Pleckstrin Homology Domains: Two Halves Make a Hole?

In a recent issue of Nature, van Rossum et al. report binding of a “split” pleckstrin homology (PH) domain from phospholipase C-γ1 to the TRPC3 ion channel. Through sequence analyses and in vitro studies, they suggest a novel mode of protein-protein interaction in which two PH domain fragments in distinct proteins associate to form an “intermolecular” PH domain that binds inositol phospholipids and is required for ion channel location and function

Cell Signaling by Receptor Tyrosine Kinases

Recent structural studies of receptor tyrosine kinases (RTKs) have revealed unexpected diversity in the mechanisms of their activation by growth factor ligands. Strategies for inducing dimerization by ligand binding are surprisingly diverse, as are mechanisms that couple this event to activation of the intracellular tyrosine kinase domains. As our understanding of these details becomes increasingly sophisticated, it provides an important context for therapeutically countering the effects of pathogenic RTK mutations in cancer and other diseases. Much remains to be learned, however, about the complex signaling networks downstream from RTKs and how alterations in these networks are translated into cellular responses

Ala-insertion scanning mutagenesis of the glycophorin A transmembrane helix

Alanine insertions into the glycophorinA transmembrane helix are found to disrupthelix-helix dimerization in a way thatis fully consistentwith earlier saturation mutagenesis datas,uggesting that Ala-insertion scanning can be used to rapidly map the approximate locatiofn structurally and/or functionally importantsegments in trans-membrane helice

The Role of the Pleckstrin Homology Domain in Membrane Targeting and Activation of Phospholipase Cβ1

Current studies involve an investigation of the role of the pleckstrin homology (PH) domain in membrane targeting and activation of phospholipase Cbeta(1) (PLCbeta(1)). Here we report studies on the membrane localization of the isolated PH domain from the amino terminus of PLCbeta(1) (PLCbeta(1)-PH) using fluorescence microscopy of a green fluorescent protein fusion protein. Whereas PLCbeta(1)-PH does not localize to the plasma membrane in serum-starved cells, it undergoes a rapid but transient migration to the plasma membrane upon stimulation of cells with serum or lysophosphatidic acid (LPA). Regulation of the plasma membrane localization of PLCbeta(1)-PH by phosphoinositides was also investigated. PLCbeta(1)-PH was found to bind phosphatidylinositol 3-phosphate most strongly, whereas other phosphoinositides were bound with lower affinity. The plasma membrane localization of PLCbeta(1)-PH induced by serum and LPA was blocked by wortmannin pretreatment and by LY294002. In parallel, activation of PLCbeta by LPA was inhibited by wortmannin, by LY294002, or by the overexpression of PLCbeta(1)-PH. Microinjection of betagamma subunits of G proteins in serum-starved cells induced the translocation of PLCbeta(1)-PH to the plasma membrane. These results demonstrate that a cooperative mechanism involving phosphatidylinositol 3-phosphate and the Gbetagamma subunit regulates the plasma membrane localization and activation of PLCbeta(1)-PH

Live cell imaging with protein domains capable of recognizing phosphatidylinositol 4,5-bisphosphate; a comparative study

<p>Abstract</p> <p>Background</p> <p>Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)<it>P</it>₂] is a critically important regulatory phospholipid found in the plasma membrane of all eukaryotic cells. In addition to being a precursor of important second messengers, PtdIns(4,5)<it>P</it>₂also regulates ion channels and transporters and serves the endocytic machinery by recruiting clathrin adaptor proteins. Visualization of the localization and dynamic changes in PtdIns(4,5)<it>P</it>₂levels in living cells is critical to understanding the biology of PtdIns(4,5)<it>P</it>₂. This has been mostly achieved with the use of the pleckstrin homology (PH) domain of PLCδ1 fused to GFP. Here we report on a comparative analysis of several recently-described yeast PH domains as well as the mammalian Tubby domain to evaluate their usefulness as PtdIns(4,5)<it>P</it>₂imaging tools.</p> <p>Results</p> <p>All of the yeast PH domains that have been previously shown to bind PtdIns(4,5)<it>P</it>₂showed plasma membrane localization but only a subset responded to manipulations of plasma membrane PtdIns(4,5)<it>P</it>₂. None of these domains showed any advantage over the PLCδ1PH-GFP reporter and were compromised either in their expression levels, nuclear localization or by causing peculiar membrane structures. In contrast, the Tubby domain showed high membrane localization consistent with PtdIns(4,5)<it>P</it>₂binding and displayed no affinity for the soluble headgroup, Ins(1,4,5)P₃. Detailed comparison of the Tubby and PLCδ1PH domains showed that the Tubby domain has a higher affinity for membrane PtdIns(4,5)<it>P</it>₂and therefore displays a lower sensitivity to report on changes of this lipid during phospholipase C activation.</p> <p>Conclusion</p> <p>These results showed that both the PLCδ1PH-GFP and the GFP-Tubby domain are useful reporters of PtdIns(4,5)<it>P</it>₂changes in the plasma membrane, with distinct advantages and disadvantages. While the PLCδ1PH-GFP is a more sensitive reporter, its Ins(1,4,5)P₃binding may compromise its accuracy to measure PtdIns(4,5)<it>P</it>₂changes. The Tubby domain is more accurate to report on PtdIns(4,5)<it>P</it>₂but its higher affinity and lower sensitivity may limit its utility when phospholipase C activation is only moderate. These studies also demonstrated that similar changes in PtdIns(4,5)<it>P</it>₂levels in the plasma membrane can differentially regulate multiple effectors if they display different affinities to PtdIns(4,5)<it>P</it>₂.</p

