Putative Pleckstrin Homology Domain Research Articles

Role of IGSF3 in Lung Epithelial Barrier Disruption Induced by Cigarette Smoke Exposure

RationaleCigarette smoke (CS) exposure disrupts lung epithelial and endothelial cell barrier function which is linked to lung injury and inflammation. We identified a tetraspanin‐interacting protein, IGSF3, with roles in cell proliferation, adhesion, and migration via unclear mechanisms. We have shown that lung IGSF3 is decreased by cigarette smoke (CS) exposure and that loss of IGSF3 can disrupt sphingolipid metabolites. Of these, sphingosine‐1 phosphate (S1P) protects endothelial barrier function but may disrupt epithelial barrier via distinct receptors. We investigated if IGSF3 and S1P are required for maintaining the lung epithelial barrier in response to CS.MethodsHuman bronchial lung epithelial cells (Beas2b) were stably transfected with lentivirus control or IGSF3 shRNA (shIGSF3). Trans‐epithelial resistance (TER) was measured using electric cell‐substrate impedance sensing. CS extract was prepared using research cigarettes (3R4F, University of KY). Phosphatidylinositol lipid strips (Echelon) were used to study IGSF3‐lipid binding.ResultsCompared to control shRNA, knockdown of IGSF3 did not alter epithelial barrier function as measured by TER. Exposure to CS (7.5%), as expected, decreased epithelial cell monolayers TER in control cells by 35%. CS‐induced effects on TER were attenuated by treatment with N‐acetylcysteine or with (R)‐FTY720‐phosphonate (1R), a sphingosine‐1 phosphate receptor 1 (S1PR1) agonist, and were worsened by sphingosine kinase‐1 inhibition using SKI2. The CS‐induced decrease in TER was significantly worsened (by 55%, p<0.01 vs. control cells exposed to CS) in shIGSF3 ‐ transfected cells which responded in turn to the barrier‐enhancing effect of N‐acetylcysteine treatment (by 30%, p<0.05). A putative Pleckstrin‐homology (PH) domain was identified in the C‐terminal cytoplasmic domain of IGSF3 which predicted binding to PI(3,4)P2, which was then experimentally confirmed. Further, site directed mutagenesis within PH domain disrupted binding of IGSF3 with tetraspanin CD81.ConclusionsIGSF3 is required for partial maintenance of the lung epithelial barrier function in response to CS injury. Loss of IGSF3 may worsen CS injury of the airways, contributing to inflammation, an effect that could be alleviated by antioxidants and S1P1‐mediated signaling. The mechanisms of IGSF3 barrier effects may include binding to and tethering PI(3,4)P2 and CD81 at the cell membrane via its PH domain.Support or Funding InformationFunding: Wollowick Chair of Pulmonary Research (IP), Department of Medicine Research Microgrant (KSS) from National Jewish Health.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Coupling of septins to the axial landmark by Bud4 in budding yeast

Cells of the budding yeast Saccharomyces cerevisiae select a site for polarized growth in a specific pattern that depends on their cell type. Haploid a and α cells bud in the axial budding pattern, which requires assembly of a landmark that includes the Bud4 protein. To understand how an axial bud site is established, we performed a structure-function analysis of Bud4. Bud4 contains DUF1709 (domain of unknown function), which is similar to a part of the anillin-homology domain, and a putative Pleckstrin homology (PH) domain near to its C terminus. Although its localization depends on septins, a conserved family of GTP-binding proteins, Bud4 is necessary for the stable inheritance of septin rings during cell division. Although some anillins interact directly with septins, we find that neither DUF1709 nor the PH domain is necessary for targeting Bud4 to the mother-bud neck. Instead, this C-terminal region is crucial for association of Bud4 with Bud3 and other components of the axial landmark. Remarkably, septins colocalize with Bud4 mutant proteins that lack these C-terminal domains, forming an arc or a single ring instead of a double ring during and after cytokinesis. Interestingly, overexpression of Bud4 also induces formation of extra Bud4 rings and arcs that are associated with septins. Analyses of a series of bud4 truncation mutants suggest that at least two domains in the central region play a redundant role in targeting Bud4 to the mother-bud neck and are thus likely to interact with septins. Taken together, these results indicate that Bud4 functions as a platform that links septins to the axial landmark.

