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Phospho-proteomic analyses of B-Raf protein complexes reveal new regulatory principles.

ABSTRACT: 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.

SUBMITTER: Eisenhardt AE

PROVIDER: S-EPMC5042004 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Phospho-proteomic analyses of B-Raf protein complexes reveal new regulatory principles.

Eisenhardt Anja E AE Sprenger Adrian A Röring Michael M Herr Ricarda R Weinberg Florian F Köhler Martin M Braun Sandra S Orth Joachim J Diedrich Britta B Lanner Ulrike U Tscherwinski Natalja N Schuster Simon S Dumaz Nicolas N Schmidt Enrico E Baumeister Ralf R Schlosser Andreas A Dengjel Jörn J Brummer Tilman T

Oncotarget 20160501 18

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 m ...[more]

PMID: 27034005

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Project description:One of the barriers to successful nonviral gene delivery is the crowded cytoplasm, which plasmids need to actively traverse for gene expression. Relatively little is known about how this process occurs, but our lab and others have shown that the microtubule network and motors are required for plasmid movement to the nucleus. To further investigate how plasmids exploit normal physiological processes to transfect cells, we have taken a proteomics approach to identify the proteins that comprise the plasmid-trafficking complex. We have developed a live cell DNA-protein pull-down assay to isolate complexes at certain time points post-transfection (15 minutes to 4 hours) for analysis by mass spectrometry (MS). Plasmids containing promoter sequences bound hundreds of unique proteins as early as 15 minutes post-electroporation, while a plasmid lacking any eukaryotic sequences failed to bind many of the proteins. Specific proteins included microtubule-based motor proteins (e.g., kinesin and dynein), proteins involved in protein nuclear import (e.g., importin 1, 2, 4, and 7, Crm1, RAN, and several RAN-binding proteins), a number of heterogeneous nuclear ribonucleoprotein (hnRNP)- and mRNA-binding proteins, and transcription factors. The significance of several of the proteins involved in protein nuclear localization and plasmid trafficking was determined by monitoring movement of microinjected fluorescently labeled plasmids via live cell particle tracking in cells following protein knockdown by small-interfering RNA (siRNA) or through the use of specific inhibitors. While importin ?1 was required for plasmid trafficking and subsequent nuclear import, importin ?1 played no role in microtubule trafficking but was required for optimal plasmid nuclear import. Surprisingly, the nuclear export protein Crm1 also was found to complex with the transfected plasmids and was necessary for plasmid trafficking along microtubules and nuclear import. Our results show that various proteins involved in nuclear import and export influence intracellular trafficking of plasmids and subsequent nuclear accumulation.

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Phospho-proteomic analyses of B-Raf protein complexes reveal new regulatory principles.

Publications

Phospho-proteomic analyses of B-Raf protein complexes reveal new regulatory principles.

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