Identification of ELK1 interacting peptide segments in the androgen receptor.

Prostate cancer (PCa) growth requires tethering of the androgen receptor (AR) to chromatin by the ETS domain transcription factor ELK1 to coactivate critical cell proliferation genes. Disruption of the ELK1-AR complex is a validated potential means of therapeutic intervention in PCa. AR associates with ELK1 by coopting its two ERK docking sites, through the amino-terminal domain (A/B domain) of AR. Using a mammalian two-hybrid assay, we have now functionally mapped amino acids within the peptide segments 358-457 and 514-557 in the A/B domain as required for association with ELK1. The mapping data were validated by GST (glutathione S-transferase)-pulldown and BRET (bioluminescence resonance energy transfer) assays. Comparison of the relative contributions of the interacting motifs/segments in ELK1 and AR to coactivation of ELK1 by AR suggested a parallel mode of binding of AR and ELK1 polypeptides. Growth of PCa cells was partially inhibited by deletion of the upstream segment in AR and nearly fully inhibited by deletion of the downstream segment. Our studies have identified two peptide segments in AR that mediate the functional association of AR with its two docking sites in ELK1. Identification of the ELK1 recognition sites in AR should enable further structural studies of the ELK1-AR interaction and rational design of small molecule drugs to disrupt this interaction.

Methane leak detection by tunable laser spectroscopy and mid-infrared imaging.

Tunable laser spectroscopy (TLS) combined with mid-infrared imaging is a powerful tool for a sensitive and quantitative visualization of gas leaks. This work deals with standoff methane leak detection within 2 m by an interband cascade laser (3270 nm wavelength) and an infrared camera. The concept demonstrates visualization of methane leakage rates down to 2 ml/min by images and sequences at frame rates up to 125 Hz. The gas plume and leak can be localized and quantified within a single image by direct absorption spectroscopy (DAS). The HITRAN database allows a calibration-free, pixelwise determination of the concentration in ppm*m. The active optical imaging concept showed pixelwise sensitivities around 1 ppm*m.

De-ubiquitination of ELK-1 by USP17 potentiates mitogenic gene expression and cell proliferation.

ELK-1 is a transcription factor involved in ERK-induced cellular proliferation. Here, we show that its transcriptional activity is modulated by ubiquitination at lysine 35 (K35). The level of ubiquitinated ELK-1 rises in mitogen-deprived cells and falls upon mitogen stimulation or oncogene expression. Ectopic expression of USP17, a cell cycle-dependent deubiquitinase, decreases ELK-1 ubiquitination and up-regulates ELK-1 target-genes with a concomitant increase in cyclin D1 expression. In contrast, USP17 depletion attenuates ELK-1-dependent gene expression and slows cell proliferation. The reduced rate of proliferation upon USP17 depletion appears to be a direct effect of ELK-1 ubiquitination because it is rescued by an ELK-1(K35R) mutant refractory to ubiquitination. Overall, our results show that ubiquitination of ELK-1 at K35, and its reversal by USP17, are important mechanisms in the regulation of nuclear ERK signalling and cellular proliferation. Our findings will be relevant for tumours that exhibit elevated USP17 expression and suggest a new target for intervention.

Gas Leak Detection by Dilution of Atmospheric Oxygen.

Gas leak detection is an important issue in infrastructure monitoring and industrial production. In this context, infrared (IR) absorption spectroscopy is a major measurement method. It can be applied in an extractive or remote detection scheme. Tunable laser spectroscopy (TLS) instruments are able to detect CH4 leaks with column densities below 10 ppm·m from a distance of 30 m in less than a second. However, leak detection of non-IR absorbing gases such as N2 is not possible in this manner. Due to the fact that any leaking gas displaces or dilutes the surrounding background gas, an indirect detection is still possible. It is shown by sensitive TLS measurements of the ambient background concentration of O2 that N2 leaks can be localized with extractive and standoff methods for distances below 1 m. Minimum leak rates of 0.1 mbar·L/s were determined. Flow simulations confirm that the leakage gas typically effuses in a narrow jet. The sensitivity is mainly determined by ambient flow conditions. Compared to TLS detection of CH4 at 1651 nm, the indirect method using O2 at 761 nm is experimentally found to be less sensitive by a factor of 100. However, the well-established TLS of O2 may become a universal tool for rapid leakage screening of vessels that contain unknown or inexpensive gases, such as N2.

