR. Several years ago, we obtained a full-length cDNA for one such molecule we designated "SH2 domain containing leukocyte phosphoprotein of 76 kD" or SLP-76. SLP-76 is a 533 amino acid protein whose expression is restricted to cells of hematopoietic origin. The primary sequence of the amino acids that make up SLP-76 reveals three domains capable of mediating interactions between proteins. Initial studies demonstrated that overexpression of SLP-76 dramatically enhances efficiency of TCR signaling. This observation, coupled with our work demonstrating that SLP-76 protein levels are regulated during T cell development and T cell activation, suggested to us that SLP-76 may play a critical role as an integrator of signaling pathways important for T cell activation. Support for this was obtained with the generation of SLP-76 deficient mice which demonstrate a complete lack of T cell development, presumably due to a defect in signal transduction mediated via the pre T cell receptor.
Current studies in the laboratory are aimed towards understanding the molecular mechanisms by which SLP-76 regulates T cell activation. Approaches we are taking include biochemical analyses to investigate the structural features of SLP-76 required for mediating intermolecular interactions, as well as genetic approaches expressing mutant variants of SLP-76 in animals to determine the impact on function. These in vivo studies make use of retroviral gene transfer gene technology, transgenic mice, and mice with mutations of SLP-76 "knocked-in" to endogenous locus.
Our studies of the SLP-76 deficient mice have revealed unexpected roles of this adapter protein in other hematopoietic lineages. We have found that in addition to defects in T cell development and activation, mast cells (those cells responsible for allergic reactions) also fail to function normally in the absence of this adapter. Neutrophils, a key component of the innate immune response, also require SLP-76 for their optimal activity as do platelets, a cell type critical for hemostasis. Projects in the laboratory are pursuing the molecular mechanism by which SLP-76 functions in each of these cell types. Interestingly, our data suggest that the structural features of SLP-76 most important in T cells, do not overlap completely with those regions of SLP-76 most critical in other cell types.
In addition to SLP-76 our laboratory is actively investigating the role of other adapter molecules in hematopoietic cell development and function. These include adhesion and degranulating promoting adapter protein (ADAP) a cytosolic adapter expressed coordinately with SLP-76. We identified ADAP initially as a binder of SLP-76 and have pursued the role of this molecule through a parallel series of investigations. The phenotype of ADAP deficient animals is particularly intriguing in that although many signaling cascades following T cell receptor (TCR) engagement are intact, the TCR fails to couple to adhesion molecules in ADAP deficient animals. Thus whereas in normal mice, engagement of the TCR increases stickiness of T cells due to increased avidity of various adhesions receptors, this response is lost in the absence of ADAP. To our knowledge, this is the first example of the role of an adapter molecule in upregulating adhesion receptor function. The laboratory is actively pursuing the molecular basis for this signaling pathway.
A third novel adapter molecule investigated by our laboratory is PML-RARalpha regulated adapter molecule 1 (PRAM-1). PRAM-1 was identified by virtue of homology to ADAP, but unlike ADAP, its expression is highest in myeloid cells. Interestingly, it appears that PRAM-1 associates with many of the same molecules which bind to ADAP. We are interested in uncovering the function of PRAM-1 in myeloid cells and have thus far generated mice heterozygous for PRAM-1 deletion.
Over the next several years our laboratory will continue to focus on the regulation of signal transduction in hematopoietic cells. In addition to the study of adapter proteins, we are interested in the role of enzymes which regulate cellular activation. A new project for the laboratory involves evaluation of regulators of the phosphotidylinositol second messenger cascade, a second messenger system required for activation in many cell types. Other projects are designed to translate the work occurring at the bench to more clinically relevant situations as it is becoming increasingly clear that the regulation of signal transduction is critical for understanding both basic biological processes as well as how these events may go awry leading to disease. |
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