scholarly journals High-affinity interaction of the N-terminal myristoylation motif of the neuronal calcium sensor protein hippocalcin with phosphatidylinositol 4,5-bisphosphate

2005 ◽  
Vol 391 (2) ◽  
pp. 231-238 ◽  
Author(s):  
Dermott W. O'Callaghan ◽  
Lee P. Haynes ◽  
Robert D. Burgoyne
Keyword(s):  
Fluorescent Protein ◽  
Adaptor Protein ◽  
Live Cells ◽  
K Channel ◽  
Pleckstrin Homology Domain ◽  
Membrane Targeting ◽  
Calcium Sensor ◽  
Ph Domain ◽  
Neuronal Calcium Sensor ◽  
High Affinity

Many proteins are associated with intracellular membranes due to their N-terminal myristoylation. Not all myristoylated proteins have the same localization within cells, indicating that other factors must determine their membrane targeting. The NCS (neuronal calcium sensor) proteins are a family of Ca2+-binding proteins with diverse functions. Most members of the family are N-terminally myristoylated and are either constitutively membrane-bound or have a Ca2+/myristoyl switch that allows their reversible membrane association in response to Ca2+ signals. In the case of hippocalcin and NCS-1, or alternatively KChIP1 (K+ channel-interacting protein 1), their N-terminal myristoylation motifs are sufficient for targeting to distinct organelles. We have shown that an N-terminal myristoylated hippocalcin peptide is able to specifically reproduce the membrane targeting of hippocalcin/NCS-1 when introduced into permeabilized cells. The peptide binds to liposomes containing phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] with high affinity (Kd 50 nM). Full-length hippocalcin also bound preferentially to liposomes supplemented with PtdIns(4,5)P2. Co-expression of hippocalcin-(1–14)–ECFP (enhanced cyan fluorescent protein) or NCS-1–ECFP partially displaced the expressed PH (pleckstrin homology) domain of phospholipase δ1 from the plasma membrane in live cells, indicating that they have a higher affinity for PtdIns(4,5)P2 than does this PH domain. The Golgi localization of the PH domain of FAPP1 (four-phosphate-adaptor protein 1), which binds to phosphatidylinositol 4-phosphate, was unaffected. The localization of NCS-1 and hippocalcin is likely to be determined, therefore, by their interaction with PtdIns(4,5)P2.

Regulation of B-lymphocyte activation by the PH domain adaptor protein Bam32/DAPP1

2007 ◽  
Vol 35 (2) ◽  
pp. 181-182 ◽  
Author(s):  
A.J. Marshall ◽  
T. Zhang ◽  
M. Al-Alwan
Keyword(s):  
B Cell ◽  
B Lymphocyte ◽  
Second Messengers ◽  
Lymphocyte Activation ◽  
Adaptor Protein ◽  
Cell Receptor ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
B Lymphocyte Activation ◽  
Signal Transduction Proteins

PI3Ks (phosphoinositide 3-kinases) play critical roles in BCR (B-cell receptor) signalling via the generation of 3-phosphoinositide second messengers. Recruitment of PH domain (pleckstrin homology domain)-containing signal transduction proteins to the plasma membrane through binding to 3-phosphoinositide second messengers represents a major effector mechanism for PI3Ks. Here, we review data on the PH domain-containing adaptor protein Bam32 (B-cell adaptor molecule of 32 kDa)/DAPP1 (dual adaptor for phosphotyrosine and 3-phosphoinositides 1), focusing on its functions in B-lymphocyte activation. Present results support the view that Bam32/DAPP1 mediates multiple PI3K-dependent responses in B-cells through membrane-proximal mechanisms involving Src kinases, Rac1, F-actin and mitogen-activated protein kinases, resulting in selective effects on BCR-mediated proliferation, antigen presentation and generation of antibody responses.

scholarly journals Neuronal Calcium Sensor-1 and Phosphatidylinositol 4-Kinase β Stimulate Extracellular Signal-regulated Kinase 1/2 Signaling by Accelerating Recycling through the Endocytic Recycling Compartment

2006 ◽  
Vol 17 (9) ◽  
pp. 4130-4141 ◽  
Author(s):  
Yaara Kapp-Barnea ◽  
Lihi Ninio-Many ◽  
Koret Hirschberg ◽  
Mitsunori Fukuda ◽  
Andreas Jeromin ◽  
...  
Keyword(s):  
Mast Cells ◽  
Nuclear Translocation ◽  
Calcium Sensor ◽  
Ph Domain ◽  
Endocytic Recycling ◽  
Inflammatory Reactions ◽  
Extracellular Signal Regulated Kinase ◽  
Neuronal Calcium Sensor ◽  
Extracellular Signal ◽  
Phosphatidylinositol 4