Photoacoustic methane detection inside a MEMS microphone.

An innovative laser based photoacoustic (PA) gas sensing concept with intrinsic miniaturization potential was developed and investigated for methane trace gas detection. An interband cascade laser (ICL) with an optical power of 8.5 mW targets a methane (CH4) absorption line feature around 3057.7 cm-1 (or 3270 nm). The ICL was focused into the sound port of a MEMS microphone, where the PA signal was generated and detected using a wavelength modulation concept (2f-WMS-PAS). The MEMS microphone was successfully implemented as an intrinsically miniaturized PA cell being gas sensing volume, acoustic resonator and sound transducer at once. Frequencies between 2 kHz and 100 kHz were investigated and used for methane detection. A sensitive and resonant methane detection at 41.8 kHz was investigated by concentration variations between 0 and 10 ppm CH4 in N2. A limit of detection ( 3 σ -LOD) of 329 ppb was estimated. The long term stability of this sensor was investigated by the measurement of methane in ambient air. A noise equivalent concentration (NEC) of 14 ppb (parts per billion) at an average time of 10 s was estimated. This value corresponds to a normalized noise equivalent absorption (NNEA) of 2 ⋅ 1 0 - 8 W cm-1 Hz-1/2. Using the MEMS microphone directly as PA cell offers the possibility for an extremely miniaturized, highly sensitive and very cost-efficient photoacoustic trace gas sensor.

Structure of a Ca2+-myristoyl switch protein that controls activation of a phosphatidylinositol 4-kinase in fission yeast.

Neuronal calcium sensor (NCS) proteins transduce Ca2+ signals and are highly conserved from yeast to humans. We determined NMR structures of the NCS-1 homolog from fission yeast (Ncs1), which activates a phosphatidylinositol 4-kinase. Ncs1 contains an α-NH2-linked myristoyl group on a long N-terminal arm and four EF-hand motifs, three of which bind Ca2+, assembled into a compact structure. In Ca2+-free Ncs1, the N-terminal arm positions the fatty acyl chain inside a cavity near the C terminus. The C14 end of the myristate is surrounded by residues in the protein core, whereas its amide-linked (C1) end is flanked by residues at the protein surface. In Ca2+-bound Ncs1, the myristoyl group is extruded (Ca2+-myristoyl switch), exposing a prominent patch of hydrophobic residues that specifically contact phosphatidylinositol 4-kinase. The location of the buried myristate and structure of Ca2+-free Ncs1 are quite different from those in other NCS proteins. Thus, a unique remodeling of each NCS protein by its myristoyl group, and Ca2+-dependent unmasking of different residues, may explain how each family member recognizes distinct target proteins.

Yeast phosphatidylinositol 4-kinase, Pik1, has essential roles at the Golgi and in the nucleus.

Phosphatidylinositol 4-kinase, Pik1, is essential for viability. GFP-Pik1 localized to cytoplasmic puncta and the nucleus. The puncta colocalized with Sec7-DsRed, a marker of trans-Golgi cisternae. Kap95 (importin-beta) was necessary for nuclear entry, but not Kap60 (importin-alpha), and exportin Msn5 was required for nuclear exit. Frq1 (frequenin orthologue) also is essential for viability and binds near the NH2 terminus of Pik1. Frq1-GFP localized to Golgi puncta, and Pik1 lacking its Frq1-binding site (or Pik1 overexpressed in frq1Delta cells) did not decorate the Golgi, but nuclear localization was unperturbed. Pik1(Delta10-192), which lacks its nuclear export sequence, displayed prominent nuclear accumulation and did not rescue inviability of pik1Delta cells. A Pik1-CCAAX chimera was excluded from the nucleus and also did not rescue inviability of pik1Delta cells. However, coexpression of Pik1(Delta10-192) and Pik1-CCAAX in pik1Delta cells restored viability. Catalytically inactive derivatives of these compartment-restricted Pik1 constructs indicated that PtdIns4P must be generated both in the nucleus and at the Golgi for normal cell function.

