This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Specimen-1 Autosomal dominant polycystic kidney disease (ADPKD or PKD) is the most common, monogenic cause of kidney failure in humans occurring in 1:1000 live births. It affects about 500,000 people in the United States and 4 to 6 million worldwide. PKD is caused by mutations in either of two genes, PKD1 and PKD2, with virtually the same clinical manifestations: the presence of hundreds of fluid filled cysts in kidneys, liver, pancreas and intestines. Renal cysts can grow up to 20 cm in size, causing gross enlargement of both kidneys and ultimately leading to end-stage renal failure. PKD is a systematic disorder often involving hypertension, mitral valve prolapse, intracranial aneurisms, hematuria, polyuria, predispostion to kidney stones, and urinary tract infections. The molecular mechanisms underlying PKD are unknown despite identification of the genes and mutations involved. PKD1 and PKD2, encode for the proteins, polycystin-1 (PC1) and polycystin-2 (PC2), respectively. PC1 is a large transmembrane protein possibly involved in cell-matrix and/or cell-cell interactions, while PC2 is a calcium (Ca2+) permeable cation channel of the transient receptor potential (TRP) family. There have to date been no crystal structures solved for either of these proteins. We will determine the structure of portions of these proteins to move towards a more complete understanding of the molecular basis for polycystic kidney disease. We are able to reproducibly grow three-dimensional crystals and have determined cryoprotectant conditions. At the home source our crystals have not diffracted, therefore, synchrotron data collection is important for these crystals. Specimen-2 Proper function of cytokine signaling pathways is critical for immunoregulation, hematopoiesis, and cytokine-directed inflammation and growth. Most signals from type I and type II cytokines are mediated by the Janus kinase (Jak) family of non-receptor tyrosine kinases. Our plan is to discover the molecular basis for cytoplasmic signal transduction from the interleukin-2 family cytokines through Jak3. We will use structural biology to provide the first description of Jak interactions with cytokine receptor cytoplasmic tails, to investigate Jak kinase domain specificity, regulation and inhibition, and to describe the role of the FERM domain in kinase activation. The Jak kinases (Janus) are dysregulated in multiple leukemias and lymphomas by point mutations and gene translocations (e.g. Tel-Jak2). They have also recently been shown to play an important, and perhaps critical, role in the acquisition of classical myeloproliferative disorders. Jak kinases are additionally abnormally activated by oncogenic tyrosine kinases (e.g. Bcr-Abl in chronic myelogenous leukemia) and by increased cytokine expression in certain malignancies (e.g. interleukin-6 overexpression in myeloma increases Jak activity). As these aberrant activations of Jak kinases are important steps in the propagation of deleterious signaling cascades there is potential for Jak-specific inhibitors to treat multiple cancers and other disorders. I solved the crystal structure of the kinase domain of Jak3, this was the first example of a crystal structure for any domain of any member of the Jak family. We are continuing this work by focusing on two areas, the determination of crystal structures of other domains of the Jak kinases and the determination of further kinase domain crystal structures. We are able to reproducibly grow three-dimensional crystals and have determined cryoprotectant conditions. At the home source our crystals have yielded diffraction to approximately 2.7 resolution. We would like to increase the resolution of these diffraction data.
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