We demonstrate that recycling through the endocytic recycling compartment (ERC) is an essential step in FcεRI-induced activation of extracellular signal-regulated kinase (ERK)1/2. We show that ERK1/2 acquires perinuclear localization and colocalizes with Rab 11 and internalized transferrin in FcεRI-activated cells. Moreover, a close correlation exists between the amount of ERC-localized ERK1/2 and the amount of phospho-ERK1/2 that resides in the nucleus. We further show that by activating phosphatidylinositol 4-kinase β (PI4Kβ) and increasing the cellular level of phosphatidylinositol(4) phosphate, neuronal calcium sensor-1 (NCS-1), a calmodulin-related protein, stimulates recycling and thereby enhances FcεRI-triggered activation and nuclear translocation of ERK1/2. Conversely, NCS-1 short hairpin RNA, a kinase dead (KD) mutant of PI4Kβ (KD-PI4Kβ), the pleckstrin homology (PH) domain of FAPP1 as well as RNA interference of synaptotagmin IX or monensin, which inhibit export from the ERC, abrogate FcεRI-induced activation of ERK1/2. Consistently, NCS-1 also enhances, whereas both KD-PI4Kβ and FAPP1-PH domain inhibit, FcεRI-induced release of arachidonic acid/metabolites, a downstream target of ERK1/2 in mast cells. Together, our results demonstrate a novel role for NCS-1 and PI4Kβ in regulating ERK1/2 signaling and inflammatory reactions in mast cells. Our results further identify the ERC as a crucial determinant in controlling ERK1/2 signaling.

scholarly journals Role of Phosphatidylinositol 3′ Kinase and a Downstream Pleckstrin Homology Domain–Containing Protein in Controlling Chemotaxis inDictyostelium

2001 ◽  
Vol 153 (4) ◽  
pp. 795-810 ◽  
Author(s):  
Satoru Funamoto ◽  
Kristina Milan ◽  
Ruedi Meili ◽  
Richard A. Firtel
Keyword(s):  
Actin Polymerization ◽  
Adaptor Protein ◽  
Leading Edge ◽  
Membrane Lipid ◽  
Lipid Binding ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Wild Type ◽  
Pi3k Inhibitor Ly294002 ◽  
Effector Pathways

We show that cells lacking two Dictyostelium class I phosphatidylinositol (PI) 3′ kinases (PI3K and pi3k1/2-null cells) or wild-type cells treated with the PI3K inhibitor LY294002 are unable to properly polarize, are very defective in the temporal, spatial, and quantitative regulation of chemoattractant-mediated filamentous (F)-actin polymerization, and chemotax very slowly. PI3K is thought to produce membrane lipid-binding sites for localization of PH domain–containing proteins. We demonstrate that in response to chemoattractants three PH domain–containing proteins do not localize to the leading edge in pi3k1/2-null cells, and the translocation is blocked in wild-type cells by LY294002. Cells lacking one of these proteins, phdA-null cells, exhibit defects in the level and kinetics of actin polymerization at the leading edge and have chemotaxis phenotypes that are distinct from those described previously for protein kinase B (PKB) (pkbA)-null cells. Phenotypes of PhdA-dominant interfering mutations suggest that PhdA is an adaptor protein that regulates F-actin localization in response to chemoattractants and links PI3K to the control of F-actin polymerization at the leading edge during pseudopod formation. We suggest that PKB and PhdA lie downstream from PI3K and control different downstream effector pathways that are essential for proper chemotaxis.

scholarly journals Membrane Targeting of Grb2-associated Binder-1 (Gab1) Scaffolding Protein through Src Myristoylation Sequence Substitutes for Gab1 Pleckstrin Homology Domain and Switches an Epidermal Growth Factor Response to an Invasive Morphogenic Program

2003 ◽  
Vol 14 (4) ◽  
pp. 1691-1708 ◽  
Author(s):  
Christiane R. Maroun ◽  
Monica A. Naujokas ◽  
Morag Park
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Epithelial Cells ◽  
Subcellular Localization ◽  
Epithelial Morphogenesis ◽  
Pleckstrin Homology Domain ◽  
Membrane Targeting ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Epidermal Growth

