Structural Insight into the Mode of Action of a Direct Inhibitor of Coregulator Binding to the Thyroid Hormone Receptor.

The development of nuclear hormone receptor antagonists that directly inhibit the association of the receptor with its essential coactivators would allow useful manipulation of nuclear hormone receptor signaling. We previously identified 3-(dibutylamino)-1-(4-hexylphenyl)-propan-1-one (DHPPA), an aromatic β-amino ketone that inhibits coactivator recruitment to thyroid hormone receptor β (TRβ), in a high-throughput screen. Initial evidence suggested that the aromatic β-enone 1-(4-hexylphenyl)-prop-2-en-1-one (HPPE), which alkylates a specific cysteine residue on the TRβ surface, is liberated from DHPPA. Nevertheless, aspects of the mechanism and specificity of action of DHPPA remained unclear. Here, we report an x-ray structure of TRβ with the inhibitor HPPE at 2.3-Å resolution. Unreacted HPPE is located at the interface that normally mediates binding between TRβ and its coactivator. Several lines of evidence, including experiments with TRβ mutants and mass spectroscopic analysis, showed that HPPE specifically alkylates cysteine residue 298 of TRβ, which is located near the activation function-2 pocket. We propose that this covalent adduct formation proceeds through a two-step mechanism: 1) β-elimination to form HPPE; and 2) a covalent bond slowly forms between HPPE and TRβ. DHPPA represents a novel class of potent TRβ antagonist, and its crystal structure suggests new ways to design antagonists that target the assembly of nuclear hormone receptor gene-regulatory complexes and block transcription

Toxoplasma and Plasmodium protein kinases: roles in invasion and host cell remodelling

Some apicomplexan parasites have evolved distinct protein kinase families to modulate host cell structure and function. Toxoplasma gondii rhoptry protein kinases and pseudokinases are involved in virulence and modulation of host cell signalling. The proteome of Plasmodium falciparum contains a family of putative kinases called FIKKs, some of which are exported to the host red blood cell and might play a role in erythrocyte remodelling. In this review we will discuss kinases known to be critical for host cell invasion, intracellular growth and egress, focusing on (i) calcium-dependent protein kinases and (ii) the secreted kinases that are unique to Toxoplasma (rhoptry protein kinases and pseudokinases) and Plasmodium (FIKKs)

AMPK:an energy-sensing pathway with multiple inputs and outputs

AMP-activated protein kinase (AMPK) is a key regulator of energy balance expressed ubiquitously in eukaryotic cells. Here we review the canonical adenine nucleotide-dependent mechanism that activates AMPK when cellular energy status is compromised, as well as other, noncanonical activation mechanisms. Once activated, AMPK acts to restore energy homeostasis by promoting catabolic pathways, resulting in ATP generation, and inhibiting anabolic pathways that consume ATP. We also review the various hypothesis-driven and unbiased approaches that have been used to identify AMPK substrates and have revealed substrates involved in both metabolic and non-metabolic processes. We particularly focus on methods for identifying the AMPK target recognition motif and how it can be used to predict new substrates. AMPK is an energy-sensing protein kinase activated by phosphorylation of Thr172 within its catalytic α subunit. It binds AMP and/or ADP, both signals of energy stress, via its regulatory γ subunit. This activates the kinase by promoting Thr172 phosphorylation, inhibiting Thr172 dephosphorylation and allosteric activation.AMP binding to the γ subunit causes its interaction with the α linker region of the α subunit. This pulls the autoinhibitory domain on the α subunit away from the kinase domain, triggering activation.AMPK is also activated by the binding of synthetic and naturally occurring drug-like molecules that bind in the allosteric drug and metabolite (ADaM) site between the α and β subunits.AMPK has a well-defined recognition motif that has been established both by hypothesis-driven approaches and by various unbiased screens. It now has over 60 well-validated substrates.</p

A chemical-genetics approach to study the role of atypical Protein Kinase C in Drosophila

