title: Optogenetic clustering of CNK1 reveals mechanistic insights in RAF and AKT signalling controlling cell fate decisions.
abstract: Scaffold proteins such as the multidomain protein CNK1 orchestrate the signalling network by integrating and controlling the underlying pathways. Using an optogenetic approach to stimulate CNK1 uncoupled from upstream effectors, we identified selective clusters of CNK1 that either stimulate RAF-MEK-ERK or AKT signalling depending on the light intensity applied. OptoCNK1 implemented in MCF7 cells induces differentiation at low light intensity stimulating ERK activity whereas stimulation of AKT signalling by higher light intensity promotes cell proliferation. CNK1 clustering in response to increasing EGF concentrations revealed that CNK1 binds to RAF correlating with ERK activation at low EGF dose. At higher EGF dose active AKT binds to CNK1 and phosphorylates and inhibits RAF. Knockdown of CNK1 protects CNK1 from this AKT/RAF crosstalk. In C2 skeletal muscle cells CNK1 expression is induced with the onset of differentiation. Hence, AKT-bound CNK1 counteracts ERK stimulation in differentiated but not in proliferating cells. Ectopically expressed CNK1 facilitates C2 cell differentiation and knockdown of CNK1 impaired the transcriptional network underlying C2 cell differentiation. Thus, CNK1 expression, CNK1 clustering and the thereto related differential signalling processes decide on proliferation and differentiation in a cell type- and cell stage-dependent manner by orchestrating AKT and RAF signalling.
authors.affiliation: Department of Biochemistry and Synthetic Biology, Faculty of Biology, University of Freiburg, Schänzlestr. 18, 79104 Freiburg, Germany.|Department of Biochemistry and Functional Proteomics, Faculty of Biology, University of Freiburg, Schänzlestr. 1, 79104 Freiburg, Germany.|BIOSS - Centre for Biological Signalling Studies, University of Freiburg, Schänzlestr. 1, 79104 Freiburg, Germany.
authors.forename: Adrian
authors.initials: A
authors.lastname: Fischer
authors.affiliation: Department of Biochemistry and Functional Proteomics, Faculty of Biology, University of Freiburg, Schänzlestr. 1, 79104 Freiburg, Germany.|BIOSS - Centre for Biological Signalling Studies, University of Freiburg, Schänzlestr. 1, 79104 Freiburg, Germany.
authors.forename: Bettina
authors.initials: B
authors.lastname: Warscheid
authors.affiliation: Department of Biochemistry and Synthetic Biology, Faculty of Biology, University of Freiburg, Schänzlestr. 18, 79104 Freiburg, Germany.|BIOSS - Centre for Biological Signalling Studies, University of Freiburg, Schänzlestr. 1, 79104 Freiburg, Germany.
authors.forename: Wilfried
authors.initials: W
authors.lastname: Weber
authors.affiliation: Department of Biochemistry and Synthetic Biology, Faculty of Biology, University of Freiburg, Schänzlestr. 18, 79104 Freiburg, Germany.|Department of Biochemistry and Functional Proteomics, Faculty of Biology, University of Freiburg, Schänzlestr. 1, 79104 Freiburg, Germany.|BIOSS - Centre for Biological Signalling Studies, University of Freiburg, Schänzlestr. 1, 79104 Freiburg, Germany.
authors.forename: Gerald
authors.initials: G
authors.lastname: Radziwill
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000235:genetics*; D002460:Cell Line; D049109:Cell Proliferation / Q000235:genetics*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D061986:MCF-7 Cells; D051379:Mice; D062308:Optogenetics; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics* / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D015398:Signal Transduction / Q000235:genetics
chemical_list: C500833:CNKSR1 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D051057:Proto-Oncogene Proteins c-akt; D019908:Proto-Oncogene Proteins c-raf