TY  - JOUR
A1  - Frank, Daniel O.
A1  - Dengjel, Jörn
A1  - Wilfling, Florian
A1  - Kozjak-Pavlovic, Vera
A1  - Häcker, Georg
A1  - Weber, Arnim
T1  - The Pro-Apoptotic BH3-Only Protein Bim Interacts with Components of the Translocase of the Outer Mitochondrial Membrane (TOM)
JF  - PLoS ONE
N2  - The pro-apoptotic Bcl-2-family protein Bim belongs to the BH3-only proteins known as initiators of apoptosis. Recent data show that Bim is constitutively inserted in the outer mitochondrial membrane via a C-terminal transmembrane anchor from where it can activate the effector of cytochrome c-release, Bax. To identify regulators of Bim-activity, we conducted a search for proteins interacting with Bim at mitochondria. We found an interaction of Bim with Tom70, Tom20 and more weakly with Tom40, all components of the Translocase of the Outer Membrane (TOM). In vitro import assays performed on tryptically digested yeast mitochondria showed reduced Bim insertion into the outer mitochondrial membrane (OMM) indicating that protein receptors may be involved in the import process. However, RNAi against components of TOM (Tom40, Tom70, Tom22 or Tom20) by siRNA, individually or in combination, did not consistently change the amount of Bim on HeLa mitochondria, either at steady state or upon de novo-induction. In support of this, the individual or combined knockdowns of TOM receptors also failed to alter the susceptibility of HeLa cells to Bim-induced apoptosis. In isolated yeast mitochondria, lack of Tom70 or the TOM-components Tom20 or Tom22 alone did not affect the import of Bim into the outer mitochondrial membrane. In yeast, expression of Bim can sensitize the cells to Bax-dependent killing. This sensitization was unaffected by the absence of Tom70 or by an experimental reduction in Tom40. Although thus the physiological role of the Bim-TOM-interaction remains unclear, TOM complex components do not seem to be essential for Bim insertion into the OMM. Nevertheless, this association should be noted and considered when the regulation of Bim in other cells and situations is investigated.
KW  - bax
KW  - preproteins
KW  - phosphorylation
KW  - proteomics
KW  - degradation
KW  - cells
KW  - family
KW  - import
KW  - BH3 domains
KW  - Bcl-2 proteins
Y1  - 2015
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-143301
VL  - 10
IS  - 4
ER  - 
TY  - JOUR
A1  - Eisenhardt, Anja E.
A1  - Sprenger, Adrian
A1  - Röring, Michael
A1  - Herr, Ricarda
A1  - Weinberg, Florian
A1  - Köhler, Martin
A1  - Braun, Sandra
A1  - Orth, Joachim
A1  - Diedrich, Britta
A1  - Lanner, Ulrike
A1  - Tscherwinski, Natalja
A1  - Schuster, Simon
A1  - Dumaz, Nicolas
A1  - Schmidt, Enrico
A1  - Baumeister, Ralf
A1  - Schlosser, Andreas
A1  - Dengjel, Jörn
A1  - Brummer, Tilman
T1  - Phospho-proteomic analyses of B-Raf protein complexes reveal new regulatory principles
JF  - Oncotarget
N2  - B-Raf represents a critical physiological regulator of the Ras/RAF/MEK/ERK-pathway and a pharmacological target of growing clinical relevance, in particular in oncology. To understand how B-Raf itself is regulated, we combined mass spectrometry with genetic approaches to map its interactome in MCF-10A cells as well as in B-Raf deficient murine embryonic fibroblasts (MEFs) and B-Raf/Raf-1 double deficient DT40 lymphoma cells complemented with wildtype or mutant B-Raf expression vectors. Using a multi-protease digestion approach, we identified a novel ubiquitination site and provide a detailed B-Raf phospho-map. Importantly, we identify two evolutionary conserved phosphorylation clusters around T401 and S419 in the B-Raf hinge region. SILAC labelling and genetic/biochemical follow-up revealed that these clusters are phosphorylated in the contexts of oncogenic Ras, sorafenib induced Raf dimerization and in the background of the V600E mutation. We further show that the vemurafenib sensitive phosphorylation of the T401 cluster occurs in trans within a Raf dimer. Substitution of the Ser/Thr-residues of this cluster by alanine residues enhances the transforming potential of B-Raf, indicating that these phosphorylation sites suppress its signaling output. Moreover, several B-Raf phosphorylation sites, including T401 and S419, are somatically mutated in tumors, further illustrating the importance of phosphorylation for the regulation of this kinase.
KW  - BRAF
KW  - proteomics
KW  - phosphorylation
KW  - sorafenib
KW  - protein-protein interaction
Y1  - 2016
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166529
VL  - 7
IS  - 18
ER  -