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

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.

[1]  C. Pritchard,et al.  Regulation of BRAF protein stability by a negative feedback loop involving the MEK–ERK pathway but not the FBXW7 tumour suppressor , 2016, Cellular signalling.

[2]  S. Fröhling,et al.  Cooperation of BRAFF595L and mutant HRAS in histiocytic sarcoma provides new insights into oncogenic BRAF signaling , 2016, Leukemia.

[3]  S. Braun,et al.  Activation loop phosphorylation regulates B‐Raf in vivo and transformation by B‐Raf mutants , 2016, The EMBO journal.

[4]  M. Reth,et al.  Kidins220/ARMS binds to the B cell antigen receptor and regulates B cell development and activation , 2015, The Journal of experimental medicine.

[5]  M. Therrien,et al.  Regulation of RAF protein kinases in ERK signalling , 2015, Nature Reviews Molecular Cell Biology.

[6]  E. Schadt,et al.  Massive parallel sequencing uncovers actionable FGFR2–PPHLN1 fusion and ARAF mutations in intrahepatic cholangiocarcinoma , 2015, Nature Communications.

[7]  N. Carragher,et al.  Paradox-Breaking RAF Inhibitors that Also Target SRC Are Effective in Drug-Resistant BRAF Mutant Melanoma , 2015, Cancer cell.

[8]  Marc Therrien,et al.  Crystal structure of a BRAF kinase domain monomer explains basis for allosteric regulation , 2014, Nature Structural &Molecular Biology.

[9]  P. Poulikakos,et al.  Targeting RAS–ERK signalling in cancer: promises and challenges , 2014, Nature Reviews Drug Discovery.

[10]  A. Shoushtari,et al.  GNAQ and GNA11 mutations in uveal melanoma , 2014, Melanoma research.

[11]  K. Aktories,et al.  Noncanonical G-Protein-Dependent Modulation of Osteoclast Differentiation and Bone Resorption Mediated by Pasteurella multocida Toxin , 2014, mBio.

[12]  S. Fröhling,et al.  BRAF inhibitor-associated ERK activation drives development of chronic lymphocytic leukemia. , 2014, The Journal of clinical investigation.

[13]  Ariana Peck,et al.  Structure of the BRAF-MEK complex reveals a kinase activity independent role for BRAF in MAPK signaling. , 2014, Cancer cell.

[14]  M. Baccarini,et al.  “RAF” neighborhood: Protein–protein interaction in the Raf/Mek/Erk pathway , 2014, FEBS letters.

[15]  Frank McCormick,et al.  Targeting RAF kinases for cancer therapy: BRAF-mutated melanoma and beyond , 2014, Nature Reviews Cancer.

[16]  Christina Kiel,et al.  Structure‐energy‐based predictions and network modelling of RASopathy and cancer missense mutations , 2014, Molecular systems biology.

[17]  M. Meyerson,et al.  Oncogenic and sorafenib-sensitive ARAF mutations in lung adenocarcinoma. , 2014, The Journal of clinical investigation.

[18]  T. Gibson,et al.  Elution Profile Analysis of SDS-induced Subcomplexes by Quantitative Mass Spectrometry* , 2014, Molecular & Cellular Proteomics.

[19]  Andrew R. Jones,et al.  ProteomeXchange provides globally co-ordinated proteomics data submission and dissemination , 2014, Nature Biotechnology.

[20]  A. Bowcock,et al.  Combined PKC and MEK inhibition for treating metastatic uveal melanoma , 2014, Oncogene.

[21]  Yang Yu,et al.  Lys63-linked polyubiquitination of BRAF at lysine 578 is required for BRAF-mediated signaling , 2013, Scientific Reports.

[22]  David R. Croucher,et al.  Functional characterization of cancer-associated Gab1 mutations , 2013, Oncogene.

[23]  F. McCormick,et al.  RAF inhibitors activate the MAPK pathway by relieving inhibitory autophosphorylation. , 2013, Cancer cell.

[24]  B. Diedrich,et al.  Alterations of Gab2 signalling complexes in imatinib and dasatinib treated chronic myeloid leukaemia cells , 2013, Cell Communication and Signaling.

[25]  Ashley M. Zehnder,et al.  IQGAP1 scaffold-kinase interaction blockade selectively targets RAS-MAP kinase–driven tumors , 2013, Nature Medicine.

[26]  B. Kholodenko,et al.  It takes two to tango--signalling by dimeric Raf kinases. , 2013, Molecular bioSystems.

[27]  A. Meyer,et al.  Kidins220/ARMS Associates with B-Raf and the TCR, Promoting Sustained Erk Signaling in T Cells , 2013, The Journal of Immunology.

