SUMMARY The RAS-stimulated RAF-MEK-ERK pathway confers epithelial cells with critical motile and invasive capacities during embryonic development, tissue regeneration and carcinoma progression. Yet many mechanisms by which ERK exerts this control remain elusive. Here, we demonstrate that the ERK-activated kinase RSK is necessary to induce motility and invasive capacities in non-transformed epithelial cells and carcinoma cells. RSK is moreover sufficient to induce certain motile responses. Expression profiling analysis revealed that a primary role of RSK is to induce transcription of potent pro-motile/invasive gene program by FRA1-dependent and independent mechanisms. Strikingly, the program enables RSK to coordinately modulate the extracellular environment, the intracellular motility apparatus, and receptors mediating communication between these compartments to stimulate motility and invasion. These findings uncover a general mechanism whereby the RAS-ERK pathway controls epithelial cell motility by identifying RSK as a key effector, from which emanates multiple highly coordinate transcription-dependent mechanisms for stimulation of motility and invasive properties.

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The nuclease-based gene editing tools are rapidly transforming capabilities for altering the genome of cells and organisms with great precision and in high throughput studies. A major limitation in application of precise gene editing lies in lack of sensitive and fast methods to detect and characterize the induced DNA changes. Precise gene editing induces double-stranded DNA breaks that are repaired by error-prone non-homologous end joining leading to introduction of insertions and deletions (indels) at the target site. These indels are often small and difficult and laborious to detect by traditional methods. Here we present a method for fast, sensitive and simple indel detection that accurately defines indel sizes down to ±1 bp. The method coined IDAA for Indel Detection by Amplicon Analysis is based on tri-primer amplicon labelling and DNA capillary electrophoresis detection, and IDAA is amenable for high throughput analysis.

The growth factor/insulin-stimulated AGC kinases share an activation mechanism based on three phosphorylation sites. Of these, only the role of the activation loop phosphate in the kinase domain and the hydrophobic motif (HM) phosphate in a C-terminal tail region are well characterized. We investigated the role of the third, socalled turn motif phosphate, also located in the tail, in the AGC kinases PKB, S6K, RSK, MSK, PRK and PKC. We report cooperative action of the HM phosphate and the turn motif phosphate, because it binds a phosphoSer/ Thr-binding site above the glycine-rich loop within the kinase domain, promoting zipper-like association of the tail with the kinase domain, serving to stabilize the HM in its kinase-activating binding site. We present a molecular model for allosteric activation of AGC kinases by the turn motif phosphate via HM-mediated stabilization of the aC helix. In S6K and MSK, the turn motif phosphate thereby also protects the HM from dephosphorylation. Our results suggest that the mechanism described is a key feature in activation of upto 26 human AGC kinases.

The 90-kDa ribosomal S6 kinases (RSK1-3) are important mediators of growth factor stimulation of cellular proliferation, survival, and differentiation and are activated via coordinated phosphorylation by ERK and 3-phosphoinositide-dependent protein kinase-1 (PDK1). Here we performed the functional characterization of a predicted new human RSK homologue, RSK4. We showed that RSK4 is a predominantly cytosolic protein with very low expression and several characteristics of the RSK family kinases, including the presence of two functional kinase domains and a C-terminal docking site for ERK. Surprisingly, however, in all cell types analyzed, endogenous RSK4 was maximally ( cdc2 -inhibitory kinase Myt1 (7) and the protein kinase Bub1 (8), respectively. In somatic cell types, RSK may stimulate cell division through phosphorylation of substrates like p27 Kip1 (9) and glycogen synthase kinase-3 (10 -12); protein synthesis and cell growth through substrates like elongation factor 2 kinase (13), glycogen synthase kinase-3 (10 -12), transcription initiation factor IA (14), and tuberous sclerosis complex-2 protein (15); cell survival through Bad (16); and transcription through substrates like c-Fos (17) and estrogen receptor (18). Experiments with PC12 cells suggest that RSK is a key mediator of ERK in neurotrophin-induced neuronal differentiation (19). Finally genetic evidence from human and mouse has identified an important role for RSK2 in osteoblast differentiation and function through phosphorylation of activating transcription factor-4 (20) and in stimulation of white adipose tissue mass via an unknown mechanism (21). RSK is related to the mitogen-and stress-activated protein kinases (MSK1 and MSK2), which also contain two kinase domains. MSK, however, is activated by the ERK family as well as by p38 family MAP kinases and thereby functions in signal transduction of growth factors as well as of cellular stress stimuli like UV/radioactive irradiation and proinflammatory cytokines (22,23).The substrates of RSK are phosphorylated by the N-terminal kinase (NTK) domain (24 -27) whose activity is regulated by

Histone methyl marks contribute to the regulation of chromatin architecture and of DNA-based processes such as replication, repair and gene transcription. There is increasing evidence for alterations in histone methylation patterns in cancer and other human diseases. Importantly, aberrant methylation can contribute to deregulated gene expression and thereby to excessive proliferation, survival and metastasis of cancer cells. Histone methyl transferases and demethylases establish a dynamic equilibrium of histone modifications, and the expression and activity of several members of these enzyme families are deregulated in human cancer. The jumonji C-domain histone H3K4me3/2 demethylase KDM5B/PLU1/JARID1B as well as its close relative KDM5A/JARID1A are potential oncogenes. Both proteins are overexpressed in human tumors and the gene encoding KDM5B is e.g. amplified in invasive breast cancer cells. Results obtained with RNA interference and targeted deletions in mice have provided additional support for a critical role of KDM5 demethylases in cancer cells. EpiTherapeutics is developing novel cancer drugs targeting the enzymatic activity of histone demethylases. Here we present our recent progress in obtaining selective, potent, and cell-permeable inhibitors of the KDM5 demethylases. Biochemical and cellular activities, pharmacological data, and in vivo antitumor activity will be discussed. Citation Format: Heidi R. Hudlebusch, Bo Heinemann, Jesper M. Nielsen, Daniela Kleine-Kohlbrecher, Camilla Hauge, Thomas Boesen, Marc Labelle, Lars-Ole Gerlach, Peter Staller, Peter Birk. The development of therapeutic inhibitors of the KDM5 histone demethylases. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5161. doi:10.1158/1538-7445.AM2014-5161