Inhibition of Ca2+ signalling by p130, a phospholipase-C-related catalytically inactive protein: critical role of the p130 pleckstrin homology domain

2000 ◽  
Vol 349 (1) ◽  
pp. 357-368 ◽  
Author(s):  
Hiroshi TAKEUCHI ◽  
Masahiro OIKE ◽  
Hugh F. PATERSON ◽  
Victoria ALLEN ◽  
Takashi KANEMATSU ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Cellular Localization ◽  
Critical Role ◽  
Inhibitory Effect ◽  
Pleckstrin Homology Domain ◽  
Pleckstrin Homology ◽  
Ligand Complex ◽  
Epidermal Growth ◽  
In Cells

p130 was originally identified as an Ins(1,4,5)P3-binding protein similar to phospholipase C-∆ but lacking any phospholipase activity. In the present study we have further analysed the interactions of p130 with inositol compounds in vitro. To determine which of the potential ligands interacts with p130 in cells, we performed an analysis of the cellular localization of this protein, the isolation of a protein-ligand complex from cell lysates and studied the effects of p130 on Ins(1,4,5)P3-mediated Ca2+ signalling by using permeabilized and transiently or stably transfected COS-1 cells (COS-1p130). In vitro, p130 bound Ins(1,4,5)P3 with a higher affinity than that for phosphoinositides. When the protein was isolated from COS-1p130 cells by immunoprecipitation, it was found to be associated with Ins(1,4,5)P3. Localization studies demonstrated the presence of the full-length p130 in the cytoplasm of living cells, not at the plasma membrane. In cell-based assays, p130 had an inhibitory effect on Ca2+ signalling. When fura-2-loaded COS-1p130 cells were stimulated with bradykinin, epidermal growth factor or ATP, it was found that the agonist-induced increase in free Ca2+ concentration, observed in control cells, was inhibited in COS-1p130. This inhibition was not accompanied by the decreased production of Ins(1,4,5)P3; the intact p130 pleckstrin homology domain, known to be the ligand-binding site in vitro, was required for this effect in cells. These results suggest that Ins(1,4,5)P3 could be the main p130 ligand in cells and that this binding has the potential to inhibit Ins(1,4,5)P3-mediated Ca2+ signalling.

scholarly journals Myo1c Binds Phosphoinositides through a Putative Pleckstrin Homology Domain

2006 ◽  
Vol 17 (11) ◽  
pp. 4856-4865 ◽  
Author(s):  
David E. Hokanson ◽  
Joseph M. Laakso ◽  
Tianming Lin ◽  
David Sept ◽  
E. Michael Ostap
Keyword(s):  
Binding Site ◽  
Membrane Trafficking ◽  
Cellular Localization ◽  
Membrane Binding ◽  
Pleckstrin Homology Domain ◽  
Dependent Manner ◽  
Ph Domain ◽  
Pleckstrin Homology

Myo1c is a member of the myosin superfamily that binds phosphatidylinositol-4,5-bisphosphate (PIP2), links the actin cytoskeleton to cellular membranes and plays roles in mechano-signal transduction and membrane trafficking. We located and characterized two distinct membrane binding sites within the regulatory and tail domains of this myosin. By sequence, secondary structure, and ab initio computational analyses, we identified a phosphoinositide binding site in the tail to be a putative pleckstrin homology (PH) domain. Point mutations of residues known to be essential for polyphosphoinositide binding in previously characterized PH domains inhibit myo1c binding to PIP2 in vitro, disrupt in vivo membrane binding, and disrupt cellular localization. The extended sequence of this binding site is conserved within other myosin-I isoforms, suggesting they contain this putative PH domain. We also characterized a previously identified membrane binding site within the IQ motifs in the regulatory domain. This region is not phosphoinositide specific, but it binds anionic phospholipids in a calcium-dependent manner. However, this site is not essential for in vivo membrane binding.

scholarly journals Phospholipase C δ1 requires a pleckstrin homology domain for interaction with the plasma membrane

1995 ◽  
Vol 312 (3) ◽  
pp. 661-666 ◽  
Author(s):  
H F Paterson ◽  
J W Savopoulos ◽  
O Perisic ◽  
R Cheung ◽  
M V Ellis ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Phospholipase C ◽  
Living Cells ◽  
Pleckstrin Homology Domain ◽  
Deletion Mutants ◽  
Pleckstrin Homology ◽  
Structural Requirements ◽  
Inositol 1,4,5 Trisphosphate ◽  
Membrane Attachment

The structural requirements of phospholipase C delta 1 for interaction with the plasma membrane were analysed by immunofluorescence after microinjection into living cells. Microinjection of deletion mutants revealed that the region required for membrane attachment and binding of inositol 1,4,5-trisphosphate in vitro corresponded to the pleckstrin homology domain, a structural module described in more than 90 proteins.