[28]  D. Morrison,et al.  Effects of Raf dimerization and its inhibition on normal and disease-associated Raf signaling. , 2013, Molecular cell.

[29]  I. Greenwald,et al.  SEL-10/Fbw7-dependent negative feedback regulation of LIN-45/Braf signaling in C. elegans via a conserved phosphodegron. , 2012, Genes & development.

[30]  A. Kusumi,et al.  Raf Inhibitors Target Ras Spatiotemporal Dynamics , 2012, Current Biology.

[31]  A. von Deimling,et al.  Distinct requirement for an intact dimer interface in wild‐type, V600E and kinase‐dead B‐Raf signalling , 2012, The EMBO journal.

[32]  E. Choi,et al.  Ring Finger Protein 149 Is an E3 Ubiquitin Ligase Active on Wild-type v-Raf Murine Sarcoma Viral Oncogene Homolog B1 (BRAF)* , 2012, The Journal of Biological Chemistry.

[33]  A. Eggermont,et al.  Skin Tumors Induced by Sorafenib; Paradoxic RAS–RAF Pathway Activation and Oncogenic Mutations of HRAS, TP53, and TGFBR1 , 2011, Clinical Cancer Research.

[34]  S. Pfister,et al.  Functional characterization of a BRAF insertion mutant associated with pilocytic astrocytoma , 2011, International journal of cancer.

[35]  G. Merlino,et al.  Protein Arginine Methyltransferase 5 Regulates ERK1/2 Signal Transduction Amplitude and Cell Fate Through CRAF , 2011, Science Signaling.

[36]  D. Morrison,et al.  14-3-3 Proteins: diverse functions in cell proliferation and cancer progression. , 2011, Seminars in cell & developmental biology.

[37]  Nils Blüthgen,et al.  Strong negative feedback from Erk to Raf confers robustness to MAPK signalling , 2011, Molecular systems biology.

[38]  N. Dumaz,et al.  ERK and PDE4 cooperate to induce RAF isoform switching in melanoma , 2011, Nature Structural &Molecular Biology.

[39]  M. Farquhar,et al.  Molecular basis of a novel oncogenic mutation in GNAO1 , 2011, Oncogene.

[40]  W. Kolch,et al.  Raf family kinases: old dogs have learned new tricks. , 2011, Genes & cancer.

[41]  M. Cobb,et al.  Calcineurin increases glucose activation of ERK1/2 by reversing negative feedback , 2010, Proceedings of the National Academy of Sciences.

[42]  S. Whittaker,et al.  Gatekeeper Mutations Mediate Resistance to BRAF-Targeted Therapies , 2010, Science Translational Medicine.

[43]  M. Belvin,et al.  RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth , 2010, Nature.

[44]  Chao Zhang,et al.  RAF inhibitors transactivate RAF dimers and ERK signaling in cells with wild-type BRAF , 2010, Nature.

[45]  Gabriele Gillessen-Kaesbach,et al.  Molecular and clinical analysis of RAF1 in Noonan syndrome and related disorders: dephosphorylation of serine 259 as the essential mechanism for mutant activation , 2010, Human mutation.

[46]  J. Reis-Filho,et al.  Kinase-Dead BRAF and Oncogenic RAS Cooperate to Drive Tumor Progression through CRAF , 2010, Cell.

[47]  D. Morrison,et al.  Impact of Feedback Phosphorylation and Raf Heterodimerization on Normal and Mutant B-Raf Signaling , 2009, Molecular and Cellular Biology.

[48]  Marc Therrien,et al.  A dimerization-dependent mechanism drives RAF catalytic activation , 2009, Nature.

[49]  D. Morrison,et al.  Signaling dynamics of the KSR1 scaffold complex , 2009, Proceedings of the National Academy of Sciences.

[50]  T. Höfer,et al.  Multisite protein phosphorylation – from molecular mechanisms to kinetic models , 2009, The FEBS journal.

[51]  B. Wilson,et al.  Pasteurella multocida toxin activation of heterotrimeric G proteins by deamidation , 2009, Proceedings of the National Academy of Sciences.

[52]  K. Patterson,et al.  Dual-specificity phosphatases: critical regulators with diverse cellular targets. , 2009, The Biochemical journal.

[53]  U. Rapp,et al.  Regulation of RAF Activity by 14-3-3 Proteins , 2009, Journal of Biological Chemistry.

[54]  E. Simpson,et al.  Frequent somatic mutations of GNAQ in uveal melanoma and blue nevi , 2008, Nature.

[55]  R. Zahedi,et al.  Positive Regulation of A-RAF by Phosphorylation of Isoform-specific Hinge Segment and Identification of Novel Phosphorylation Sites* , 2008, Journal of Biological Chemistry.