Studying the function of proteins using genetics in cycling cells is complicated by the fact that there is often a delay between gene inactivation and the time point of phenotypic analysis. This is particularly true when studying kinases that have pleiotropic functions and multiple substrates. Drosophila neuroblasts (NBs) are rapidly dividing stem cells and an important model system for the study of cell polarity. Mutations in multiple kinases cause NB polarity defects, but their precise functions at particular time points in the cell cycle are unknown. Here, we use chemical genetics and report the generation of an analogue-sensitive allele of Drosophila atypical Protein Kinase C (aPKC). We demonstrate that the resulting mutant aPKC kinase can be specifically inhibited in vitro and in vivo Acute inhibition of aPKC during NB polarity establishment abolishes asymmetric localization of Miranda, whereas its inhibition during NB polarity maintenance does not in the time frame of normal mitosis. However, aPKC helps to sharpen the pattern of Miranda, by keeping it off the apical and lateral cortex after nuclear envelope breakdown.</p

The serine/threonine kinase Par1b regulates epithelial lumen polarity via IRSp53-mediated cell–ECM signaling

Par1b regulates cell–ECM signaling and dictates epithelial lumenal organization by targeting IRSp53, a Rho GTPase adaptor and scaffolding protein

Modular Mass Spectrometric Tool for Analysis of Composition and Phosphorylation of Protein Complexes

The combination of high accuracy, sensitivity and speed of single and multiple-stage mass spectrometric analyses enables the collection of comprehensive sets of data containing detailed information about complex biological samples. To achieve these properties, we combined two high-performance matrix-assisted laser desorption ionization mass analyzers in one modular mass spectrometric tool, and applied this tool for dissecting the composition and post-translational modifications of protein complexes. As an example of this approach, we here present studies of the Saccharomyces cerevisiae anaphase-promoting complexes (APC) and elucidation of phosphorylation sites on its components. In general, the modular concept we describe could be useful for assembling mass spectrometers operating with both matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) ion sources into powerful mass spectrometric tools for the comprehensive analysis of complex biological samples

Caenorhabditis elegans Cyclin B3 Is Required for Multiple Mitotic Processes Including Alleviation of a Spindle Checkpoint–Dependent Block in Anaphase Chromosome Segregation

The master regulators of the cell cycle are cyclin-dependent kinases (Cdks), which influence the function of a myriad of proteins via phosphorylation. Mitotic Cdk1 is activated by A-type, as well as B1- and B2-type, cyclins. However, the role of a third, conserved cyclin B family member, cyclin B3, is less well defined. Here, we show that Caenorhabditis elegans CYB-3 has essential and distinct functions from cyclin B1 and B2 in the early embryo. CYB-3 is required for the timely execution of a number of cell cycle events including completion of the MII meiotic division of the oocyte nucleus, pronuclear migration, centrosome maturation, mitotic chromosome condensation and congression, and, most strikingly, progression through the metaphase-to-anaphase transition. Our experiments reveal that the extended metaphase delay in CYB-3–depleted embryos is dependent on an intact spindle assembly checkpoint (SAC) and results in salient defects in the architecture of holocentric metaphase chromosomes. Furthermore, genetically increasing or decreasing dynein activity results in the respective suppression or enhancement of CYB-3–dependent defects in cell cycle progression. Altogether, these data reveal that CYB-3 plays a unique, essential role in the cell cycle including promoting mitotic dynein functionality and alleviation of a SAC–dependent block in anaphase chromosome segregation

The mechanics of mitotic cell rounding

When animal cells enter mitosis, they round up to become spherical. This shape change is accompanied by changes in mechanical properties. Multiple studies using different measurement methods have revealed that cell surface tension, intracellular pressure and cortical stiffness increase upon entry into mitosis. These cell-scale, biophysical changes are driven by alterations in the composition and architecture of the contractile acto-myosin cortex together with osmotic swelling and enable a mitotic cell to exert force against the environment. When the ability of cells to round is limited, for example by physical confinement, cells suffer severe defects in spindle assembly and cell division. The requirement to push against the environment to create space for spindle formation is especially important for cells dividing in tissues. Here we summarize the evidence and the tools used to show that cells exert rounding forces in mitosis in vitro and in vivo, review the molecular basis for this force generation and discuss its function for ensuring successful cell division in single cells and for cells dividing in normal or diseased tissues