The hepatocyte growth factor receptor tyrosine kinase Met promotes cell dissociation and the inherent morphogenic program of epithelial cells. In a search for substrates downstream from Met, we have previously identified the Grb2-associated binder-1 (Gab1) as critical for the morphogenic program. Gab1 is a scaffold protein that acts to diversify the signal downstream from the Met receptor through its ability to couple with multiple signal transduction pathways. Gab1 contains a pleckstrin homology (PH) domain with specificity for phosphatidylinositol 3,4,5-trisphosphate. The phospholipid binding capacity of the Gab1 PH domain is required for the localization of Gab1 at sites of cell-cell contact in colonies of epithelial cells and for epithelial morphogenesis, suggesting that PH domain-dependent subcellular localization of Gab1 is a prerequisite for function. We have investigated the requirement for membrane localization of Gab1 for biological activity. We show that substitution of the Gab1 PH domain with the myristoylation signal from the c-Src protein is sufficient to replace the Gab1 PH domain for epithelial morphogenesis. The membrane targeting of Gab1 enhances Rac activity in the absence of stimulation and switches a nonmorphogenic noninvasive response to epidermal growth factor to a morphogenic invasive program. These results suggest that the subcellular localization of Gab1 is a critical determinant for epithelial morphogenesis and invasiveness.

scholarly journals Alteration of Epithelial Structure and Function Associated with PtdIns(4,5)P2 Degradation by a Bacterial Phosphatase

2007 ◽  
Vol 129 (4) ◽  
pp. 267-283 ◽  
Author(s):  
David Mason ◽  
Gustavo V. Mallo ◽  
Mauricio R. Terebiznik ◽  
Bernard Payrastre ◽  
B. Brett Finlay ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Fluorescent Protein ◽  
Fluid Loss ◽  
Pleckstrin Homology Domain ◽  
High Avidity ◽  
Ph Domain ◽  
Cytosolic Ph ◽  
Functional Changes ◽  
Epithelial Cultures ◽  
Green Fluorescent

Elucidation of the role of PtdIns(4,5)P2 in epithelial function has been hampered by the inability to selectively manipulate the cellular content of this phosphoinositide. Here we report that SigD, a phosphatase derived from Salmonella, can effectively hydrolyze PtdIns(4,5)P2, generating PtdIns(5)P. When expressed by microinjecting cDNA into epithelial cells forming confluent monolayers, wild-type SigD induced striking morphological and functional changes that were not mimicked by a phosphatase-deficient SigD mutant (C462S). Depletion of PtdIns(4,5)P2 in intact SigD-injected cells was verified by detachment from the membrane of the pleckstrin homology domain of phospholipase Cδ, used as a probe for the phosphoinositide by conjugation to green fluorescent protein. Single-cell measurements of cytosolic pH indicated that the Na+/H+ exchange activity of epithelia was markedly inhibited by depletion of PtdIns(4,5)P2. Similarly, anion permeability, measured using two different halide-sensitive probes, was depressed in cells expressing SigD. Depletion of PtdIns(4,5)P2 was associated with marked alterations in the actin cytoskeleton and its association with the plasma membrane. The junctional complexes surrounding the injected cells gradually opened and the PtdIns(4,5)P2-depleted cells eventually detached from the monolayer, which underwent rapid restitution. Similar observations were made in intestinal and renal epithelial cultures. In addition to its effects on phosphoinositides, SigD has been shown to convert inositol 1,3,4,5,6-pentakisphosphate (IP5) into inositol 1,4,5,6-tetrakisphosphate (IP4), and the latter has been postulated to mediate the diarrhea caused by Salmonella. However, the effects of SigD on epithelial cells were not mimicked by microinjection of IP4. In contrast, the cytoskeletal and ion transport effects were replicated by hydrolyzing PtdIns(4,5)P2 with a membrane-targeted 5-phosphatase or by occluding the inositide using high-avidity tandem PH domain constructs. We therefore suggest that opening of the tight junctions and inhibition of Na+/H+ exchange caused by PtdIns(4,5)P2 hydrolysis combine to account, at least in part, for the fluid loss observed during Salmonella-induced diarrhea.

scholarly journals Signal-dependent membrane targeting by pleckstrin homology (PH) domains

2000 ◽  
Vol 350 (1) ◽  
pp. 1-18 ◽  
Author(s):  
Mark A. LEMMON ◽  
Kathryn M. FERGUSON
Keyword(s):  
Fluorescent Protein ◽  
Cell Signalling ◽  
Physiological Role ◽  
Structural Basis ◽  
Membrane Targeting ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Oligomeric State ◽  
Ph Domains ◽  
Green Fluorescent

