Regulation of Puma by degradation, complex formation, posttranslational modifications and cooperation with other BH3-only proteins

Abstract

It is widely accepted that apoptosis induced by genotoxic or ER stress as well as by cytokine deprivation requires the transcriptional up-regulation of the BH3-only protein Puma. Puma then acts in two ways. It either binds with high affinity to the hydrophobic pocket of Bcl-2-like survival factors or it directly activates Bax/Bak by transient interaction, leading to their oligomerization and pore-forming activity on the outer mitochondrial membrane (MOMP), a prerequisite for cytochrome c release, caspase activation and apoptosis. However, we and others found that Puma is not only regulated on the transcriptional but also on the posttranscriptional/posttranslational level. In this case, the question is whether Puma is only sequestered by Bcl-2-like survival factors, which prevent its direct Bax/Bak activating function, or whether other cellular proteins neutralize Puma. Gel filtration analysis and blue native gel electrophoresis have shown that in healthy cells Puma forms high molecular mass complexes of up to 400 kD which largely exceed the predicted molecular masses of a Puma/Bcl-2 dimer. Hence, additional, so far unknown proteins must keep Puma in check in healthy cells. One of our goals is therefore to identify these proteins by co-immunoprecipitation/mass spectrometry analysis, determine their binding properties /domains and stoichiometry in the Puma complex and unravel their functional impact on Puma and Bax/Bak activation. Another way to regulate Puma is by posttranslational modifications (PTMs). So far only two PTMs have been described for Puma in healthy cells, a single phosphorylation at S10 by IKK2/Nemo and a triple phosphorylation at Y58/Y152/Y172 by HER2. Both modifications destabilize the Puma protein leading to its degradation by the proteasome. However, it is still unclear whether the degradation occurs through ubiquitination (Puma does not contain any lysines) and which enzymes are involved in this process. Finally, PTMs that would stabilize/activate Puma, such as phosphorylation at S100/T112/T114 on Bim have not yet been described either. We are therefore in the process of identifying new PTMs of Puma by mass spectrometry analysis, mutate the sites and study their impact on Puma complex formation and Bax/Bak activation in different apoptosis setting in which Puma is crucial.

Scientific Goals

  1. Identify Puma binding partners in healthy and apoptotic cells and decipher their functions
  2. Identify posttranslational modifications (PTMs) of Puma in healthy and apoptotic cells and decipher their functions
  3. Identify the degradation mechanism of Puma in healthy cells

  4. Unravel the mechanism by which Puma binds to and is released from Bcl-2-like survival factors in apoptotic cells

Strategies to identify novel Puma binding proteins by the SILAC (A) or BioID (B) methods followed by mass spectrometry. The lower panel in (B) is anti-Puma or anti-Mcl-1 Western blot analysis of a streptavidin pull down of an extract from Puma-/- MEFs reconstituted with either the expression vector (ev), BirA* alone or a BirA*-Puma fusion protein. Details of the methods are described in the text.

People

Christoph Borner (PI, Professor)

Simon Neumann (Postdoc)

Anusha Venkatramen (PhD student)

Cooperation within FOR2036

Georg Häcker, Ana Garcia-Saez, Markus Morrison, Thomas Kaufmann, Thomas Brunner, Andreas Villunger


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  • Christoph Borner