scholarly journals The Lipid Binding Pleckstrin Homology Domain in UNC-104 Kinesin is Necessary for Synaptic Vesicle Transport in Caenorhabditis elegans

2004 ◽  
Vol 15 (8) ◽  
pp. 3729-3739 ◽  
Author(s):  
Dieter R. Klopfenstein ◽  
Ronald D. Vale
Keyword(s):  
Caenorhabditis Elegans ◽  
Synaptic Vesicles ◽  
Critical Role ◽  
Lipid Binding ◽  
Neuronal Cell Body ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Pleckstrin Homology

UNC-104 (KIF1A) is a kinesin motor that transports synaptic vesicles from the neuronal cell body to the terminal. Previous in vitro studies have shown that a Dictyostelium relative of UNC-104 transports liposomes containing acidic phospholipids, but whether this interaction is needed for the recognition and transport of synaptic vesicles in metazoans remains unexplored. Here, we have introduced mutations in the nonmotor domain of UNC-104 and examined whether these mutant motors can rescue an unc-104 Caenorhabditis elegans strain. We show that a pleckstrin homology (PH) domain in UNC-104 is essential for membrane transport in living C. elegans, that this PH domain binds specifically to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), and that point mutants in the PH domain that interfere with PI(4,5)P2 binding in vitro also interfere with UNC-104 function in vivo. Several other lipid-binding modules could not effectively substitute for the UNC-104 PH domain in this in vivo assay. Real time imaging also revealed that a lipid-binding point mutation in the PH domain reduced movement velocity and processivity of individual UNC-104::GFP punctae in neurites. These results reveal a critical role for PI(4,5)P2 binding in UNC-104–mediated axonal transport and shows that the cargo-binding properties of the distal PH domain can affect motor output.

scholarly journals Inhibition of Ca2+ signalling by p130, a phospholipase-C-related catalytically inactive protein: critical role of the p130 pleckstrin homology domain

2000 ◽  
Vol 349 (1) ◽  
pp. 357 ◽  
Author(s):  
Hiroshi TAKEUCHI ◽  
Masahiro OIKE ◽  
Hugh F. PATERSON ◽  
Victoria ALLEN ◽  
Takashi KANEMATSU ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Critical Role ◽  
Pleckstrin Homology Domain ◽  
Pleckstrin Homology ◽  
Inactive Protein

scholarly journals Critical Role for Kalirin in Nerve Growth Factor Signaling through TrkA

2005 ◽  
Vol 25 (12) ◽  
pp. 5106-5118 ◽  
Author(s):  
Kausik Chakrabarti ◽  
Rong Lin ◽  
Noraisha I. Schiller ◽  
Yanping Wang ◽  
David Koubi ◽  
...  
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Pc12 Cells ◽  
Critical Role ◽  
Neuronal Morphology ◽  
Neurotrophin Receptor ◽  
General Mechanism ◽  
Pleckstrin Homology Domain ◽  
Pleckstrin Homology ◽  
Nerve Growth

ABSTRACT Kalirin is a multidomain guanine nucleotide exchange factor (GEF) that activates Rho proteins, inducing cytoskeletal rearrangement in neurons. Although much is known about the effects of Kalirin on Rho GTPases and neuronal morphology, little is known about the association of Kalirin with the receptor/signaling systems that affect neuronal morphology. Our experiments demonstrate that Kalirin binds to and colocalizes with the TrkA neurotrophin receptor in neurons. In PC12 cells, inhibition of Kalirin expression using antisense RNA decreased nerve growth factor (NGF)-induced TrkA autophosphorylation and process extension. Kalirin overexpression potentiated neurotrophin-stimulated TrkA autophosphorylation and neurite outgrowth in PC12 cells at a low concentration of NGF. Furthermore, elevated Kalirin expression resulted in catalytic activation of TrkA, as demonstrated by in vitro kinase assays and increased NGF-stimulated cellular activation of Rac, Mek, and CREB. Domain mapping demonstrated that the N-terminal Kalirin pleckstrin homology domain mediates the interaction with TrkA. The effects of Kalirin on TrkA provide a molecular basis for the requirement of Kalirin in process extension from PC12 cells and for previously observed effects on axonal extension and dendritic maintenance. The interaction of TrkA with the pleckstrin homology domain of Kalirin may be one example of a general mechanism whereby receptor/Rho GEF pairings play an important role in receptor tyrosine kinase activation and signal transduction.

