18 research outputs found
Phosphoinositide 3-Kinase C2β Regulates Cytoskeletal Organization and Cell Migration via Rac-dependent Mechanisms
Receptor-linked class I phosphoinositide 3-kinases (PI3Ks) induce assembly of signal transduction complexes through protein–protein and protein–lipid interactions that mediate cell proliferation, survival, and migration. Although class II PI3Ks have the potential to make the same phosphoinositides as class I PI3Ks, their precise cellular role is currently unclear. In this report, we demonstrate that class II phosphoinositide 3-kinase C2β (PI3KC2β) associates with the Eps8/Abi1/Sos1 complex and is recruited to the EGF receptor as part of a multiprotein signaling complex also involving Shc and Grb2. Increased expression of PI3KC2β stimulated Rac activity in A-431 epidermoid carcinoma cells, resulting in enhanced membrane ruffling and migration speed of the cells. Conversely, expression of dominant negative PI3KC2β reduced Rac activity, membrane ruffling, and cell migration. Moreover, PI3KC2β-overexpressing cells were protected from anoikis and displayed enhanced proliferation, independently of Rac function. Taken together, these findings suggest that PI3KC2β regulates the migration and survival of human tumor cells by distinct molecular mechanisms
Evaluation of cell-based assays for steroid nuclear receptors delivered by recombinant baculoviruses
Evaluation of Cell-Based Assays for Steroid Nuclear Receptors Delivered by Recombinant Baculoviruses
Recommended from our members
A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response
The jumonji (JMJ) family of histone demethylases are Fe2+- and α-ketoglutarate-dependent oxygenases that are essential components of regulatory transcriptional chromatin complexes. These enzymes demethylate lysine residues in histones in a methylation-state and sequence-specific context. Considerable effort has been devoted to gaining a mechanistic understanding of the roles of histone lysine demethylases in eukaryotic transcription, genome integrity and epigenetic inheritance, as well as in development, physiology and disease. However, because of the absence of any selective inhibitors, the relevance of the demethylase activity of JMJ enzymes in regulating cellular responses remains poorly understood. Here we present a structure-guided small-molecule and chemoproteomics approach to elucidating the functional role of the H3K27me3-specific demethylase subfamily (KDM6 subfamily members JMJD3 and UTX). The liganded structures of human and mouse JMJD3 provide novel insight into the specificity determinants for cofactor, substrate and inhibitor recognition by the KDM6 subfamily of demethylases. We exploited these structural features to generate the first small-molecule catalytic site inhibitor that is selective for the H3K27me3-specific JMJ subfamily. We demonstrate that this inhibitor binds in a novel manner and reduces lipopolysaccharide-induced proinflammatory cytokine production by human primary macrophages, a process that depends on both JMJD3 and UTX. Our results resolve the ambiguity associated with the catalytic function of H3K27-specific JMJs in regulating disease-relevant inflammatory responses and provide encouragement for designing small-molecule inhibitors to allow selective pharmacological intervention across the JMJ family
Cell penetrant inhibitors of the KDM4 and KDM5 families of histone lysine demethylases. 2. Pyrido[3,4-d]pyrimidin-4(3H)-one derivatives
Following the discovery of cell penetrant pyridine-4-carboxylate inhibitors of the KDM4 (JMJD2) and KDM5 (JARID1) families of histone lysine demethylases (e.g., 1), further optimization led to the identification of non-carboxylate inhibitors derived from pyrido[3,4-d]pyrimidin-4(3H)-one. A number of exemplars such as compound 41 possess interesting activity profiles in KDM4C and KDM5C biochemical and target-specific, cellular mechanistic assays
Light it up: Highly efficient multigene delivery in mammalian cells
Multigene delivery and expression systems are emerging as key technologies for many applications in contemporary biology. We have developed new methods for multigene delivery and expression in eukaryotic hosts for a variety of applications, including production of protein complexes for structural biology and drug development, provision of multicomponent protein biologics and for cell-based assays. We implemented tandem recombineering to facilitate rapid generation of multicomponent gene expression constructs for efficient transformation of mammalian cells, resulting in homogenous cell populations. Analysis of multiple parameters in living cells may require co-expression of fluorescently tagged sensors simultaneously in a single cell, at defined and ideally controlled ratios. Our method enables such applications by overcoming currently limiting challenges.
Here, we review recent multigene delivery and expression strategies and their exploits in mammalian cells. We discuss applications in drug discovery assays, interaction studies and biologics production, which may benefit in the future from our novel approach