Asymmetric Motility Powered by Myo1c on Lipid Membranes

Class-I myosins are single-headed motors that link cell membranes to the underlying actin cytoskeleton. Actin binding occurs via the motor domain, while membrane binding has been proposed to occur via the tail domain that, in some isoforms, has been shown to interact with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) through a putative pleckstrin homology domain. To study the interplay between myosin-I, actin, and membranes, we reconstituted actin gliding motility on a membrane using full-length vertebrate myo1c bound to fluid (D ∼ 1 μm2/s) supported lipid bilayers (SLBs) composed of 2% PtdIns(4,5)P2, 97.9% DOPC, and 0.1% TRITC-PE. In this system, myo1c dynamically attaches and detaches to/from the lipid bilayer and actin filaments. The actin-gliding velocity on SLBs at 22 °C (∼ 20 nm/s) is comparable to that of myo1c rigidly anchored to a non-fluid surface via a tail-binding monoclonal antibody. Strikingly, the gliding of actin filaments on SLBs occurs along curved paths in a counterclockwise fashion (i.e., the actin filaments turn left) when viewed from the side of the membrane. This striking asymmetric motility was not observed when the myo1c was rigidly anchored to the surface via the antibody. The tail domain was not required for filament turning, as asymmetric motility was observed with a motor domain construct (no tail) attached to fluid biotinylated SLBs via a biotin-streptavidin linkage. A slight leftward bias was also observed when full-length myo1c dynamically interacts to Ins(1,4,5)P3 that is attached to the coverslip by a flexible 3-carbon linker. We conclude that class-I myosins can produce asymmetric motility on surfaces to which they can dynamically reorient. This asymmetric motility appears to be a fundamental property of the motor domain.

Myo1e Binds Anionic Phospholipids with High Affinity

Myo1e is a single-headed motor protein that has been shown to play roles in clathrin-mediated endocytosis in HeLa cells and podocyte function in the kidney. The myo1e C-terminal tail domain includes a basic region that is required for localization to clathrin-coated vesicles and contains a putative pleckstrin-homology (PH) domain that has been shown to play a role in phospholipid binding in other myosin-I proteins. We used sedimentation assays, stopped-flow fluorescence, and fluorescence microscopy to determine the membrane binding affinities, kinetics, and in vivo localization of fluorescently labeled recombinant myo1e-tail constructs. We found that the myo1e tail binds tightly to large unilamellar vesicles (LUVs) containing physiological concentrations of the anionic phospholipids phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) or phosphatidylserine. The rate of myo1e attachment to LUVs nears the diffusion limit while the calculated rate of detachment from LUVs is slow (k(diss) ≤ 0.4 s(-1)). Mutation of conserved residues in the myo1e PH domain has little effect on lipid binding in vitro or membrane localization in vivo. Soluble inositol phosphate headgroups, such as inositol 1,4,5-trisphosphate, can compete with PtdIns(4,5)P(2) for binding, but the apparent affinity for the soluble inositol phosphate is substantially lower than that for PtdIns(4,5)P(2). These results suggest that myo1e binds lipids through nonspecific electrostatic interactions rather than a stereospecific protein-phosphoinositide interaction.

