Publikationsserver der RWTH Aachen University
Implication of Bcl-2 family members in apoptosis signalling pathways triggered by growth factor deprivation
Abstract
dc:descriptionApoptosis or programmed cell death is a conserved cellular suicide program that selectively eliminates excess or potentially dangerous cells. To elucidate the signalling pathways implicated in the regulation of apoptosis and proliferation, cytokine withdrawal was used as a physiological stimulus to trigger programmed cell death. By means of representational difference analysis, the short isoform of the integral membrane protein (ITM2Bs) was identified in the present study as a novel regulator of cell death that was upregulated upon IL-2 deprivation. ITM2Bs was shown to belong to the BH3-only members of the Bcl-2 family and promoted apoptosis by antagonizing the pro-survival function of Bcl-2 protein. Biochemical and confocal studies demonstrated that association of ITM2Bs to mitochondria correlated with loss of mitochondrial membrane potential, release of cytochrome c into the cytosol as well as activation of up- and downstream caspases suggesting signalling via mi! tochondrial-mediated pathways. Functional interactions between proteins play a pivotal role in signal transduction. In this work, a novel function has been uncovered for the lymphoid-specific transcription factor Aiolos that was found to be associated with the pro-survival Bcl-xL protein in IL-4-deprived, but not IL-4-stimulated T-lymphocytes. The phosphorylation status appeared to be important since dephosphorylation of Aiolos and Bcl-xL increased their binding affinity following IL-4 withdrawal. These data propose a model, in which the anti-apoptotic effects of Bcl-xL are blocked through its sequestration by unphosphorylated Aiolos. Moreover, the anti-apoptotic proteins Bcl-xL and Bcl-w were identified as targeting subunits of the serine/threonine phosphatase PP1alpha in IL-4-stimulated T-lymphocytes as well as in freshly isolated thymocytes, replacing Bcl-2 as regulatory subunit of PP1alpha in IL-2-stimulated cells. Using co-immunoprecipitation assays, Bcl-xL or Bcl-w were shown to interact with PP1alpha and Bad forming two types of trimolecular complexes, Bcl-xL/PP1alpha/Bad and Bcl-w/PP1alpha/Bad. Depletion of Bcl-xL and Bcl-w resulted in loss of the interaction of PP1alpha and its substrate Bad, thus preventing dephosphorylation of Bad and subsequently programmed cell death. In addition to phosphorylation, the function of the pro-apoptotic Bad is also regulated by subcellular localization. It was demonstrated that a major fraction of Bad was localized in rafts upon IL-4 stimulation. This pool of Bad was dephosphorylated, but it was unable to exert its cell death-inducing function due to its sequestration in these plasma membrane microdomains. Confocal microscopy studies provided evidence that IL-4 deprivation triggered disorganization of lipids rafts, resulting in the segregation of dephosphorylated Bad from the plasma membrane and subsequent translocation to mitochondria correlating with T-lymphocyte death.
Degree
thesis:*- Grantor dc:publisher
- Publikationsserver der RWTH Aachen University
- Year dc:date
- 2003
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Fleischer, Aarne
- Contributors dc:contributor
-
- Rebollo, Angelita
Subjects
dc:subject × 7Rights
dc:rights- Statement dc:rights
-
- info:eu-repo/semantics/openAccess
- Language dc:language
- eng