scholarly journals Membrane activity of the phospholipase C-δ1 pleckstrin homology (PH) domain

2005 ◽  
Vol 389 (2) ◽  
pp. 435-441 ◽  
Author(s):  
Frits M. Flesch ◽  
Jong W. Yu ◽  
Mark A. Lemmon ◽  
Koert N. J. Burger
Keyword(s):  
Binding Site ◽  
Phospholipase C ◽  
Membrane Surface ◽  
Pleckstrin Homology Domain ◽  
Membrane Insertion ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Lipid Mixture ◽  
Membrane Activity ◽  
Binding Domains

PH-PLCδ1 [the PH domain (pleckstrin homology domain) of PLCδ1 (phospholipase C-δ1)] is among the best-characterized phosphoinositide-binding domains. PH-PLCδ1 binds with high specificity to the headgroup of PtdIns(4,5)P2, but little is known about its interfacial properties. In the present study, we show that PH-PLCδ1 is also membrane-active and can insert significantly into PtdIns(4,5)P2-containing monolayers at physiological (bilayer-equivalent) surface pressures. However, this membrane activity appears to involve interactions distinct from those that target PH-PLCδ1 to the PtdIns(4,5)P2 headgroup. Whereas the majority of PtdIns(4,5)P2-bound PH-PLCδ1 can be displaced by adding excess of soluble headgroup [Ins(1,4,5)P3], membrane activity of PH-PLCδ1 cannot. PH-PLCδ1 differs from other phosphoinositide-binding domains in that its membrane insertion does not require that the phosphoinositide-binding site be occupied. Significant monolayer insertion remains when the phosphoinositide-binding site is mutated, and PH-PLCδ1 can insert into monolayers that contain no PtdIns(4,5)P2 at all. Our results suggest a model in which reversible membrane binding of PH-PLCδ1, mediated by PtdIns(4,5)P2 or other acidic phospholipids, occurs without membrane insertion. Accumulation of the PH domain at the membrane surface enhances the efficiency of insertion, but does not significantly affect its extent, whereas the presence of phosphatidylethanolamine and cholesterol in the lipid mixture promotes the extent of insertion. This is the first report of membrane activity in an isolated PH domain and has implications for understanding the membrane targeting by this common type of domain.

Molecular modelling and site-directed mutagenesis of the inositol 1,3,4,5-tetrakisphosphate-binding pleckstrin homology domain from the Ras GTPase-activating protein GAP1IP4BP

2000 ◽  
Vol 349 (1) ◽  
pp. 333-342 ◽  
Author(s):  
Gyles COZIER ◽  
Richard SESSIONS ◽  
Joanna R. BOTTOMLEY ◽  
Jon S. REYNOLDS ◽  
Peter J. CULLEN
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Inositol Phosphate ◽  
Site Directed Mutagenesis ◽  
Pleckstrin Homology Domain ◽  
Directed Mutagenesis ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Gtpase Activating Protein ◽  
Ras Gtpase

GAP1IP4BP is a Ras GTPase-activating protein (GAP) that in vitro is regulated by the cytosolic second messenger inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. We have studied Ins(1,3,4,5)P4 binding to GAP1IP4BP, and shown that the inositol phosphate specificity and binding affinity are similar to Ins(1,3,4,5)P4 binding to Bruton's tyrosine kinase (Btk), evidence which suggests a similar mechanism for Ins(1,3,4,5)P4 binding. The crystal structure of the Btk pleckstrin homology (PH) domain in complex with Ins(1,3,4,5)P4 has shown that the binding site is located in a partially buried pocket between the β1/β2- and β3/β4-loops. Many of the residues involved in the binding are conserved in GAP1IP4BP. Therefore we generated a model of the PH domain of GAP1IP4BP in complex with Ins(1,3,4,5)P4 based on the Btk-Ins(1,3,4,5)P4 complex crystal structure. This model had the typical PH domain fold, with the proposed binding site modelling well on the Btk structure. The model has been verified by site-directed mutagenesis of various residues in and around the proposed binding site. These mutations have markedly reduced affinity for Ins(1,3,4,5)P4, indicating a specific and tight fit for the substrate. The model can also be used to explain the specificity of inositol phosphate binding.

scholarly journals Substitution of the myristoylation signal of human immunodeficiency virus type 1 Pr55Gag with the phospholipase C-δ1 pleckstrin homology domain results in infectious pseudovirion production

2008 ◽  
Vol 89 (12) ◽  
pp. 3144-3149 ◽  
Author(s):  
Emiko Urano ◽  
Toru Aoki ◽  
Yuko Futahashi ◽  
Tsutomu Murakami ◽  
Yuko Morikawa ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Phospholipase C ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Viral Production ◽  
Immunodeficiency Virus

