We use fission yeast (Schizosaccharo, nyces pombe) as a genetic tool to study conserved mechanisms that govern tumorigenesis in humans. In a study of the yeast Rasi G-protein, we have identified a yeast homolog of the mammalian 1NT6 gene. which we named yin6. INT6 was first identified as one of the key loci involved in the formation of breast cancer as it is a frequent site of integration by the mouse mammary tumor virus. Studies of other LVT genes have led to the discovery of three families of very important signaling proteins: WntI/IntI, Notch/Intl. and FGF3/lnt2, and contributed significantly to the understanding of tumorigenesis at the molecular level. Among all the INT genes, the function of INT6 is the least understood. Our data show that one of the key functions of Yin6 is to cooperate with Ras to regulate chromosome segregation: yin6 deletion (yin6) or ras1 alone weakly affects chromosome segregation, but yin6 together with ras1 induces severe chromosome missegregation. The function of Yin6 is conserved evolutionarily: full-length human Int6 rescues phenotypes of yin6 cells, while truncated Int6 proteins found in tumors do not. These data provided a novel insight into Int6 function and support a model whereby Int6 acts as a tumor suppressor, and its absence (alone or together with RAS conserved mechanisms by which Yin6 affects chromosome segregation in yeast. Our preliminary data demonstrate that Yin6 can genetically and physically interact with components of the proteasome and APC (Anaphase Promoting Complex), which are part of a critical and conserved machinery for controlling chromosome segregation. Thus, in Aim 1, we plan to determine whether the phenotypes caused by yin6 or ras1 indeed affect proteolysis and polyubiquitination.
In Aim 2, we plan to reveal the mechanisms by which Yin6 and Ras1 affect proteolysis. Our preliminary data indicate that yin6 causes at least one component in the proteasome to improperly assemble and to decrease in protein levels. Thus, we will investigate whether Yin6 and Ras1 can globally affect assembly, transport, or the protein levels of the proteasome and APC. Lastly, in Aim 3, we will perform genetic screens to isolate components in the proteasome and APC that interact with Yin6 in a cooperative fashion and to identify proteins whose degradation is Yin6-dependent. The results of our studies are intended to provide a thorough understanding of how Yin6 interacts with components that regulate chromosome segregation, and insight into the mechanism which Int6 may be involved in breast carcinogenesis.
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