Localization of Myosin 1b to Actin Protrusions Requires Phosphoinositide Binding

Myosin 1b (Myo1b), a class I myosin, is a widely expressed, single-headed, actin-associated molecular motor. Transient kinetic and single-molecule studies indicate that it is kinetically slow and responds to tension. Localization and subcellular fractionation studies indicate that Myo1b associates with the plasma membrane and certain subcellular organelles such as endosomes and lysosomes. Whether Myo1b directly associates with membranes is unknown. We demonstrate here that full-length rat Myo1b binds specifically and with high affinity to phosphatidylinositol 4,5-bisphosphate (PIP(2)) and phosphatidylinositol 3,4,5-triphosphate (PIP(3)), two phosphoinositides that play important roles in cell signaling. Binding is not Ca(2+)-dependent and does not involve the calmodulin-binding IQ region in the neck domain of Myo1b. Furthermore, the binding site is contained entirely within the C-terminal tail region, which contains a putative pleckstrin homology domain. Single mutations in the putative pleckstrin homology domain abolish binding of the tail domain of Myo1b to PIP(2) and PIP(3) in vitro. These same mutations alter the distribution of Myc-tagged Myo1b at membrane protrusions in HeLa cells where PIP(2) localizes. In addition, we found that motor activity is required for Myo1b localization in filopodia. These results suggest that binding of Myo1b to phosphoinositides plays an important role in vivo by regulating localization to actin-enriched membrane projections.

Kinetics of the Interaction of myo1c with Phosphoinositides

myo1c is a single-headed myosin that dynamically links membranes to the actin cytoskeleton. A putative pleckstrin homology domain has been identified in the myo1c tail that binds phosphoinositides and soluble inositol phosphates with high affinity. However, the kinetics of association and dissociation and the influence of phospholipid composition on the kinetics have not been determined. Stopped-flow spectroscopy was used to measure the binding and dissociation of a recombinant myo1c construct containing the tail and regulatory domains (myo1c(IQ-tail)) to and from 100-nm diameter large unilamellar vesicles (LUVs). We found the time course of association of myo1c(IQ-tail) with LUVs containing 2% phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) followed a two-exponential time course, and the rate of the predominant fast phase depended linearly upon the total lipid concentration. The apparent second-order rate constant was approximately diffusion-limited. Increasing the molar ratio of anionic phospholipid by adding phosphatidylserine, additional PtdIns(4,5)P(2), or by situating PtdIns(4,5)P(2) in a more physiologically relevant lipid background increased the apparent association rate constant less than 2-fold. myo1c(IQ-tail) dissociated from PtdIns(4,5)P(2) at a slower rate (2.0 s(-1)) than the pleckstrin homology domain of phospholipase C-delta (13 s(-1)). The presence of additional anionic phospholipid reduced the myo1c(IQ-tail) dissociation rate constant >50-fold but marginally changed the dissociation rate of phospholipase C-delta, suggesting that additional electrostatic interactions in myo1c(IQ-tail) help to stabilize binding. Remarkably, high concentrations of soluble inositol phosphates induce dissociation of myo1c(IQ-tail) from LUVs, suggesting that phosphoinositides are able to bind to and dissociate from myo1c(IQ-tail) as it remains bound to the membrane.

Kinetics Of Myo1c Association To And Dissociation From Phosphoinositide-containing Vesicles

