The long term goal of this renewal grant is to understand the role of RhoGTPases and their regulators in hematopoiesis, hematopoietic cell function and hematopoietic malignancies. RhoGTPases (Rho/Rac/CDC42) have emerged as a family of proteins of central importance. Together, they regulate a diverse array of fundamental cellular functions including cytoskeletal organization, membrane-trafficking, motility, gene transcription, cell division and differentiation. A key molecular mechanism upon which rests the operation of a GTPase is its cycling between an inactive GDP-bound an active GTP-bound state. GTPases, activated by external cellular cues, must be directed to proper intracellular locations where they act transiently followed by a switching to an inactive state again. Little is known about how these signals are transmitted and coordinated. GDP-Dissociation Inhibitors are one of the three key regulators of the molecular switch. As powerful inhibitors of the dissociation of GDP, RhoGDIs play critical role in modulating levels and location of GTPases. The objectives of the previous grant were a) to disrupt RhoGDIbeta, a GDI which is preferentially expressed in hematopoietic cells, and to study the consequences on ES cell differentiation; b) to search for other GDI interacting proteins; and c) to investigate the possible tumor suppressor effect of RhoGDIs. Phagocytes derived from RhoGDIbeta-/- ES were found to have a defect in their superoxide production. RhoGDIbeta- /- animals have now been generated. A putative RhoGDI-interacting protein has been identified that is identical to a novel serine/ threonine phosphatase of unknown function. The gene for RhoGDI- interacting protein has been identified that is identical to a novel serine/threonine phosphatase of unknown function. The gene for Rho H, a new hematopoietic specific RhoGTPase, has been found to be involved in non-random translocations in lymphoma and myeloma. This is the first example of a RhoGTPase mutation in human disease and raises the question whether there may be other examples.
The specific aims i n this renewal proposal are:
Aim 1) to phenotype RhoGDIbeta-/- animals, to compare superoxide production in tissues of wildtype versus mutant animals and to test for the effect of such differences on oncogenic challenge; to disrupt the ubiquitous RhoGDIalpha gene and phenotype the animals; to plan to use the two mutants to derive animals with dual-GDI knockout;
Aim 2) to decipher the function of the novel serine/threonine phosphatases that was found to interact with RhoGDIs; 3) to further investigate how GDIs affect cell division and transformation by oncogenes, to test whether RhoH can transform factor dependent hematopoietic cells, and to initiate a search in data base for more evidence of involvement of RhoGTPase and their regulators in human diseases. The immediate aims of this renewal proposal will continue to inform us about how RhoGTPase pathways converge upon the biochemical steps of carcinogenesis and metastasis. The expectations and long term goals are to build upon this information and identify specific steps and molecules that promote growth and dissipation of cancer cells and to design drugs that will stop this pathological process.
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