Chromosomal rearrangements that fuse NH2-terminal sequences of the Bcr protein to COOH-terminal sequences of the Abl tyrosine kinase (Bcr-Abl) are associated with virtually all cases of chronic myelogenous leukemia (CML). In preliminary studies we have identified and verified an interaction between Bcr and the TSG101 tumor suppressor protein. TSG101 is a component of the ESCRT1 complex which recognizes and partitions endosomal cargo into the multivesicular body. The association with TSG101 suggests that Bcr may also be a component of the endosomal trafficking machinery. Since defects in endosomal trafficking have been described in the context of transformation, disruption of the Bcr-TSG101 association may be relevant to CML.
The aim of this proposal is to determine whether Bcr is a bona fide regulator of endosomal trafficking, and to determine whether this process is disrupted in the context of Bcr-Abl transformed cells. Additionally, we will examine the possibility that Bcr regulates the steady-state levels of TSG101. Using a combination of GST-based in vitro binding assays, and indirect immunofluorescence, we will determine whether Bcr physically localizes with the endosomal sorting machinery. To examine the functional consequences of this interaction, we will suppress Bcr expression by siRNAs and determine whether endosomal sorting is disrupted in HeLa cells, as well as in Bcr-Abl positive cells. Finally, we will determine if TSG101 steady state levels are sensitive to Bcr dosage, and whether this effect is mediated by ubiquitination. If the interaction between Bcr and TSG101 is physiologically relevant, these studies may uncover a mitigating factor in Bcr-Abl-mediated transformationthat was not previously appreciated.
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