Myo1c is a single-headed unconventional myosin that associates with negatively-charged lipids through electrostatic interactions. A putative pleckstrin homology (PH) domain has been identified in the myo1c tail that binds PI(4,5)P2 with high affinity. However, the kinetics of association and dissociation, as well as the influence of membrane phospholipid composition and Mg2+ on the kinetics, remain unknown. Stopped-flow measurements were made using the increase in light scattering that occurred upon myo1c-tail binding to 100 nm diameter large unilamellar vesicles (LUVs).We found that the association of myo1c-tail with phosphatidylcholine (PC) LUVs containing 2% PI(4,5)P2 followed a 2-exponential time-course. The rate of the predominant, fast phase depended linearly upon the total lipid concentration. The apparent second order rate constant in the absence of Mg2+ was approximately diffusion-limited, indicating that no conformational change occurs upon binding. The molar ratio of anionic lipid was increased by adding phosphatidylserine (PS) or additional PI(4,5)P2 to LUVs or by situating PI(4,5)P2 in a more physiologically relevant lipid background (phosphatidylethanolamine, PC, PS, phosphatidylinositol, and sphingomyelin). None of these conditions increased the apparent association rate constant much more than two-fold. Dissociation of myo1c-tail was measured by chasing with excess inositol 1,4,5-trisphosphate (InsP3). The presence of additional anionic phospholipid reduced the observed dissociation rate constant by orders of magnitude (3.2 s-1 vs. 0.03 s-1). This suggests that once myo1c-tail interacts via its putative PH domain with PI(4,5)P2, additional electrostatic interactions between positively-charged regions of the tail and negatively-charged lipids help to stabilize binding. The presence of Mg2+, known to interact with polyvalent anions, did not alter these trends, though it did increase the dissociation rate for all lipid compositions. Finally, we measured the dissociation rate of myo1c-tail from InsP3 and found that it approximates the dissociation rate from PI(4,5)P2.

Acanthamoeba Myosin IC Colocalizes with Phosphatidylinositol 4,5-Bisphosphate at the Plasma Membrane Due to the High Concentration of Negative Charge

The tail of Acanthamoeba myosin IC (AMIC) has a basic region (BR), which contains a putative pleckstrin homology (PH) domain, followed by two Gly/Pro/Ala (GPA)-rich regions separated by a Src homology 3 (SH3) domain. Cryoelectron microscopy had shown that the tail is folded back on itself at the junction of BR and GPA1, and nuclear magnetic resonance spectroscopy indicated that the SH3 domain may interact with the putative PH domain. The BR binds to acidic phospholipids, and the GPA region binds to F-actin. We now show that the folded tail does not affect the affinity of AMIC for acidic phospholipids. AMIC binds phosphatidylinositol 4,5-bisphosphate (PIP2) with high affinity (approximately 1 microm), but binding is not stereospecific. When normalized to net negative charge, AMIC binds with equal affinity to phosphatidylserine (PS) and PIP2. This and other data show that the putative PH domain of AMIC is not a typical PIP2-specific PH domain. We have identified a 13-residue sequence of basic-hydrophobic-basic amino acids within the putative PH domain that may be a major determinant of binding of AMIC to acidic phospholipids. Despite the lack of stereospecificity, AMIC binds 10 times more strongly to vesicles containing 5% PIP2 plus 25% PS than to vesicles containing only 25% PS, suggesting that AMIC may be targeted to PIP2-enriched regions of the plasma membrane. In agreement with this, AMIC colocalizes with PIP2 at dynamic, protrusive regions of the plasma membrane. We discuss the possibility that AMIC binding to PIP2 may initiate the formation of a multiprotein complex at the plasma membrane.

Intramolecular interaction in the tail of Acanthamoeba myosin IC between the SH3 domain and a putative pleckstrin homology domain

The 466-aa tail of the heavy chain of Acanthamoeba myosin IC (AMIC) comprises an N-terminal 220-residue basic region (BR) followed by a 56-residue Gly/Pro/Ala-rich region (GPA1), a 55-residue Src homology 3 (SH3) domain, and a C-terminal 135-residue Gly/Pro/Ala-rich region (GPA2). Cryo-electron microscopy of AMIC had shown previously that the AMIC tail is folded back on itself, suggesting the possibility of interactions between its N- and C-terminal regions. We now show specific differences between the NMR spectrum of bacterially expressed full-length tail and the sum of the spectra of individually expressed BR and GPA1-SH3-GPA2 (GSG) regions. These results are indicative of interactions between the two subdomains in the full-length tail. From the NMR data, we could assign many of the residues in BR and GSG that are involved in these interactions. By combining homology modeling with the NMR data, we identify a putative pleckstrin homology (PH) domain within BR, and show that the PH domain interacts with the SH3 domain.

