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Bad Serine 170 - regulation and cellular effects

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Title: Bad Serine 170 - regulation and cellular effects
Author: Waissbluth, Ivan
Degree Doctor of Philosophy - PhD
Program Experimental Medicine
Copyright Date: 2009
Publicly Available in cIRcle 2009-11-12
Abstract: The balance between cell proliferation and cell death is imperative for homeostasis in multicellular organisms. This homeostasis had long been thought to be the result of two separate processes, but there is recent evidence indicating that the processes of proliferation and apoptosis are coupled. Here we demonstrate that Bad, a proapoptotic member of the Bcl-2 family of proteins that is thought to exert a deathpromoting effect by heterodimerization with Bcl-xL, is able to interact directly with the cell cycle machinery. Immunoprecipitation experiments indicate that Bad interacts with both Cdk2 and its late Gi cyclin partner, Cyclin E. This finding is relevant as we also demonstrate that prior to the conversion of Bad into a death factor; Bad’s phosphorylation state in healthy mammalian cells, specifically at Ser- 170, is able to influence cell cycle progression. Here we show that transfection with Bad Si 70A, a mutant form of Bad which mimics the unphosphorylated form, results in a prolonged S phase during cell cycle. We also show that the kinase activity towards Bad Ser- 170 increases in S phase of the cell cycle. Together this suggests that Bad Ser-170 is a phosphorylation site which is targeted during S phase and is able to interfere with the normal progression through S phase of the cell cycle. From this, it was of interest to elucidate the kinase responsible for phosphorylating Bad at Ser-170 as it may provide insight into signal transduction pathways that converge in terms of controlling both survival and cell proliferation, and ultimately cell expansion. Through a process of column purification, usage of chemical inhibitors, and gene knockdown, we show evidence that CaMKII-γ mediates the phosphorylation of Bad Ser 170, thus establishing a novel connection between CaMKII signaling and apoptosis in hematopoietic cells. We hypothesize that CaMKII-γ plays a major role in controlling Bad’s ability to induce apoptosis and to affect cell cycle progression, by controlling Bad’s phosphorylation state at Ser-170.
URI: http://hdl.handle.net/2429/14847

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