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Pleckstrin Homology Domain (pleckstrin + homology_domain)
Selected AbstractsDynamin 2 mutations associated with human diseases impair clathrin-mediated receptor endocytosis,HUMAN MUTATION, Issue 10 2009Marc Bitoun Abstract Dynamin 2 (DNM2) is a large GTPase involved in the release of nascent vesicles during endocytosis and intracellular membrane trafficking. Distinct DNM2 mutations, affecting the middle domain (MD) and the Pleckstrin homology domain (PH), have been identified in autosomal dominant centronuclear myopathy (CNM) and in the intermediate and axonal forms of the Charcot-Marie-Tooth peripheral neuropathy (CMT). We report here the first CNM mutation (c.1948G>A, p.E650,K) in the DNM2 GTPase effector domain (GED), leading to a slowly progressive moderate myopathy. COS7 cells transfected with DNM2 constructs harboring a disease-associated mutation in MD, PH, or GED show a reduced uptake of transferrin and low-density lipoprotein (LDL) complex, two markers of clathrin-mediated receptor endocytosis. A decrease in clathrin-mediated endocytosis was also identified in skin fibroblasts from one CNM patient. We studied the impact of DNM2 mutant overexpression on epidermal growth factor (EGF)-induced extracellular signal-regulated kinase 1 (ERK1) and ERK2 activation, known to be an endocytosis- and DNM2-dependent process. Activation of ERK1/2 was impaired for all the transfected mutants in COS7 cells, but not in CNM fibroblasts. Our results indicate that impairment of clathrin-mediated endocytosis may play a role in the pathophysiological mechanisms leading to DNM2-related diseases, but the tissue-specific impact of DNM2 mutations in both diseases remains unclear. Hum Mutat 30:1,9, 2009. © 2009 Wiley-Liss, Inc. [source] Expression of PI(4,5)P2 -binding proteins lowers the PI(4,5)P2 level and inhibits Fc,RIIA-mediated cell spreading and phagocytosisEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 1 2008Ewelina Szyma Abstract We found that Fc,RII-mediated cell spreading and phagocytosis were correlated with an increase of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] level in cells. During the spreading, a long-lasting elevation of PI(4,5)P2 and concomitant actin polymerization occurred. Filopodia and lamellae of spreading cells were enriched in phosphatidylinositol 4-phosphate 5-kinase I, (PIP5-kinase I,) that colocalized with PI(4,5)P2 and actin filaments. Both spreading and phagocytosis were inhibited by expression of the C374,440 fragment of PIP5-kinase I, or the pleckstrin homology domain of phospholipase C,1 (PLC,1 -PH), two probes binding PI(4,5)P2. These probes reduced the amount of PI(4,5)P2 in the cells, evoked reorganization of the actin cytoskeleton and abolished PI(4,5)P2 elevation during phagocytosis. Simultaneously, PLC,1 -PH-GFP reduced the amount of PIP5-kinase I, associated with the plasma membrane. In vitro studies demonstrated that PIP5-kinase I,-GST bound PI(4,5)P2, phosphatidylinositol 4-monophosphate, and less efficiently, phosphatidic acid. The data suggest that the PLC,1 -PH domain, and possibly also the C374,440 fragment, when expressed in cells, can compete with endogenous PIP5-kinase I, for PI(4,5)P2 binding in the plasma membrane leading eventually to PI(4,5)P2 depletion. [source] Schwann cells and the pathogenesis of inherited motor and sensory neuropathies (Charcot-Marie-Tooth disease)GLIA, Issue 4 2006Philipp Berger Abstract Over the last 15 years, a number of mutations in a variety of genes have been identified that lead to inherited motor and sensory neuropathies (HMSN), also called Charcot-Marie-Tooth disease (CMT). In this review we will focus on the molecular and cellular mechanisms that cause the Schwann cell pathologies observed in dysmyelinating and demyelinating forms of CMT. In most instances, the underlying gene defects alter primarily myelinating Schwann cells followed by secondary axonal degeneration. The first set of proteins affected by disease-causing mutations includes the myelin components PMP22, P0/MPZ, Cx32/GJB1, and periaxin. A second group contains the regulators of myelin gene transcription EGR2/Krox20 and SOX10. A third group is composed of intracellular Schwann cells proteins that are likely to be involved in the synthesis, transport and degradation of myelin components. These include the myotubularin-related lipid phosphatase MTMR2 and its regulatory binding partner MTMR13/SBF2, SIMPLE, and potentially also dynamin 2. Mutations affecting the mitochondrial fission factor GDAP1 may indicate an important contribution of mitochondria in myelination or myelin maintenance, whereas the functions