Myo1c binds phosphoinositides through a putative pleckstrin homology domain.

Myo1c is a member of the myosin superfamily that binds phosphatidylinositol-4,5-bisphosphate (PIP(2)), 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 PIP(2) 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.

Regulation of plant defense responses in Arabidopsis by EDR2, a PH and START domain‐containing protein

We have identified an Arabidopsis mutant that displays enhanced disease resistance (edr2) to the biotrophic powdery mildew pathogen Erysiphe cichoracearum. Inhibition of fungal growth on edr2 mutant leaves occurred at a late stage of the infection process and coincided with formation of necrotic lesions approximately 5 days after inoculation. Double-mutant analysis revealed that edr2-mediated resistance is suppressed by mutations that inhibit salicylic acid (SA)-induced defense signaling, including npr1, pad4 and sid2, demonstrating that edr2-mediated disease resistance is dependent on SA. However, edr2 showed normal responses to the bacterial pathogen Pseudomonas syringae pv. tomato strain DC3000. EDR2 appears to be constitutively transcribed in all tissues and organs and encodes a novel protein, consisting of a putative pleckstrin homology (PH) domain and a steroidogenic acute regulatory protein-related lipid-transfer (START) domain, and contains an N-terminal mitochondrial targeting sequence. The PH and START domains are implicated in lipid binding, suggesting that EDR2 may provide a link between lipid signaling and activation of programmed cell death mediated by mitochondria.

Point Mutations in the Split PLC-γ1 PH Domain Modulate Phosphoinositide Binding

A number of signaling molecules contain small pleckstrin homology (PH) domains capable of binding phosphoinositides or proteins. Phospholipase C (PLC)-gamma1 has two putative PH domains, an NH(2)-terminal (PH(1)) and a split PH domain (nPH(2) and cPH(2)). We previously reported that the split PH domain of PLC-gamma1 binds to phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) (Chang et al., 2002). To identify the amino acid residues responsible for binding with PI(4)P and PI(4,5)P(2), we used site-directed mutagenesis to replace each amino acid in the variable loop-1 (VL-1) region of the PLC-gamma1 nPH(2) domain with alanine (a neutral amino acid). The phosphoinositide-binding affinity of these mutant molecules was analyzed by Dot-blot assay followed by ECL detection. We found that two PLC-gamma1 nPH2 domain mutants, P500A and H503A, showed reduced affinities for phosphoinositide binding. Furthermore, these mutant PLC-gamma1 molecules showed reduced PI(4,5)P(2) hydrolysis. Using green fluorescent protein (GFP) fusion protein system, we showed that both PH(1) and nPH(2) domains are responsible for membrane-targeted translocation of PLC-gamma1 upon serum stimulation. Together, our data reveal that the amino acid residues Pro(500) and His(503) are critical for binding of PLC-gamma1 to one of its substrates, PI(4,5)P(2) in the membrane.

Phosphatidylinositol-5-Phosphate Activation and Conserved Substrate Specificity of the Myotubularin Phosphatidylinositol 3-Phosphatases

Phosphoinositides control many different processes required for normal cellular function [1, 2]. Myotubularins are a family of Phosphatidylinositol 3-phosphate (PtdIns3P) phosphatases identified by the positional cloning of the MTM1 gene in patients suffering from X-linked myotubular myopathy and the MTMR2 gene in patients suffering from the demyelinating neuropathy Charcot-Marie-Tooth disease type 4B [3–9]. MTM1 is a phosphatidylinositol phosphatase with reported specificity toward PtdIns3P [6, 7], while the related proteins MTMR2 and MTMR3 hydrolyze both PtdIns3P and PtdIns(3,5)P2 [10, 11]. We have investigated MTM1 and MTMR6 and find that they use PtdIns(3,5)P2 in addition to PtdIns3P as a substrate in vitro. The product of PtdIns(3,5)P2 hydrolysis, PtdIns5P, causes MTM1 to form a heptameric ring that is 12.5 nm in diameter, and it is a specific allosteric activator of MTM1, MTMR3, and MTMR6. A disease-causing mutation at arginine 69 of MTM1 falling within a putative pleckstrin homology domain reduces the ability of the enzyme to respond to PtdIns5P. We propose that the myotubularin family of enzymes utilize both PtdIns3P and PtdIns(3,5)P2 as substrates, and that PtdIns5P functions in a positive feedback loop controlling their activity. These findings highlight the importance of regulated phosphatase activity for the control of phosphoinositide metabolism.

