Inositol lipid binding and membrane localization of isolated pleckstrin homology (PH) domains. Studies on the PH domains of phospholipase C delta 1 and p130.

Endocrinology and Reproduction Research Branch, NICHD/National Institutes of Health, 49 Convent Drive, Bldg. 49, Bethesda, MD 20892, USA.
Journal of Biological Chemistry (Impact Factor: 4.65). 08/2002; 277(30):27412-22. DOI: 10.1074/jbc.M109672200
Source: PubMed

ABSTRACT The relationship between the ability of isolated pleckstrin homology (PH) domains to bind inositol lipids or soluble inositol phosphates in vitro and to localize to cellular membranes in live cells was examined by comparing the PH domains of phospholipase Cdelta(1) (PLCdelta(1)) and the recently cloned PLC-like protein p130 fused to the green fluorescent protein (GFP). The prominent membrane localization of PLCdelta(1)PH-GFP was paralleled with high affinity binding to inositol 1,4,5-trisphosphate (InsP(3)) as well as to phosphatidylinositol 4,5-bisphosphate-containing lipid vesicles or nitrocellulose membrane strips. In contrast, no membrane localization was observed with p130PH-GFP despite its InsP(3) and phosphatidylinositol 4,5-bisphosphate-binding properties being comparable with those of PLCdelta(1)PH-GFP. The N-terminal ligand binding domain of the type I InsP(3) receptor also failed to localize to the plasma membrane despite its 5-fold higher affinity to InsP(3) than the PH domains. By using a chimeric approach and cassette mutagenesis, the C-terminal alpha-helix and the short loop between the beta6-beta7 sheets of the PLCdelta(1)PH domain, in addition to its InsP(3)-binding region, were identified as critical components for membrane localization in intact cells. These data indicate that binding to the inositol phosphate head group is necessary but may not be sufficient for membrane localization of the PLCdelta(1)PH-GFP fusion protein, and motifs located within the C-terminal half of the PH domain provide auxiliary contacts with additional membrane components.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Phosphoinositide-specific phospholipase C δ (PLC δ) plays an important role in many cellular responses and is involved in the production of second messenger. Here, we describe the presence of novel N-terminal extended alternative splice form of PLC-δ1B in Paralichthys olivaceus, which differs from the reported mammalian PLC-δ1 isoform. The two variants PoPLC-δ1B-Lf and PoPLC-δ1B-Sf share exon 3 (including the PH domain) to exon 16, but differ at the exon 1 (Short form: Sf) and novel exon 2 (Long form: Lf) of the transcript. For the characterization of the novel duplicated gene variant of PLC-δ1B in P. olivaceus, tissue-specific expression with RT-PCR and real-time PCR, and purification and enzymatic characterization of native and recombinant protein of all the three-types of PLC-δ1 isoforms (PoPLC-δ1A, PoPLC-δ1B-Lf and PoPLC-δ1B-Sf) of P. olivaceus were studied. The PoPLC-δ1A was ubiquitously distributed in gill, kidney and spleen. The PoPLC-δ1B-Lf gene was widely detected in various tissues, especially in the digestive system, while PoPLC-δ1B-Sf was highly expressed in the stomach. The recombinant PoPLC-δ1A, PoPLC-δ1B-Lf and PoPLC-δ1B-Sf proteins were expressed as a histidine-tagged fusion protein in Escherichia coli. The PLC activity of the PoPLC-δ1 isoform proteins showed a concentration-dependent activity to phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2). Also, U73122, the PLC inhibitor, effectively inhibited PLC activities of PoPLC-δ1A, PoPLC-δ1B-Lf and PoPLC-δ1B-Sf proteins. However, PoPLC-δ1A and PoPLC-δ1B-Lf were sensitive at pH 7.5, while PoPLC-δ1B-Sf was relatively sensitive at pH 7. These results might be useful for the study of phospholipase C-mediated signal transduction in fish.
    Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 04/2013; · 1.61 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Phosphatidylinositolphosphates (PIPs) are phospholipids that contain a phosphorylated inositol head group. PIPs represent a minor fraction of the total phospholipids, yet they are involved in many regulatory processes such as cell signalling and intracellular trafficking. Membrane compartments are enriched or depleted in specific PIPs, which constitute a signature for these compartments and contribute to their identity. The precise subcellular localisation and dynamics of most PIP species is not fully understood in plants. Here, we designed genetically encoded biosensors with distinct relative affinities and expressed them stably in Arabidopsis thaliana. Analysis of this multi-affinity "PIPline" marker set revealed previously unrecognized localisation for various PIPs in root epidermis. Notably, we found that PI(4,5)P2 is able to drive PIP2 -interacting protein domains to the plasma membrane in non-stressed root epidermal cells. Our analysis further revealed that there is a gradient of PI4P, with the highest concentration at the plasma membrane, intermediate concentration in post-Golgi/endosomal compartments and lowest concentration in the Golgi. Finally, we also uncovered that there is a similar gradient of PI3P from high in late endosomes to low in the tonoplast. All together our library extends the palette of available PIP biosensors and should promote rapid progress in our understanding of PIP dynamics in plants. This article is protected by copyright. All rights reserved.
    The Plant Journal 10/2013; · 6.58 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Phosphoinositides (PIs) make up only a small fraction of cellular phospholipids, yet they control almost all aspects of a cell's life and death. These lipids gained tremendous research interest as plasma membrane signaling molecules when discovered in the 1970s and 1980s. Research in the last 15 years has added a wide range of biological processes regulated by PIs, turning these lipids into one of the most universal signaling entities in eukaryotic cells. PIs control organelle biology by regulating vesicular trafficking, but they also modulate lipid distribution and metabolism via their close relationship with lipid transfer proteins. PIs regulate ion channels, pumps, and transporters and control both endocytic and exocytic processes. The nuclear phosphoinositides have grown from being an epiphenomenon to a research area of its own. As expected from such pleiotropic regulators, derangements of phosphoinositide metabolism are responsible for a number of human diseases ranging from rare genetic disorders to the most common ones such as cancer, obesity, and diabetes. Moreover, it is increasingly evident that a number of infectious agents hijack the PI regulatory systems of host cells for their intracellular movements, replication, and assembly. As a result, PI converting enzymes began to be noticed by pharmaceutical companies as potential therapeutic targets. This review is an attempt to give an overview of this enormous research field focusing on major developments in diverse areas of basic science linked to cellular physiology and disease.
    Physiological Reviews 07/2013; 93(3):1019-137. · 30.17 Impact Factor


Available from
May 28, 2014