Quantitative analysis of the binding of ezrin to large unilamellar vesicles containing phosphatidylinositol 4,5 bisphosphate

The plasma membrane-cytoskeleton interface is a dynamic structure participating in a variety of cellular events. Among the proteins involved in the direct linkage between the cytoskeleton and the plasma membrane is the ezrin/radixin/moesin (ERM) family. The FERM (4.1 ezrin/radixin/moesin) domain in their N-terminus contains a phosphatidylinositol 4,5 bisphosphate (PIP₂) (membrane) binding site whereas their C-terminus binds actin. In this work, our aim was to quantify the interaction of ezrin with large unilamellar vesicles (LUVs) containing PIP₂. For this purpose, we produced human recombinant ezrin bearing a cysteine residue at its C-terminus for subsequent labeling with Alexa488 maleimide. The functionality of labeled ezrin was checked by comparison with that of wild-type ezrin. The affinity constant between ezrin and LUVs was determined by cosedimentation assays and fluorescence correlation spectroscopy. The affinity was found to be ∼5 μM for PIP₂-LUVs and 20-to 70-fold lower for phosphatidylserine-LUVs. These results demonstrate, as well, that the interaction between ezrin and PIP₂-LUVs is not cooperative. Finally, we found that ezrin FERM domain (area of ∼30 nm²) binding to a single PIP₂ can block access to neighboring PIP₂ molecules and thus contributes to lower the accessible PIP₂ concentration. In addition, no evidence exists for a clustering of PIP₂ induced by ezrin addition.     

WAVE2 targeting to phosphatidylinositol 3,4,5-triphosphate mediated by insulin receptor substrate p53 through a complex with WAVE2

Membrane targeting of WAVE2 along microtubules to phosphatidylinositol 3,4,5-triphosphate (PIP₃) in response to an extracellular stimulus requires Rac1, Pak1, stathmin, and EB1. However, whether WAVE2 interacts directly with PIP₃ or not remains unclear. We demonstrate that insulin-like growth factor I (IGF-I) induces WAVE2 membrane targeting, accompanied by phosphorylation of Pak1 at serine 199/204 (Ser199/204) and stathmin at Ser38 in the inner cytoplasmic region. This is spatially independent of the membrane region where the IGF-I receptor (IGF-IR) is locally activated. WAVE2, phosphorylated Pak1, and phosphorylated stathmin located at the microtubule ends began to accumulate at the leading edge of cells in close proximity to PIP₃ that was produced in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent manner. The PIP₃-beads binding assay revealed that insulin receptor substrate p53 (IRSp53) and actin rather than WAVE2 bound to PIP₃. IRSp53 constitutively associated with WAVE2 and these two proteins colocalized with PIP₃ at the leading edge after IGF-I stimulation. Suppression of IRSp53 expression by two independent small interfering RNAs (siRNAs) completely inhibited IGF-I-induced membrane targeting and local accumulation of WAVE2 at the leading edge of cells. We propose that IRSp53 constitutively forms a complex with WAVE2 and is crucial for membrane targeting followed by local accumulation of WAVE2 at the leading edge of cells through linking WAVE2 to PIP₃ that is produced near locally activated IGF-IR in response to IGF-I.     

Cholesterol is obligatory for polarization and chemotaxis but not for endocytosis and associated signaling from chemoattractant receptors in human neutrophils

Plasma membrane cholesterol is critical for neutrophil chemotaxis, although how cholesterol affects chemotactic signaling pathway has not been clearly delineated. Here we demonstrate that cholesterol was absolutely required for polarized redistribution of key chemotactic mediators in human neutrophils in response to all chemoattractants tested (fMet-Leu-Phe, and the chemokines CXCL1, CXCL8 and CXCL12). In particular, PI3K and phosphatidylinositol-3,4,5 triphosphate (PIP₃) failed to accumulate at the front and phosphatase and tensin homolog (PTEN) at the back of chemoattractant-stimulated neutrophils after cholesterol depletion. Cholesterol depletion did not affect early chemoattractant signaling events such as G-protein activation, intracellular calcium flux or G-protein-independent endocytosis-linked signaling, including the activation of mitogen-activated protein kinase (MAPK), Hck and Fgr transduced by β-arrestin. During cell polarization, F-actin assemblies redistributed the cholesterol-rich microdomains and cytoskeleton-anchored proteins, including CD16 and CD44 from the leading edge. These data suggest that spatial polarization of chemotactic mediators is orchestrated by protein:protein interactions that organize cholesterol-rich domains of the plasma membrane.     