Titan Science with the James Webb Space Telescope (JWST)

The James Webb Space Telescope (JWST), scheduled for launch in 2018, is the successor to the Hubble Space Telescope (HST) but with a significantly larger aperture (6.5 m) and advanced instrumentation focusing on infrared science (0.6-28.0 $\mu$m ). In this paper we examine the potential for scientific investigation of Titan using JWST, primarily with three of the four instruments: NIRSpec, NIRCam and MIRI, noting that science with NIRISS will be complementary. Five core scientific themes are identified: (i) surface (ii) tropospheric clouds (iii) tropospheric gases (iv) stratospheric composition and (v) stratospheric hazes. We discuss each theme in depth, including the scientific purpose, capabilities and limitations of the instrument suite, and suggested observing schemes. We pay particular attention to saturation, which is a problem for all three instruments, but may be alleviated for NIRCam through use of selecting small sub-arrays of the detectors - sufficient to encompass Titan, but with significantly faster read-out times. We find that JWST has very significant potential for advancing Titan science, with a spectral resolution exceeding the Cassini instrument suite at near-infrared wavelengths, and a spatial resolution exceeding HST at the same wavelengths. In particular, JWST will be valuable for time-domain monitoring of Titan, given a five to ten year expected lifetime for the observatory, for example monitoring the seasonal appearance of clouds. JWST observations in the post-Cassini period will complement those of other large facilities such as HST, ALMA, SOFIA and next-generation ground-based telescopes (TMT, GMT, EELT).Comment: 50 pages, including 22 figures and 2 table

Dust aerosol, clouds, and the atmospheric optical depth record over 5 Mars years of the Mars Exploration Rover mission

Dust aerosol plays a fundamental role in the behavior and evolution of the Martian atmosphere. The first five Mars years of Mars Exploration Rover data provide an unprecedented record of the dust load at two sites. This record is useful for characterization of the atmosphere at the sites and as ground truth for orbital observations. Atmospheric extinction optical depths have been derived from solar images after calibration and correction for time-varying dust that has accumulated on the camera windows. The record includes local, regional, and globally extensive dust storms. Comparison with contemporaneous thermal infrared data suggests significant variation in the size of the dust aerosols, with a 1 {\mu}m effective radius during northern summer and a 2 {\mu}m effective radius at the onset of a dust lifting event. The solar longitude (LS) 20-136{\deg} period is also characterized by the presence of cirriform clouds at the Opportunity site, especially near LS=50 and 115{\deg}. In addition to water ice clouds, a water ice haze may also be present, and carbon dioxide clouds may be present early in the season. Variations in dust opacity are important to the energy balance of each site, and work with seasonal variations in insolation to control dust devil frequency at the Spirit site.Comment: 60 pages, 12 figures, to be published in Icaru

Mechanism of Activation and Inhibition of the HER4/ErbB4 Kinase

SummaryHER4/ErbB4 is a ubiquitously expressed member of the EGF/ErbB family of receptor tyrosine kinases that is essential for normal development of the heart, nervous system, and mammary gland. We report here crystal structures of the ErbB4 kinase domain in active and lapatinib-inhibited forms. Active ErbB4 kinase adopts an asymmetric dimer conformation essentially identical to that observed to be important for activation of the EGF receptor/ErbB1 kinase. Mutagenesis studies of intact ErbB4 in Ba/F3 cells confirm the importance of this asymmetric dimer for activation of intact ErbB4. Lapatinib binds to an inactive form of the ErbB4 kinase in a mode equivalent to its interaction with the EGF receptor. All ErbB4 residues contacted by lapatinib are conserved in the EGF receptor and HER2/ErbB2, which lapatinib also targets. These results demonstrate that key elements of kinase activation and inhibition are conserved among ErbB family members