Pleckstrin homology (PH) domains are small protein modules of around 120 amino acids found in many proteins involved in cell signalling, cytoskeletal rearrangement and other processes. Although several different protein ligands have been proposed for PH domains, their only clearly demonstrated physiological function to date is to bind membrane phosphoinositides. The PH domain from phospholipase C-δ1 binds specifically to PtdIns(4,5)P2 and its headgroup, and has become a valuable tool for studying cellular PtdIns(4,5)P2 functions. More recent developments have demonstrated that a subset of PH domains recognizes the products of agonist-stimulated phosphoinositide 3-kinases. Fusion of these PH domains to green fluorescent protein has allowed dramatic demonstrations of their independent ability to drive signal-dependent recruitment of their host proteins to the plasma membrane. We discuss the structural basis for this 3-phosphoinoistide recognition and the role that it plays in cellular signalling. PH domains that bind specifically to phosphoinositides comprise only a minority (perhaps 15%) of those known, raising questions as to the physiological role of the remaining 85% of PH domains. Most (if not all) PH domains bind weakly and non-specifically to phosphoinositides. Studies of dynamin-1 have indicated that oligomerization of its PH domain may be important in driving membrane association. We discuss the possibility that membrane targeting by PH domains with low affinity for phosphoinositides could be driven by alteration of their oligomeric state and thus the avidity of their membrane binding.

scholarly journals The Dbs PH domain contributes independently to membrane targeting and regulation of guanine nucleotide-exchange activity

2006 ◽  
Vol 400 (3) ◽  
pp. 563-572 ◽  
Author(s):  
Mark A. Baumeister ◽  
Kent L. Rossman ◽  
John Sondek ◽  
Mark A. Lemmon
Keyword(s):  
Rho Gtpases ◽  
Regulatory Role ◽  
Pleckstrin Homology Domain ◽  
Membrane Targeting ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Ph Domain ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Dh Domain

Dbl family GEFs (guanine nucleotide-exchange factors) for the Rho GTPases almost invariably contain a PH (pleckstrin homology) domain adjacent to their DH (Dbl homology) domain. The DH domain is responsible for GEF activity, and the PH domain plays a regulatory role that remains poorly understood. We demonstrated previously that Dbl family PH domains bind phosphoinositides with low affinity and cannot function as independent membrane targeting modules. In the present study, we show that dimerization of a Dbs (Dbl's big sister) DH/PH domain fragment is sufficient to drive it to the plasma membrane through a mechanism involving PH domain–phosphoinositide interactions. Thus, the Dbs PH domain could play a significant role in membrane targeting if it co-operates with other domains in the protein. We also show that mutations that prevent phosphoinositide binding by the Dbs PH domain significantly impair cellular GEF activity even in chimaeric proteins that are robustly membrane targeted by farnesylation or by the PH domain of phospholipase C-δ1. This finding argues that the Dbs PH domain plays a regulatory role that is independent of its ability to aid membrane targeting. Thus, we suggest that the PH domain plays dual roles, contributing independently to membrane localization of Dbs (as part of a multi-domain interaction) and allosteric regulation of the DH domain.

scholarly journals Neuronal calcium sensor-1 binds to regulated secretory organelles and functions in basal and stimulated exocytosis in PC12 cells

2002 ◽  
Vol 115 (11) ◽  
pp. 2399-2412 ◽  
Author(s):  
Bethe A. Scalettar ◽  
Patrizia Rosa ◽  
Elena Taverna ◽  
Maura Francolini ◽  
Takashi Tsuboi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Pc12 Cells ◽  
Fluorescent Protein ◽  
Secretory Granules ◽  
Yellow Fluorescent Protein ◽  
Growth Cones ◽  
Neuroendocrine Cells ◽  
Beta Activity ◽  
Calcium Sensor ◽  
Neuronal Calcium Sensor