The matrix domain (MA) of human immunodeficiency virus type 1 Pr55Gag is covalently modified with a myristoyl group that mediates efficient viral production. However, the role of myristoylation, particularly in the viral entry process, remains uninvestigated. This study replaced the myristoylation signal of MA with a well-studied phosphatidylinositol 4,5-biphosphate-binding plasma membrane (PM) targeting motif, the phospholipase C-δ1 pleckstrin homology (PH) domain. PH–Gag–Pol PM targeting and viral production efficiencies were improved compared with Gag–Pol, consistent with the estimated increases in Gag–PM affinity. Both virions were recovered in similar sucrose density-gradient fractions and had similar mature virion morphologies. Importantly, PH–Gag–Pol and Gag–Pol pseudovirions had almost identical infectivity, suggesting a dispensable role for myristoylation in the virus life cycle. PH–Gag–Pol might be useful in separating the myristoylation-dependent processes from the myristoylation-independent processes. This the first report demonstrating infectious pseudovirion production without myristoylated Pr55Gag.

Phosphoinositide 3-kinase-dependent regulation of phospholipase Cγ

2007 ◽  
Vol 35 (2) ◽  
pp. 229-230 ◽  
Author(s):  
T. Maffucci ◽  
M. Falasca
Keyword(s):  
Phospholipase C ◽  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Second Messengers ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Functional Roles ◽  
Phosphoinositide 3 Kinase

Activation of the enzyme PLC (phospholipase C) leads to the formation of second messengers Ins(1,4,5)P3 and diacylglycerol. RTKs (receptor tyrosine kinases) activate this reaction through PLCγ isoenzymes. It has been shown that PI3K (phosphoinositide 3-kinase) may regulate PLCγ activity through the interaction of PI3K product PtdIns(3,4,5)P3 and the PLCγ PH domain (pleckstrin homology domain). Here, we analyse the potential functional roles of the PI3K/PLC pathway.

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 Binding of phosphatidylinositol 3,4,5-trisphosphate to the pleckstrin homology domain of protein kinase B induces a conformational change

2003 ◽  
Vol 375 (3) ◽  
pp. 531-538 ◽  
Author(s):  
Christine C. MILBURN ◽  
Maria DEAK ◽  
Sharon M. KELLY ◽  
Nick C. PRICE ◽  
Dario R. ALESSI ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Binding Site ◽  
Conformational Change ◽  
Conformational Changes ◽  
Protein Kinase B ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Variable Loop ◽  
Solution Studies

Protein kinase B (PKB/Akt) is a key regulator of cell growth, proliferation and metabolism. It possesses an N-terminal pleckstrin homology (PH) domain that interacts with equal affinity with the second messengers PtdIns(3,4,5)P3 and PtdIns(3,4)P2, generated through insulin and growth factor-mediated activation of phosphoinositide 3-kinase (PI3K). The binding of PKB to PtdIns(3,4,5)P3/PtdIns(3,4)P2 recruits PKB from the cytosol to the plasma membrane and is also thought to induce a conformational change that converts PKB into a substrate that can be activated by the phosphoinositide-dependent kinase 1 (PDK1). In this study we describe two high-resolution crystal structures of the PH domain of PKBα in a noncomplexed form and compare this to a new atomic resolution (0.98 Å, where 1 Å=0.1 nm) structure of the PH domain of PKBα complexed to Ins(1,3,4,5)P4, the head group of PtdIns(3,4,5)P3. Remarkably, in contrast to all other PH domains crystallized so far, our data suggest that binding of Ins(1,3,4,5)P4 to the PH domain of PKB, induces a large conformational change. This is characterized by marked changes in certain residues making up the phosphoinositide-binding site, formation of a short α-helix in variable loop 2, and a movement of variable loop 3 away from the lipid-binding site. Solution studies with CD also provided evidence of conformational changes taking place upon binding of Ins(1,3,4,5)P4 to the PH domain of PKB. Our data provides the first structural insight into the mechanism by which the interaction of PKB with PtdIns(3,4,5)P3/PtdIns(3,4)P2 induces conformational changes that could enable PKB to be activated by PDK1.

scholarly journals Phosphotyrosine protein of molecular mass 30 kDa binds specifically to the positively charged region of the pleckstrin N-terminal pleckstrin homology domain

1999 ◽  
Vol 342 (2) ◽  
pp. 423-430
Author(s):  
Limin LIU ◽  
Mary MAKOWSKE
Keyword(s):  
Binding Site ◽  
Inositol Phosphates ◽  
Site Directed Mutagenesis ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Glutathione S Transferase ◽  
Protein Protein Interaction ◽  
Ph Domains ◽  
Positively Charged