Specificity and Structural Requirements of Phospholipase C-β Stimulation by Rho GTPases Versus G Protein βγ Dimers

Phospholipase C-beta(2) (PLC beta(2)) is activated both by heterotrimeric G protein alpha- and beta gamma- subunits and by Rho GTPases. In this study, activated Rho GTPases are shown to stimulate PLC beta isozymes with the rank order of PLC beta(2) > PLC beta(3) > or = PLC beta(1). The sensitivity of PLC beta isozymes to Rho GTPases was clearly different from that observed for G protein beta gamma dimers, which decreased in the following order: PLC beta(3) > PLC beta(2) > PLC beta(1) for beta(1)gamma(1/2) and PLC beta(2) > PLC beta(1) >>> PLC beta(3) for beta(5)gamma(2). Rac1 and Rac2 were found to be more potent and efficacious activators of PLC beta(2) than was Cdc42Hs. The stimulation of PLC beta(2) by Rho GTPases and G protein beta gamma dimers was additive, suggesting that PLC beta(2) activation can be augmented by independent regulation of the enzyme by the two stimuli. Using chimeric PLC beta(1)-PLC beta(2) enzymes, beta gamma dimers, and Rho GTPases are shown to require different regions of PLC beta(2) to mediate efficient stimulation of the enzyme. Although the catalytic subdomains X and Y of PLC beta(2) were sufficient for efficient stimulation by beta gamma, the presence of the putative pleckstrin homology domain of PLC beta(2) was absolutely required for the stimulation of the enzyme by Rho GTPases. Taken together, these results identify Rho GTPases as novel PLC beta regulators, which mediate PLC beta isozyme-specific stimulation and are potentially involved in coordinating the activation of PLC beta(2) by extracellular mediators in intact cells.

The Intermolecular Interaction between the PH Domain and the C-terminal Domain of Arabidopsis Dynamin-like 6 Determines Lipid Binding Specificity

Dynamin and its related proteins are a group of mechanochemical proteins involved in the modulation of lipid membranes in various biological processes. Here we investigate the nature of membrane binding of the Arabidopsis dynamin-like 6 (ADL6) involved in vesicle trafficking from the trans-Golgi network to the central vacuole. Fractionation experiments by continuous sucrose gradients and gel filtration revealed that the majority of ADL6 is associated with membranes in vivo. Amino acid sequence analysis revealed that ADL6 has a putative pleckstrin homology (PH) domain. In vitro lipid binding assays demonstrated that ADL6 showed high affinity binding to phosphatidylinositol 3-phosphate (PtdIns-3-P) and that the PH domain was responsible for this interaction. However, the PH domain alone binds equally well to both PtdIns-3-P and phosphatidylinositol 4-phosphate (PtdIns-4-P). Interestingly, the high affinity binding of the PH domain to PtdIns-3-P was restored by a protein-protein interaction between the PH domain and the C-terminal region. In addition, deletion of the inserted regions within the PH domain results in high affinity binding of the PH domain to PtdIns-3-P. These results suggest that ADL6 binds specifically to PtdIns-3-P and that the lipid binding specificity is determined by the interaction between the PH domain and the C-terminal domain of ADL6.