Synthesis and function of membrane phosphoinositides in budding yeast, Saccharomyces cerevisiae.

It is now well appreciated that derivatives of phosphatidylinositol (PtdIns) are key regulators of many cellular processes in eukaryotes. Of particular interest are phosphoinositides (mono- and polyphosphorylated adducts to the inositol ring in PtdIns), which are located at the cytoplasmic face of cellular membranes. Phosphoinositides serve both a structural and a signaling role via their recruitment of proteins that contain phosphoinositide-binding domains. Phosphoinositides also have a role as precursors of several types of second messengers for certain intracellular signaling pathways. Realization of the importance of phosphoinositides has brought increased attention to characterization of the enzymes that regulate their synthesis, interconversion, and turnover. Here we review the current state of our knowledge about the properties and regulation of the ATP-dependent lipid kinases responsible for synthesis of phosphoinositides and also the additional temporal and spatial controls exerted by the phosphatases and a phospholipase that act on phosphoinositides in yeast.

Roles of phosphoinositides and of Spo14p (phospholipase D)-generated phosphatidic acid during yeast sporulation.

During yeast sporulation, internal membrane synthesis ensures that each haploid nucleus is packaged into a spore. Prospore membrane formation requires Spo14p, a phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]-stimulated phospholipase D (PLD), which hydrolyzes phosphatidylcholine (PtdCho) to phosphatidic acid (PtdOH) and choline. We found that both meiosis and spore formation also require the phosphatidylinositol (PtdIns)/PtdCho transport protein Sec14p. Specific ablation of the PtdIns transport activity of Sec14p was sufficient to impair spore formation but not meiosis. Overexpression of Pik1p, a PtdIns 4-kinase, suppressed the sec14-1 meiosis and spore formation defects; conversely, pik1-ts diploids failed to undergo meiosis and spore formation. The PtdIns(4)P 5-kinase, Mss4p, also is essential for spore formation. Use of phosphoinositide-specific GFP-PH domain reporters confirmed that PtdIns(4,5)P2 is enriched in prospore membranes. sec14, pik1, and mss4 mutants displayed decreased Spo14p PLD activity, whereas absence of Spo14p did not affect phosphoinositide levels in vivo, suggesting that formation of PtdIns(4,5)P2 is important for Spo14p activity. Spo14p-generated PtdOH appears to have an essential role in sporulation, because treatment of cells with 1-butanol, which supports Spo14p-catalyzed PtdCho breakdown but leads to production of Cho and Ptd-butanol, blocks spore formation at concentrations where the inert isomer, 2-butanol, has little effect. Thus, rather than a role for PtdOH in stimulating PtdIns(4,5)P2 formation, our findings indicate that during sporulation, Spo14p-mediated PtdOH production functions downstream of Sec14p-, Pik1p-, and Mss4p-dependent PtdIns(4,5)P2 synthesis.

Structural insights into activation of phosphatidylinositol 4-kinase (Pik1) by yeast frequenin (Frq1).