[56]  Christoph H. Emmerich,et al.  Phosphorylation‐dependent binding of 14‐3‐3 terminates signalling by the Gab2 docking protein , 2008, The EMBO journal.

[57]  Hisashi Nojima,et al.  IQGAP3 regulates cell proliferation through the Ras/ERK signalling cascade , 2008, Nature Cell Biology.

[58]  Y. Matsubara,et al.  The RAS/MAPK syndromes: novel roles of the RAS pathway in human genetic disorders , 2008, Human mutation.

[59]  S. Borysov,et al.  A novel role for Cdk1/cyclin B in regulating B-raf activation at mitosis. , 2008, Molecular biology of the cell.

[60]  C. Der.  Faculty Opinions recommendation of Differential effects of oncogenic K-Ras and N-Ras on proliferation, differentiation and tumor progression in the colon. , 2008 .

[61]  M. Baccarini,et al.  Essential role of B-Raf in oligodendrocyte maturation and myelination during postnatal central nervous system development , 2008, The Journal of cell biology.

[62]  Martin Moorhead,et al.  Rapid identification of somatic mutations in colorectal and breast cancer tissues using mismatch repair detection (MRD) , 2008, Human mutation.

[63]  P. Marin,et al.  GPCR-jacking: from a new route in RTK signalling to a new concept in GPCR activation. , 2007, Trends in pharmacological sciences.

[64]  R. Roepman,et al.  A novel tandem affinity purification strategy for the efficient isolation and characterisation of native protein complexes , 2007, Proteomics.

[65]  James E. Ferrell,et al.  Tuning Bulk Electrostatics to Regulate Protein Function , 2007, Cell.

[66]  Mike Tyers,et al.  A Mechanism for Cell-Cycle Regulation of MAP Kinase Signaling in a Yeast Differentiation Pathway , 2007, Cell.

[67]  T. Veenstra,et al.  CK2 Is a Component of the KSR1 Scaffold Complex that Contributes to Raf Kinase Activation , 2007, Current Biology.

[68]  C. Peyssonnaux,et al.  Differential Regulation of B-Raf Isoforms by Phosphorylation and AutoinhibitoryMechanisms , 2006, Molecular and Cellular Biology.

[69]  M. Reth,et al.  Functional analysis of the regulatory requirements of B-Raf and the B-RafV600E oncoprotein , 2006, Oncogene.

[70]  A. Kolbus,et al.  ERK and Beyond: Insights from B-Raf and Raf-1 Conditional Knockouts , 2006, Cell cycle.

[71]  Keiji Tanaka,et al.  Cdc37 Interacts with the Glycine-Rich Loop of Hsp90 Client Kinases , 2006, Molecular and Cellular Biology.

[72]  Pablo Rodriguez-Viciana,et al.  A phosphatase holoenzyme comprised of Shoc2/Sur8 and the catalytic subunit of PP1 functions as an M-Ras effector to modulate Raf activity. , 2006, Molecular cell.

[73]  Pablo Rodriguez-Viciana,et al.  Germline Mutations in Genes Within the MAPK Pathway Cause Cardio-facio-cutaneous Syndrome , 2006, Science.

[74]  W. Kolch,et al.  Regulation and Role of Raf-1/B-Raf Heterodimerization , 2006, Molecular and Cellular Biology.

[75]  N. Rosen,et al.  V600E B-Raf requires the Hsp90 chaperone for stability and is degraded in response to Hsp90 inhibitors. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[76]  Alcino J. Silva,et al.  Forebrain‐specific knockout of B‐raf kinase leads to deficits in hippocampal long‐term potentiation, learning, and memory , 2006, Journal of neuroscience research.

[77]  Volker Brinkmann,et al.  Prohibitin is required for Ras-induced Raf–MEK–ERK activation and epithelial cell migration , 2005, Nature Cell Biology.

[78]  Achim Kramer,et al.  Mapping of phosphorylation sites by a multi-protease approach with specific phosphopeptide enrichment and NanoLC-MS/MS analysis. , 2005, Analytical chemistry.

[79]  Ming Zhou,et al.  Regulation of Raf-1 by direct feedback phosphorylation. , 2005, Molecular cell.

[80]  L. Wennogle,et al.  Novel C‐Raf phosphorylation sites: serine 296 and 301 participate in Raf regulation , 2005, FEBS letters.

[81]  A. Bershadsky,et al.  Processive capping by formin suggests a force-driven mechanism of actin polymerization , 2004, The Journal of cell biology.

[82]  D. Auclair,et al.  BAY 43-9006 Exhibits Broad Spectrum Oral Antitumor Activity and Targets the RAF/MEK/ERK Pathway and Receptor Tyrosine Kinases Involved in Tumor Progression and Angiogenesis , 2004, Cancer Research.