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

The pleckstrin homology (PH) domain is a small motif for membrane targeting in the signaling molecules. Phospholipase C (PLC)-gamma1 has two putative PH domains, an NH(2)-terminal and a split PH domain. Here we report studies on the interaction of the PH domain of PLC-gamma1 with translational elongation factor (EF)-1alpha, which has been shown to be a phosphatidylinositol 4-kinase activator. By pull-down of cell extract with the glutathione S-transferase (GST) fusion proteins with various domains of PLC-gamma1 followed by peptide sequence analysis, we identified EF-1alpha as a binding partner of a split PH domain of PLC-gamma1. Analysis by site-directed mutagenesis of the PH domain revealed that the beta2-sheet of a split PH domain is critical for the interaction with EF-1alpha. Moreover, Dot-blot assay shows that a split PH domain specifically binds to phosphoinositides including phosphatidylinositol 4-phosphate and phosphatidylinositol 4, 5-bisphosphate (PIP(2)). So the PH domain of PLC-gamma1 binds to both EF-1alpha and PIP(2). The binding affinity of EF-1alpha to the GST.PH domain fusion protein increased in the presence of PIP(2), although PIP(2) does not bind to EF-1alpha directly. This suggests that EF-1alpha may control the binding affinity between the PH domain and PIP(2). PLC-gamma1 is substantially activated in the presence of EF-1alpha with a bell-shaped curve in relation to the molar ratio between them, whereas a double point mutant PLC-gamma1 (Y509A/F510A) that lost its binding affinity to EF-1alpha shows basal level activity. Taken together, our data show that EF-1alpha plays a direct role in phosphoinositide metabolism of cellular signaling by regulating PLC-gamma1 activity via a split PH domain.

Influence of phosphatidylinositol 4,5-bisphosphate on human phospholipase D1 wild-type and deletion mutants: is there evidence for an interaction of phosphatidylinositol 4,5-bisphosphate with the putative Pleckstrin homology domain?

Phosphatidylinositol 4,5-bisphosphate (PIP 2) is an essential cofactor of phospholipase D (PLD) enzymes. In order to further characterize its role in PLD activation, we have constructed N-terminal deletion mutants of the human PLD1 (hPLD1) and a mutant lacking the putative pleckstrin homology domain (ΔPH), which has been proposed to be involved in PIP 2 binding. For the N-terminal deletion mutants (up to 303 amino acids) and the ΔPH mutant we found no significant differences compared to the hPLD1 wild-type, except changes in the specific activities: the K m values were about 20 μM for the substrate phosphatidylcholine, and PIP 2 activated the PLD enzymes maximally between 5 and 10 μM. In contrast, preincubation of the PLD proteins with 5–10 μM PIP 2 or PIP 2-containing lipid vesicles inhibited the PLD activity. This inhibition was neither abolished by n-octyl-β- D-glucopyranoside or neomycin nor by the ADP-ribosylation factor, another activator of PLD enzymes. All tested PLD proteins were active without PIP 2 in the presence of 1 M ammonium sulfate. The 303 N-terminal amino acids of hPLD1 are not involved in substrate binding or the interaction with PIP 2. Our data indicate further that the putative PH domain of hPLD1 is not responsible for the essential effects of PIP 2 on PLD activity.

Fatty Acylation of Phospholipase D1 on Cysteine Residues 240 and 241 Determines Localization on Intracellular Membranes

We have reported previously that phospholipase D1 (PLD1) is labeled specifically with [(3)H]palmitate following transient expression and immunoprecipitation and that this modification appeared important both for membrane localization and catalytic activity. In this work we identify by mutagenesis that the acylation sites on PLD1 are cysteine residues 240 and 241, with the cysteine at position 241 accounting for most but not all of the modification. Replacement of both cysteine residues with either serines or alanines resulted in a mutant protein that contained undetectable [(3)H]palmitate. In comparison with the wild type protein, the double mutant showed reduced catalytic activity in vivo, whereas its activity in vitro was unchanged. In addition, the localization of the double mutant was altered in comparison with the wild type protein, whereas wild type PLD1 is primarily on intracellular membranes and on punctate structures, the double mutant was on plasma membrane. Because cysteines 240 and 241 lie within a putative pleckstrin homology domain of PLD1, it is likely that fatty acylation on these residues modulates the function of the PLD1 pleckstrin homology domain.

