Optogenetic clustering of CNK1 reveals mechanistic insights in RAF and AKT signalling controlling cell fate decisions

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

Mutant Actins Demonstrate a Role for Unpolymerized Actin in Control of Transcription by Serum Response Factor

Signal-induced activation of the transcription factor serum response factor (SRF) requires alterations in actin dynamics. SRF activity can be inhibited by ectopic expression of β-actin, either because actin itself participates in SRF regulation or as a consequence of cytoskeletal perturbations. To distinguish between these possibilities, we studied actin mutants. Three mutant actins, G13R, R62D, and a C-terminal VP16 fusion protein, were shown not to polymerize in vivo, as judged by two-hybrid, immunofluorescence, and cell fractionation studies. These actins effectively inhibited SRF activation, as did wild-type actin, which increased the G-actin level without altering the F:G-actin ratio. Physical interaction between SRF and actin was not detectable by mammalian or yeast two-hybrid assays, suggesting that SRF regulation involves an unidentified cofactor. SRF activity was not blocked upon inhibition of CRM1-mediated nuclear export by leptomycin B. Two actin mutants were identified, V159N and S14C, whose expression favored F-actin formation and which strongly activated SRF in the absence of external signals. These mutants seemed unable to inhibit SRF activity, because their expression did not reduce the absolute level of G-actin as assessed by DNase I binding. Taken together, these results provide strong evidence that G-actin, or a subpopulation of it, plays a direct role in signal transduction to SRF

Repressive and active histone methylation mark distinct promoters in human and mouse spermatozoa.

Contains fulltext : 89401.pdf (publisher's version ) (Closed access)In higher eukaryotes, histone methylation is involved in maintaining cellular identity during somatic development. As most nucleosomes are replaced by protamines during spermatogenesis, it is unclear whether histone modifications function in paternal transmission of epigenetic information. Here we show that two modifications important for Trithorax- and Polycomb-mediated gene regulation have methylation-specific distributions at regulatory regions in human spermatozoa. Histone H3 Lys4 dimethylation (H3K4me2) marks genes that are relevant in spermatogenesis and cellular homeostasis. In contrast, histone H3 Lys27 trimethylation (H3K27me3) marks developmental regulators in sperm, as in somatic cells. However, nucleosomes are only moderately retained at regulatory regions in human sperm. Nonetheless, genes with extensive H3K27me3 coverage around transcriptional start sites in particular tend not to be expressed during male and female gametogenesis or in preimplantation embryos. Promoters of orthologous genes are similarly modified in mouse spermatozoa. These data are compatible with a role for Polycomb in repressing somatic determinants across generations, potentially in a variegating manner.1 juni 201