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  - 
TY  - JOUR
A1  - Fagan, Jeremy K.
A1  - Dollar, Gretchen
A1  - Lu, Qiuheng
A1  - Barnett, Austen
A1  - Jorge, Joaquin Pechuan
A1  - Schlosser, Andreas
A1  - Pfleger, Cathie
A1  - Adler, Paul
A1  - Jenny, Andreas
T1  - Combover/CG10732, a Novel PCP Effector for Drosophila Wing Hair Formation
JF  - PLOS ONE
N2  - The polarization of cells is essential for the proper functioning of most organs. Planar Cell Polarity (PCP), the polarization within the plane of an epithelium, is perpendicular to apical-basal polarity and established by the non-canonical Wnt/Fz-PCP signaling pathway. Within each tissue, downstream PCP effectors link the signal to tissue specific readouts such as stereocilia orientation in the inner ear and hair follicle orientation in vertebrates or the polarization of ommatidia and wing hairs in Drosophila melanogaster. Specific PCP effectors in the wing such as Multiple wing hairs (Mwh) and Rho Kinase (Rok) are required to position the hair at the correct position and to prevent ectopic actin hairs. In a genome-wide screen in vitro, we identified Combover (Cmb)/CG10732 as a novel Rho kinase substrate. Overexpression of Cmb causes the formation of a multiple hair cell phenotype (MHC), similar to loss of rok and mwh. This MHC phenotype is dominantly enhanced by removal of rok or of other members of the PCP effector gene family. Furthermore, we show that Cmb physically interacts with Mwh, and cmb null mutants suppress the MHC phenotype of mwh alleles. Our data indicate that Cmb is a novel PCP effector that promotes to wing hair formation, a function that is antagonized by Mwh.
KW  - planar cell polarity
KW  - RHO-associated kinease
KW  - convergent extension movements
KW  - ROK-alpha
KW  - protein
KW  - phosphorylation
KW  - actin
KW  - gene
KW  - morphogenesis
KW  - localization
Y1  - 2014
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115394
SN  - 1932-6203
VL  - 9
IS  - 9
ER  - 
TY  - JOUR
A1  - Weigand, Isabel
A1  - Ronchi, Cristina L.
A1  - Vanselow, Jens T.
A1  - Bathon, Kerstin
A1  - Lenz, Kerstin
A1  - Herterich, Sabine
A1  - Schlosser, Andreas
A1  - Kroiss, Matthias
A1  - Fassnacht, Martin
A1  - Calebiro, Davide
A1  - Sbiera, Silviu
T1  - PKA Cα subunit mutation triggers caspase-dependent RIIβ subunit degradation via Ser\(^{114}\) phosphorylation
JF  - Science Advances
N2  - Mutations in the PRKACA gene are the most frequent cause of cortisol-producing adrenocortical adenomas leading to Cushing’s syndrome. PRKACA encodes for the catalytic subunit α of protein kinase A (PKA). We already showed that PRKACA mutations lead to impairment of regulatory (R) subunit binding. Furthermore, PRKACA mutations are associated with reduced RIIβ protein levels; however, the mechanisms leading to reduced RIIβ levels are presently unknown. Here, we investigate the effects of the most frequent PRKACA mutation, L206R, on regulatory subunit stability. We find that Ser\(^{114}\) phosphorylation of RIIβ is required for its degradation, mediated by caspase 16. Last, we show that the resulting reduction in RIIβ protein levels leads to increased cortisol secretion in adrenocortical cells. These findings reveal the molecular mechanisms and pathophysiological relevance of the R subunit degradation caused by PRKACA mutations, adding another dimension to the deregulation of PKA signaling caused by PRKACA mutations in adrenal Cushing’s syndrome.
KW  - mutation triggers
KW  - phosphorylation
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-270445
VL  - 7
IS  - 8
ER  -