Bcl-xL/xS phosphorylation regulates the sensitivity of PC12 cells to apoptosis
| dc.contributor.advisor | Mousseau, Darrell D. | en_US |
| dc.contributor.committeeMember | Li, Xin-Min | en_US |
| dc.contributor.committeeMember | Geyer, Ron | en_US |
| dc.contributor.committeeMember | Misra, Vikram | en_US |
| dc.contributor.committeeMember | Walz, Wolfgang | en_US |
| dc.creator | Qi, Ji | en_US |
| dc.date.accessioned | 2010-01-11T14:33:11Z | en_US |
| dc.date.accessioned | 2013-01-04T04:23:47Z | |
| dc.date.available | 2011-01-19T08:00:00Z | en_US |
| dc.date.available | 2013-01-04T04:23:47Z | |
| dc.date.created | 2009-12 | en_US |
| dc.date.issued | 2009-12-19 | en_US |
| dc.date.submitted | December 2009 | en_US |
| dc.description.abstract | The Bcl-2 family of proteins contains both anti-apoptotic (e.g.Bcl-2, Bcl-xL) and pro-apoptotic (e.g.Bad, Bcl-xS) proteins. The Bcl-xL and Bcl-xS are splice variants, but have different functions during apoptosis. The pro-survival kinase Akt can phosphorylate certain Bcl-2-related proteins, specifically on serine residues, to regulate their function and localization. This is an extension of the work from our laboratory’s finding that haloperidol induces PC12 cell death by inducing Bcl-xS which then translocates from cytosol to mitochondria where it facilitates the release of cytochrome c. The toxicity induced by Bcl-xS is reversed by expression of constitutively active Akt. I hypothesized that Akt-mediated post-translational modification may be important for regulating the function of Bcl-xS and Bcl-xL. Three specific serine residues were ultimately chosen for the characterization of Bcl-xS/xL function: Ser62 (inactivation mutant), Ser106 (putative Akt phosphorylation motif), and Ser165 in Bcl-xS (and the corresponding Ser228 in Bcl-xL) (immediately upstream of hydrophobic tail). The individual substitution of all three Serines with Alanines (which precludes phosphorylation at that site) in Bcl-xS did not affect the expression of the protein, but they did induce varying degrees of cytotoxicity in both PC12 and HEK cultures. I focused on the Ser106 substitution mutant given my hypothesis that Akt targeted this site. Overexpression of Bcl-xS(S106A) was toxic in both PC12 and HEK cultures, as expected, and this coincided with the appearance of the Bcl-xS(S106A) protein in the mitochondrial fraction. The release of cytochrome c from PC12 cell mitochondria coincided with the co-immunoprecipitation of the Bcl-xS protein with VDAC (voltage-dependent anion channel), a channel-forming protein that is known to mediate cytochrome c release, and with the initiation of caspase-dependent events. This was not the case in HEK cells, where the mitochondrial VDAC seemed to be diminished and the toxicity was cytochrome c-independent as well as caspase-independent. In addition, I was able to demonstrate that the S106A substituted protein was not able to co-immunoprecipitate with Akt, supporting Ser106 as a potential target for the Akt protein. I then studied the effects of the homologous substitutions in Bcl-xL on cell function. I chose to use treatment with the potent inducer of apoptosis, staurosporine, as a model of cytotoxicity. Again, substituted proteins exerted toxicity, but they did not potentiate the effects of staurosporine, at least not on MTT conversion. I did notice, however, that there was a clear morphological change with certain concentrations of staurosporine, and subsequently demonstrated that the Bcl-xL(S106A) protein potentiated PC12 cell differentiation induced by staurosporine. This protein also co-immunoprecipitated better with Akt, which was unexpected given my results with the Bcl-xS(S106A) protein described above. Perhaps the extra amino acids in Bcl-xL account for this. It is clear that the phosphorylation of Bcl-xS and Bcl-xL proteins is an important means of regulating their function and localization within the cell. These data support the S106 residues in both Bcl-xS and Bcl-xL as novel targets for the pro-survival Akt kinase, and indicate a role for this/these residue(s) in cellular functions as diverse as apoptosis and differentiation. | en_US |
| dc.identifier.uri | http://hdl.handle.net/10388/etd-01112010-143311 | en_US |
| dc.language.iso | en_US | en_US |
| dc.subject | Apoptosis | en_US |
| dc.subject | PC12 Cells | en_US |
| dc.subject | Staurosporine | en_US |
| dc.subject | Akt | en_US |
| dc.subject | BCL-xL/xS | en_US |
| dc.title | Bcl-xL/xS phosphorylation regulates the sensitivity of PC12 cells to apoptosis | en_US |
| dc.type.genre | Thesis | en_US |
| dc.type.material | text | en_US |
| thesis.degree.department | Psychiatry | en_US |
| thesis.degree.discipline | Psychiatry | en_US |
| thesis.degree.grantor | University of Saskatchewan | en_US |
| thesis.degree.level | Masters | en_US |
| thesis.degree.name | Master of Science (M.Sc.) | en_US |