of other identified genes, including NDRG1, KIAA1985, and the tyrosyl-tRNA synthase YARS, are not yet clear. Mutations in GDAP1, YARS, and the pleckstrin homology domain of dynamin 2 lead to an intermediate form of CMT that is characterized by moderately reduced nerve conduction velocity consistent with minor myelin deficits. Whether these phenotypes originate in Schwann cells or in neurons, or whether both cell types are directly affected, remains a challenging question. However, based on the advances in systematic gene identification in CMT and the analyses of the function and dysfunction of the affected proteins, crucially interconnected pathways in Schwann cells in health and disease have started to emerge. These networks include the control of myelin formation and stability, membrane trafficking, intracellular protein sorting and quality control, and may extend to mitochondrial dynamics and basic protein biosynthesis. © 2006 Wiley-Liss, Inc. [source] Fluorescence resonance energy transfer and anisotropy reveals both hetero- and homo-energy transfer in the pleckstrin homology-domain and the parathyroid hormone-receptorMICROSCOPY RESEARCH AND TECHNIQUE, Issue 1 2009Ralf Steinmeyer Abstract We present a method and an apparatus of polarized fluorescence resonance energy transfer (FRET) and anisotropy imaging microscopy done in parallel for improved interpretation of the photophysical interactions. We demonstrate this apparatus to better determine the protein,protein interactions in the pleckstrin homology domain and the conformational changes in the Parathyroid Hormone Receptor, a G-protein coupled receptor, both fused to the cyan and yellow fluorescent proteins for either inter- or intramolecular FRET. In both cases, the expression levels of proteins and also background autofluorescence played a significant role in the depolarization values measured in association with FRET. The system has the sensitivity and low-noise capability of single-fluorophore detection. Using counting procedures from single-molecule methods, control experiments were performed to determine number densities of green fluorescence protein variants CFP and YFP where homo resonance energy transfer can occur. Depolarization values were also determined for flavins, a common molecule of cellular background autofluorescence. From the anisotropy measurements of donor and acceptor, the latter when directly excited or when excited by energy transfer, we find that our instrumentation and method also characterizes crucial effects from homotransfer, polarization specific photobleaching and background molecules. Microsc. Res. Tech., 2009. © 2008 Wiley-Liss, Inc. [source] Absence of E17K mutation in the pleckstrin homology domain of AKT1 in gastrointestinal and liver cancers in the Korean population,APMIS, Issue 6 2008Letter to the editor First page of article [source] Purification, crystallization and preliminary X-ray diffraction of a proteolytic fragment of PDK1 containing the pleckstrin homology domainACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2004David Komander 3-Phosphoinositide-dependent protein kinase-1 (PDK1) is a Ser/Thr kinase with an essential role in insulin and growth-factor signalling. PDK1 activity towards protein kinase B (PKB) is partially regulated by its pleckstrin homology (PH) domain, which preferentially binds to 3-phosphoinositides. However, the precise molecular mechanism of this regulation is not well understood. Here, the cloning, purification and crystallization of a 150-amino-acid C-terminal region of PDK1 containing the PH domain is reported. A crystal of the PDK1 PH domain grown in the presence of inositol 1,3,4,5-tetrakisphosphate and derivatized with AuCN diffracted to 1.5,Å at a synchrotron source. Diffraction data collected near the Au edge resulted in an anomalous Patterson map with a 30, peak. [source] Molecular analysis of the serine/threonine kinase Akt and its expression in the mosquito Aedes aegyptiINSECT MOLECULAR BIOLOGY, Issue 3 2003M. A. Riehle Abstract A key component of the insulin-signalling pathway, the protein kinase Akt, was identified and cloned as a cDNA from ovaries of the mosquito Aedes aegypti. An ortholog gene was found in the Anopheles gambiae genome database, and like other Akts, both mosquito Akts possess pleckstrin homology domains for membrane binding and a serine/threonine kinase domain. When Ae. aegypti ovaries were treated with bovine insulin in vitro, a putative Akt was threonine-phosphorylated, as expected for Akts. AaegAKT was only expressed in embryos for the first 6 h after oviposition and in ovaries before and during a gonotrophic cycle. [source] | |||||||||||||