Abstract 356: Krueppel-Like Factor 15 Regulates Wnt/β-Catenin Transcription and Controls Cardiac Progenitor Cell Fate in the Postnatal Heart

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Claudia Noack ◽  
Maria P Zafiriou ◽  
Anke Renger ◽  
Hans J Schaeffer ◽  
Martin W Bergmann ◽  
...  
Keyword(s):  
Cell Fate ◽  
Transcriptional Activation ◽  
Progenitor Cell ◽  
Cellular Localization ◽  
Target Genes ◽  
Critical Role ◽  
Isolated Cells ◽  
Cardiac Progenitor Cell

Wnt/β-catenin signaling controls adult heart remodeling partly by regulating cardiac progenitor cell (CPC) differentiation. We now identified and characterized a novel cardiac interaction of the transcription factor Krueppel-like factor 15 (KLF15) with the Wnt/β-catenin signaling on adult CPCs. In vitro mutation, reporter gene assays and co-localization studies revealed that KLF15 requires two distinct domains for nuclear localization and for repression of β-catenin-mediated transcription. KLF15 had no effect on β-catenin stability or cellular localization, but interacted with its co-factor TCF4, which is required for activation of β-catenin target gene expression. Moreover, increased TCF4 ubiquitination was induced by KLF15. In line with this finding we found KLF15 to interact with the Nemo-like kinase, which was shown to phosphorylate and target TCF4 for degradation. In vivo analyses of adult Klf15 functional knock-out (KO) vs. wild-type (WT) mice showed a cardiac β-catenin-mediated transcriptional activation and reduced TCF4 degradation along with cardiac dysfunction assessed by echocardiography (n=10). FACS analysis of the CPC enriched-population of KO vs. WT mice revealed a significant reduction of cardiogenic-committed precursors identified as Sca1+/αMHC+ (0.8±0.2% vs. 1.8±0.1%) and Tbx5+ (3.5±0.3% vs. 5.2±0.5%). In contrast, endothelial Sca1+/CD31+ cells were significantly higher in KO mice (11.3±0.4% vs. 8.6±0.4%; n≥9). In addition, Sca1+ isolated cells of Klf15 KO showed increased RNA expression of endothelial markers von Willebrand Factor, CD105, and Flk1 along with upregulation of β-catenin target genes. CPCs co-cultured on adult fibroblasts resulted in increased endothelial Flk1 cells and reduction of αMHC and Hand1 cardiogenic cells in KO vs. WT CPCs (n=9). Treating these co-cultures with Quercetin, an inhibitor of nuclear β-catenin, resulted in partial rescue of the observed phenotype. This study uncovers a critical role of KLF15 for the maintenance of cardiac tissue homeostasis. Via inhibition of β-catenin transcription, KLF15 controls cardiomyogenic cell fate similar to embryonic cardiogenesis. This knowledge may provide a tool for activation of endogenous CPCs in the postnatal heart.

scholarly journals Interaction of Elongation Factor-1α and Pleckstrin Homology Domain of Phospholipase C-γ1 with Activating Its Activity

2002 ◽  
Vol 277 (22) ◽  
pp. 19697-19702 ◽  
Author(s):  
Jong-Soo Chang ◽  
Heon Seok ◽  
Taeg-Kyu Kwon ◽  
Do Sik Min ◽  
Bong-Hyun Ahn ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Elongation Factor ◽  
Pleckstrin Homology Domain ◽  
Pleckstrin Homology ◽  
Elongation Factor 1Α

scholarly journals Phosphorylation and activation of epidermal growth factor receptors in cells transformed by the src oncogene.

1991 ◽  
Vol 11 (1) ◽  
pp. 309-321 ◽  
Author(s):  
W J Wasilenko ◽  
D M Payne ◽  
D L Fitzgerald ◽  
M J Weber
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Phospholipase C ◽  
Tyrosine Phosphorylation ◽  
Egf Receptor ◽  
Growth Factor Receptors ◽  
Epidermal Growth ◽  
Phospholipase C Gamma ◽  
Signaling Activity ◽  
In Cells

Because functionally significant substrates for the tyrosyl protein kinase activity of pp60v-src are likely to include membrane-associated proteins involved in normal growth control, we have tested the hypothesis that pp60v-src could phosphorylate and alter the signaling activity of transmembrane growth factor receptors. We have found that the epidermal growth factor (EGF) receptor becomes constitutively phosphorylated on tyrosine in cells transformed by the src oncogene and in addition displays elevated levels of phosphoserine and phosphothreonine. High-performance liquid chromatography phosphopeptide mapping revealed two predominant sites of tyrosine phosphorylation, both of which differed from the major sites of receptor autophosphorylation; thus, the src-induced phosphorylation is unlikely to occur via an autocrine mechanism. To determine whether pp60v-src altered the signaling activity of the EGF receptor, we analyzed the tyrosine phosphorylation of phospholipase C-gamma, since phosphorylation of this enzyme occurs in response to activation of the EGF receptor but not in response to pp60v-src alone. We found that in cells coexpressing pp60v-src and the EGF receptor, phospholipase C-gamma was constitutively phosphorylated, a result we interpret as indicating that the signaling activity of the EGF receptor was altered in the src-transformed cells. These findings suggest that pp60v-src-induced alterations in phosphorylation and function of growth regulatory receptors could play an important role in generating the phenotypic changes associated with malignant transformation.

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