[83]  T. Pawson,et al.  Proteomic, Functional, and Domain-Based Analysis of In Vivo 14-3-3 Binding Proteins Involved in Cytoskeletal Regulation and Cellular Organization , 2004, Current Biology.

[84]  Tilman Brummer,et al.  Feedback regulation of lymphocyte signalling , 2004, Nature Reviews Immunology.

[85]  D. Barford,et al.  Mechanism of Activation of the RAF-ERK Signaling Pathway by Oncogenic Mutations of B-RAF , 2004, Cell.

[86]  L. Iakoucheva,et al.  The importance of intrinsic disorder for protein phosphorylation. , 2004, Nucleic acids research.

[87]  M. Reth,et al.  Identification of novel ERK-mediated feedback phosphorylation sites at the C-terminus of B-Raf , 2003, Oncogene.

[88]  R. Wetzker,et al.  Transactivation joins multiple tracks to the ERK/MAPK cascade , 2003, Nature Reviews Molecular Cell Biology.

[89]  M. Reth,et al.  Inducible gene deletion reveals different roles for B‐Raf and Raf‐1 in B‐cell antigen receptor signalling , 2002, The EMBO journal.

[90]  P. Khavari,et al.  Epidermal Ras blockade demonstrates spatially localized Ras promotion of proliferation and inhibition of differentiation , 2002, Oncogene.

[91]  T. Jacks,et al.  NF1 Tumor Suppressor Gene Function Narrowing the GAP , 2001, Cell.

[92]  K. Guan,et al.  Activation of B‐Raf kinase requires phosphorylation of the conserved residues Thr598 and Ser601 , 2000, The EMBO journal.

[93]  T. Barber,et al.  Negative regulation of the serine/threonine kinase B-Raf by Akt. , 2000, The Journal of biological chemistry.

[94]  U. Rapp,et al.  Overlapping and specific functions of Braf and Craf-1 proto-oncogenes during mouse embryogenesis , 2000, Mechanisms of Development.

[95]  Anthony J. Muslin,et al.  Disruption of the 14-3-3 Binding Site within the B-Raf Kinase Domain Uncouples Catalytic Activity from PC12 Cell Differentiation* , 2000, The Journal of Biological Chemistry.

[96]  Giulio Superti‐Furga,et al.  Serine and tyrosine phosphorylations cooperate in Raf‐1, but not B‐Raf activation , 1999, The EMBO journal.

[97]  Z. Songyang,et al.  Recognition and regulation of primary-sequence motifs by signaling modular domains. , 1999, Progress in biophysics and molecular biology.

[98]  T. Soderling,et al.  A structural basis for substrate specificities of protein Ser/Thr kinases: primary sequence preference of casein kinases I and II, NIMA, phosphorylase kinase, calmodulin-dependent kinase II, CDK5, and Erk1 , 1996, Molecular and cellular biology.

[99]  W. Kolch,et al.  Nerve Growth Factor-mediated Activation of the Mitogen-activated Protein (MAP) Kinase Cascade Involves a Signaling Complex Containing B-Raf and HSP90* , 1996, The Journal of Biological Chemistry.

[100]  A. Eychène,et al.  Identification of signalling proteins interacting with B-Raf in the yeast two-hybrid system. , 1996, Oncogene.

[101]  A. Ullrich,et al.  Role of transactivation of the EGF receptor in signalling by G-protein-coupled receptors , 1996, Nature.

[102]  J. Troppmair,et al.  The ins and outs of Raf kinases. , 1994, Trends in biochemical sciences.

[103]  N. Girard,et al.  Clinical characteristics and outcome of patients with lung cancer harboring BRAF mutations. , 2016, Lung cancer.

[104]  Gary D Bader,et al.  Systematic analysis of somatic mutations in phosphorylation signaling predicts novel cancer drivers , 2013 .

[105]  Jonathan W. Pillow,et al.  POSTER PRESENTATION Open Access , 2013 .

[106]  T. Brummer,et al.  Aberrant B-Raf signaling in human cancer -- 10 years from bench to bedside. , 2012, Critical reviews in oncogenesis.

[107]  P. Simister,et al.  Order and disorder in large multi-site docking proteins of the Gab family--implications for signalling complex formation and inhibitor design strategies. , 2012, Molecular bioSystems.

[108]  J. Stockman.  Mutations in GNA11 in Uveal Melanoma , 2012 .

[109]  U. Rapp,et al.  Interaction between the protein kinase B-Raf and the alpha-subunit of the 11S proteasome regulator. , 1998, Cancer research.