Yeast frequenin (Frq1), a small N-myristoylated EF-hand protein, activates phosphatidylinositol 4-kinase Pik1. The NMR structure of Ca2+-bound Frq1 complexed to an N-terminal Pik1 fragment (residues 121-174) was determined. The Frq1 main chain is similar to that in free Frq1 and related proteins in the same branch of the calmodulin superfamily. The myristoyl group and first eight residues of Frq1 are solvent-exposed, and Ca2+ binds the second, third, and fourth EF-hands, which associate to create a groove with two pockets. The Pik1 peptide forms two helices (125-135 and 156-169) connected by a 20-residue loop. Side chains in the Pik1 N-terminal helix (Val-127, Ala-128, Val-131, Leu-132, and Leu-135) interact with solvent-exposed residues in the Frq1 C-terminal pocket (Leu-101, Trp-103, Val-125, Leu-138, Ile-152, and Leu-155); side chains in the Pik1 C-terminal helix (Ala-157, Ala-159, Leu-160, Val-161, Met-165, and Met-167) contact solvent-exposed residues in the Frq1 N-terminal pocket (Trp-30, Phe-34, Phe-48, Ile-51, Tyr-52, Phe-55, Phe-85, and Leu-89). This defined complex confirms that residues in Pik1 pinpointed as necessary for Frq1 binding by site-directed mutagenesis are indeed sufficient for binding. Removal of the Pik1 N-terminal region (residues 8-760) from its catalytic domain (residues 792-1066) abolishes lipid kinase activity, inconsistent with Frq1 binding simply relieving an autoinhibitory constraint. Deletion of the lipid kinase unique motif (residues 35-110) also eliminates Pik1 activity. In the complex, binding of Ca2+-bound Frq1 forces the Pik1 chain into a U-turn. Frq1 may activate Pik1 by facilitating membrane targeting via the exposed N-myristoyl group and by imposing a structural transition that promotes association of the lipid kinase unique motif with the kinase domain.

Conservation of regulatory function in calcium-binding proteins: human frequenin (neuronal calcium sensor-1) associates productively with yeast phosphatidylinositol 4-kinase isoform, Pik1.

Frequenin, also known as neuronal calcium sensor-1 (NCS-1), is an N-myristoylated Ca2+-binding protein that has been conserved in both sequence and three-dimensional fold during evolution. We demonstrate using both genetic and biochemical approaches that the observed structural conservation between Saccharomyces cerevisiae frequenin (Frq1) and human NCS-1 is also reflected at the functional level. In yeast, the sole essential target of Frq1 is the phosphatidylinositol 4-kinase isoform, Pik1; both FRQ1 and PIK1 are indispensable for cell viability. Expression of human NCS-1 in yeast, but not a close relative (human KChIP2), rescues the inviability of frq1 cells. Furthermore, in vitro, Frq1 and NCS-1 (either N-myristoylated or unmyristoylated) compete for binding to a small 28-residue motif near the N terminus of Pik1. Site-directed mutagenesis indicates that the binding determinant in Pik1 is a hydrophobic alpha-helix and that frequenins bind to one side of this alpha-helix. We propose, therefore, that the function of NCS-1 in mammals may closely resemble that of Frq1 in S. cerevisiae and, hence, that frequenins in general may serve as regulators of certain isoforms of phosphatidylinositol 4-kinase.

Molecular interactions of yeast frequenin (Frq1) with the phosphatidylinositol 4-kinase isoform, Pik1.

Frq1, a 190-residue N-myristoylated calcium-binding protein, associates tightly with the N terminus of Pik1, a 1066-residue phosphatidylinositol 4-kinase. Deletion analysis of an Frq1-binding fragment, Pik1-(10-192), showed that residues within 80-192 are necessary and sufficient for Frq1 association in vitro. A synthetic peptide (residues 151-199) competed for binding of [(35)S]Pik1-(10-192) to bead-immobilized Frq1, whereas shorter peptides (164-199 and 174-199) did not. Correspondingly, a deletion mutant, Pik1(delta152-191), did not co-immunoprecipitate efficiently with Frq1 and did not support growth at elevated temperature. Site-directed mutagenesis of Pik1-(10-192) suggested that recognition determinants lie over an extended region. Titration calorimetry demonstrated that binding of an 83-residue fragment, Pik1-(110-192), or the 151-199 peptide to Frq1 shows high affinity (K(d) approximately 100 nm) and is largely entropic, consistent with hydrophobic interaction. Stoichiometry of Pik1-(110-192) binding to Frq1 was 1:1, as judged by titration calorimetry, by changes in NMR spectrum and intrinsic tryptophan fluorescence, and by light scattering. In cell extracts, Pik1 and Frq1 exist mainly in a heterodimeric complex, as shown by size exclusion chromatography. Cys-15 in Frq1 is not S-palmitoylated, as assessed by mass spectrometry; a Frq1(C15A) mutant and even a non-myristoylated Frq1(G2A,C15A) double mutant rescued the inviability of frq1Delta cells. This study defines the segment of Pik1 required for high affinity binding of Frq1.