"Signal integration by phosphorylation and complex formation of Bim and Puma"People involved: Christoph Borner, Simon Neumann
The exact mechanism by which the two pro-apoptotic BH3-only proteins, Bim and Puma induce Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP) is still unclear. While it is known that they bind to Bcl-2-like survival factors with high affinity, there has been emerging genetic, biochemical and recent structural evidence that they can directly interact with Bax or Bak to stimulate their MOMP formation capacity. The details of this interaction and how it is mechanistically performed inside cells have however remained elusive. Apart from transcriptional induction by stimuli like growth factor deprivation or genotoxic stress, both Bim and Puma are present in high molecular protein complexes whose components, apart from Bcl-2-like survival factors, have not yet been identified. In addition, Bim and Puma are both phosphorylated in healthy and apoptotic cells but the phosphorylation sites, the protein kinases and their implications in apoptotic responses are either unknown or need to be better characterized. Here we propose to identify phosphorylation sites of Bim and Puma by biochemical and established proteomics methods, generate mutants of these sites and test them for their binding efficiency to Bcl-2-like survival factors as well as to pro-apoptotic Bax, Bak and Bok. Moreover, their apoptotic potential will be tested in various cellular systems in vitro, and as an outlook to the second funding phase, their impact on hematopoietic neoplasm formation (CLL and sAML) will be studied in vivo. In addition, we want to identify novel binding partners of Puma in healthy and apoptotic cells and test by overexpression and shRNA-mediated knockdown strategies as well by using an in vitro mitochondrial MOMP assay if they act as sequestering proteins keeping Puma inactive at particular subcellular sites or as auxiliary factors for the proapoptotic function of Puma. Our analysis on Puma phosphorylation will tell us if this posttranslational modification modulates the interaction(s) between Puma and its binding partners, just as it was shown for Bim and dynein light chain 1 (DLC1).
Co-operations within FOR2036:
Georg Haecker, Arnim Weber, Freiburg
Thomas Kaufmann, Bern
Tancred Frickey, Konstanz
Thomas Brunner, Konstanz
Ana Garcia-Saez, Tuebingen