Gβγ Stimulates Phosphoinositide 3-Kinase-γ by Direct Interaction with Two Domains of the Catalytic p110 Subunit

Class I phosphoinositide 3-kinases (PI3Ks) regulate important cellular processes such as mitogenesis, apoptosis, and cytoskeletal functions. They include PI3Kalpha, -beta, and -delta isoforms coupled to receptor tyrosine kinases and a PI3Kgamma isoform activated by receptor-stimulated G proteins. This study examines the direct interaction of purified recombinant PI3Kgamma catalytic subunit (p110gamma) and Gbetagamma complexes. When phosphatidylinositol was used as a substrate, Gbetagamma stimulated p110gamma lipid kinase activity more than 60-fold (EC50, approximately 20 nM). Stimulation was inhibited by Galphao-GDP or wortmannin in a concentration-dependent fashion. Stoichiometric binding of a monoclonal antibody to the putative pleckstrin homology domain of p110gamma did not affect Gbetagamma-mediated enzymatic stimulation, whereas incubation of Gbetagamma with a synthetic peptide resembling a predicted Gbetagamma effector domain of type 2 adenylyl cyclase selectively inhibited activation of p110gamma. Gbetagamma complexes bound to N- as well as C-terminal deletion mutants of p110gamma. Correspondingly, these enzymatically inactive N- and C-terminal mutants inhibited Gbetagamma-mediated activation of wild type p110gamma. Our data suggest that (i) p110gamma directly interacts with Gbetagamma, (ii) the pleckstrin homology domain is not the only region important for Gbetagamma-mediated activation of the lipid kinase, and (iii) Gbetagamma binds to at least two contact sites of p110gamma, one of which is close to or within the catalytic core of the enzyme.

Myotonic dystrophy kinase-related Cdc42-binding kinase acts as a Cdc42 effector in promoting cytoskeletal reorganization.

The Rho GTPases play distinctive roles in cytoskeletal reorganization associated with growth and differentiation. The Cdc42/Rac-binding p21-activated kinase (PAK) and Rho-binding kinase (ROK) act as morphological effectors for these GTPases. We have isolated two related novel brain kinases whose p21-binding domains resemble that of PAK whereas the kinase domains resemble that of myotonic dystrophy kinase-related ROK. These approximately 190-kDa myotonic dystrophy kinase-related Cdc42-binding kinases (MRCKs) preferentially phosphorylate nonmuscle myosin light chain at serine 19, which is known to be crucial for activating actin-myosin contractility. The p21-binding domain binds GTP-Cdc42 but not GDP-Cdc42. The multidomain structure includes a cysteine-rich motif resembling those of protein kinase C and n-chimaerin and a putative pleckstrin homology domain. MRCK alpha and Cdc42V12 colocalize, particularly at the cell periphery in transfected HeLa cells. Microinjection of plasmid encoding MRCK alpha resulted in actin and myosin reorganization. Expression of kinase-dead MRCK alpha blocked Cdc42V12-dependent formation of focal complexes and peripheral microspikes. This was not due to possible sequestration of the p21, as a kinase-dead MRCK alpha mutant defective in Cdc42 binding was an equally effective blocker. Coinjection of MRCK alpha plasmid with Cdc42 plasmid, at concentrations where Cdc42 plasmid by itself elicited no effect, led to the formation of the peripheral structures associated with a Cdc42-induced morphological phenotype. These Cdc42-type effects were not promoted upon coinjection with plasmids of kinase-dead or Cdc42-binding-deficient MRCK alpha mutants. These results suggest that MRCK alpha may act as a downstream effector of Cdc42 in cytoskeletal reorganization.