Neuronal calcium sensor-1 (NCS-1) and its non-mammalian homologue,frequenin, have been implicated in a spectrum of cellular processes, including regulation of stimulated exocytosis of synaptic vesicles and secretory granules (SGs) in neurons and neuroendocrine cells and regulation of phosphatidylinositol 4-kinase beta activity in yeast. However, apart from these intriguing putative functions, NCS-1 and frequenin are relatively poorly understood. Here, the distribution, dynamics and function of NCS-1 were studied using PC12 cells that stably express NCS-1-EYFP (NCS-1 fused to enhanced yellow fluorescent protein) or that stably overexpress NCS-1. Fluorescence and electron microscopies show that NCS-1-EYFP is absent from SGs but is present on small clear organelles, some of which are just below the plasma membrane. Total internal reflection fluorescence microscopy shows that NCS-1-EYFP is associated with synaptic-like microvesicles (SLMVs) in growth cones. Overexpression studies show that NCS-1 enhances exocytosis of synaptotagmin-labeled regulated secretory organelles (RSOs) under basal conditions and during stimulation by UTP. Significantly, these studies implicate NCS-1 in the enhancement of both basal and stimulated phosphoinositide-dependent exocytosis of RSOs in PC12 cells, and they show that NCS-1 is distributed strategically to interact with putative targets on the plasma membrane and on SLMVs. These studies also reveal that SLMVs undergo both fast directed motion and highly hindered diffusive motion in growth cones, suggesting that cytoskeletal constituents can both facilitate and hinder SLMV motion. These results also reveal interesting similarities and differences between transport organelles in differentiated neuroendocrine cells and neurons.

scholarly journals FAPP2 is involved in the transport of apical cargo in polarized MDCK cells

2005 ◽  
Vol 170 (4) ◽  
pp. 521-526 ◽  
Author(s):  
Otilia V. Vieira ◽  
Paul Verkade ◽  
Aki Manninen ◽  
Kai Simons
Keyword(s):  
Fluorescent Protein ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Adaptor Protein ◽  
Pleckstrin Homology Domain ◽  
Transport Pathways ◽  
Protein Encoding ◽  
Phosphatidylinositol 4 ◽  
Golgi Network ◽  
Darby Canine Kidney

Phosphatidylinositol-4-phosphate (PI(4)P) is the main phosphoinositide in the Golgi complex and has been reported to play a pleiotropic role in transport of cargo from the trans-Golgi network to the plasma membrane (PM) in polarized Madin–Darby canine kidney (MDCK) cells. Overexpression of the chimeric fluorescent protein encoding the pleckstrin homology domain, which is specific for PI(4)P, inhibited both apical and basolateral transport pathways. The transport of apical cargo from the Golgi was shown to be specifically decreased by adenovirus-mediated RNA interference directed against PI(4)P adaptor protein (FAPP) 2. FAPP1 depletion had no effect on transport. On the other hand, FAPP2 was not involved in the Golgi-to-PM transport of cargo that was targeted to the basolateral membrane domain. Thus, we conclude that FAPP2 plays a specific role in apical transport in MDCK cells.

scholarly journals Binding of a Pleckstrin Homology Domain Protein to Phosphoinositide in Membranes: A Miniaturized FRET-Based Assay for Drug Screening

2002 ◽  
Vol 7 (1) ◽  
pp. 45-55 ◽  
Author(s):  
Brian D. Hamman ◽  
Brian A. Pollok ◽  
Todd Bennett ◽  
Janet Allen ◽  
Roger Heim
Keyword(s):  
Fluorescent Protein ◽  
Cell Transformation ◽  
Membrane Surface ◽  
Resonance Energy ◽  
Cell Receptor ◽  
Biochemical Assay ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Pleckstrin Homology

Pleckstrin homology (PH) domains are present in key proteins involved in many vital cell processes. For example, the PH domain of Bruton’s tyrosine kinase (Btk) binds to phosphatidylinositol triphosphate (PIP3) in the plasma membrane after stimulation of the B-cell receptor in B cells. Mutations in the Btk PH domain result in changes in its affinity for PIP3, with higher binding leading to cell transformation in vitro and lower binding leading to antibody deficiencies in both humans and mice. We describe here a fluorescence resonance energy transfer (FRET)-based biochemical assay that directly monitors the interaction of a PH domain with PIP3 at a membrane surface. We overexpressed a fusion protein consisting of an enhanced green fluorescent protein (GFP) and the N-terminal 170 amino acids of a Tec family kinase that contains its PH domain (PH170). Homogeneous unilamellar vesicles were made that contained PIP3 and octadecylrhodamine (OR), a lipophilic FRET acceptor for GFP. After optimization of both protein and vesicle components, we found that binding of the GFP-PH170 protein to PIP3 in vesicles that contain OR results in about a 90% reduction of GFP fluorescence. Using this assay to screen 1440 compounds, we identified three that efficiently inhibited binding of GFP-PH170 to PIP3 in vesicles. This biochemical assay readily miniaturized to 1.8-μl reaction volumes and was validated in a 3456-well screening format.

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