It has been proposed that phosphoinositides and inositol phosphates serve as general ligands for members of the structurally related pleckstrin homology (PH) domain family. The N-terminal PH domain of pleckstrin (N-PH), in contrast with other PH domains, does not bind to any of these ligands with the high affinity expected for a physiological interaction. To examine whether N-PH might instead mediate protein-protein interaction, a fusion protein with glutathione S-transferase (GST) expressing N-PH (GST-N-PH) was used to screen [35S]methionine metabolically labelled HL-60 and Bac1.2F5 cell lysates for potential binding partners. A 30 kDa binding protein was identified in both cell lines. Binding to N-PH demonstrated specificity, because binding was approx. 10-fold higher than when an equimolar amount of pleckstrin C-terminal PH domain (GST-C-PH) was used as probe. The 30 kDa protein could also be metabolically labelled with [32P]Pi and proved to be a tyrosine-phosphorylated protein. Binding to N-PH could be specifically inhibited with phosphotyrosine but not with phosphothreonine; the inhibition was concentration-dependent. Site-directed mutagenesis indicated that a positively charged region previously identified as the phosphoinositide-binding site in N-PH and other PH domains, rather than a putative phosphotyrosine-binding region previously identified in structurally similar phosphotyrosine-binding (PTB) domains, served as the binding site. These results suggest that the positively charged region of N-PH has the potential to interact with a protein ligand that contains phosphotyrosine.

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.

scholarly journals Membrane Insertion of the Pleckstrin Homology Domain Variable Loop 1 Is Critical for Dynamin-catalyzed Vesicle Scission

2009 ◽  
Vol 20 (22) ◽  
pp. 4630-4639 ◽  
Author(s):  
Rajesh Ramachandran ◽  
Thomas J. Pucadyil ◽  
Ya-Wen Liu ◽  
Sharmistha Acharya ◽  
Marilyn Leonard ◽  
...  
Keyword(s):  
Membrane Curvature ◽  
Pleckstrin Homology Domain ◽  
Membrane Insertion ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Coated Pits ◽  
Variable Loop ◽  
Membrane Fission

The GTPase dynamin catalyzes the scission of deeply invaginated clathrin-coated pits at the plasma membrane, but the mechanisms governing dynamin-mediated membrane fission remain poorly understood. Through mutagenesis, we have altered the hydrophobic nature of the membrane-inserting variable loop 1 (VL1) of the pleckstrin homology (PH) domain of dynamin-1 and demonstrate that its stable insertion into the lipid bilayer is critical for high membrane curvature generation and subsequent membrane fission. Dynamin PH domain mutants defective in curvature generation regain function when assayed on precurved membrane templates in vitro, but they remain defective in the scission of clathrin-coated pits in vivo. These results demonstrate that, in concert with dynamin self-assembly, PH domain membrane insertion is essential for fission and vesicle release in vitro and for clathrin-mediated endocytosis in vivo.

scholarly journals Alternate pleckstrin homology domain orientations regulate dynamin-catalyzed membrane fission

2014 ◽  
Vol 25 (6) ◽  
pp. 879-890 ◽  
Author(s):  
Niharika Mehrotra ◽  
Justin Nichols ◽  
Rajesh Ramachandran
Keyword(s):  
Self Assembly ◽  
Molecular Mechanisms ◽  
Membrane Surface ◽  
Resonance Energy ◽  
Membrane Curvature ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Long Distance ◽  
Membrane Fission

The self-assembling GTPase dynamin catalyzes endocytic vesicle scission via membrane insertion of its pleckstrin homology (PH) domain. However, the molecular mechanisms underlying PH domain–dependent membrane fission remain obscure. Membrane-curvature–sensing and membrane-curvature–generating properties have been attributed, but it remains to be seen whether the PH domain is involved in either process independent of dynamin self-assembly. Here, using multiple fluorescence spectroscopic and microscopic techniques, we demonstrate that the isolated PH domain does not act to bend membranes but instead senses high membrane curvature through hydrophobic insertion into the membrane bilayer. Furthermore, we use a complementary set of short- and long-distance Förster resonance energy transfer approaches to distinguish PH-domain orientation from proximity at the membrane surface in full-length dynamin. We reveal, in addition to the GTP-sensitive “hydrophobic mode,” the presence of an alternate, GTP-insensitive “electrostatic mode” of PH domain–membrane interactions that retains dynamin on the membrane surface during the GTP hydrolysis cycle. Stabilization of this alternate orientation produces dramatic variations in the morphology of membrane-bound dynamin spirals, indicating that the PH domain regulates membrane fission through the control of dynamin polymer dynamics.

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