Changes in phosphoinositide metabolism with days in culture affect signal transduction pathways in galdieria sulphuraria

The metabolism of phosphatidylinositol-4,5-bisphosphate (PIP₂) changed during the culture period of the thermoacidophilic red alga Galdieria sulphuraria. Seven days after inoculation, the amount of PIP₂ in the cells was 910 ± 100 pmol g⁻¹ fresh weight; by 12 d, PIP₂ levels increased to 1200 ± 150 pmol g⁻¹ fresh weight. In vitro assays indicated that phosphatidylinositol monophosphate (PIP) kinase specific activity increased from 75 to 230 pmol min⁻¹ mg⁻¹ protein between d 7 and 12. When G. sulphuraria cells were osmostimulated, transient increases of up to 4-fold could be observed in inositol-1,4,5-trisphosphate (IP₃) levels within 90 s, regardless of the age of the cells. In d-12 cells, the increase in IP₃ was preceded by a transient increase of up to 5-fold in specific PIP kinase activity, whereas no such increase was detected after osmostimulation of d-7 cells. The increase in PIP kinase activity before IP₃ signaling in d-12 cells indicates that there is an additional pathway for regulation of phosphoinositide metabolism after stimulation other than an initial activation of phospholipase C. Also, the rapid activation of PIP₂ biosynthesis in cells with already-high PIP₂ levels suggests that the PIP₂ present was not available for signal transduction. By comparing the response of the cells at d 7 and 12, we have identified two potentially distinct pools of PIP₂.     

Synergistic Activation of p21-activated Kinase 1 by Phosphatidylinositol 4,5-Bisphosphate and Rho GTPases

Autoinhibited p21-activated kinase 1 (Pak1) can be activated in vitro by the plasma membrane-bound Rho GTPases Rac1 and Cdc42 as well as by the lipid phosphatidylinositol (4,5)-bisphosphate (PIP₂). Activator binding is mediated by a GTPase-binding motif and an adjacent phosphoinositide-binding motif. Whether these two classes of activators play alternative, additive, or synergistic roles in Pak1 activation is unknown, as is their contributions to Pak1 activation in vivo. To address these questions, we developed a system to mimic the membrane anchoring of Rho GTPases by creating liposomes containing both PIP₂ and a Ni²⁺-NTA modified lipid capable of binding hexahistidine-tagged Cdc42. We find that among all biologically relevant phosphoinositides, only PIP₂ is able to synergistically activate Pak1 in concert with Cdc42. Membrane binding of the kinase was highly sensitive to the spatial density of PIP₂ and Pak1 demonstrated dramatically enhanced affinity for Cdc42 anchored in a PIP₂ environment. To validate these findings in vivo, we utilized an inducible recruitment system to drive the ectopic synthesis of PIP₂ on Golgi membranes, which normally have active Cdc42 but lack significant concentrations of PIP₂. Pak1 was recruited to PIP₂-containing membranes in a manner dependent on the ability of Pak1 to bind to both PIP₂ and Cdc42. These findings provide a mechanistic explanation for the essential role of both phosphoinositides and GTPases in Pak1 recruitment and activation. In contrast, Ack, another Cdc42 effector kinase that lacks an analogous phosphoinositide-binding motif, fails to show the same enhancement of membrane binding and activation by PIP₂, thus indicating that regulation by PIP₂ and Cdc42 could provide a combinatorial code for activation of different GTPase effectors in different subcellular locations.     

Transition between conformational states of the TREK-1 K2P channel promoted by interaction with PIP2

Members of the TREK family of two-pore domain potassium channels are highly sensitive to regulation by membrane lipids, including phosphatidylinositol-4,5-bisphosphate (PIP₂). Previous studies have demonstrated that PIP₂ increases TREK-1 channel activity; however, the mechanistic understanding of the conformational transitions induced by PIP₂ remain unclear. Here, we used coarse-grained molecular dynamics and atomistic molecular dynamics simulations to model the PIP₂-binding site on both the up and down state conformations of TREK-1. We also calculated the free energy of PIP₂ binding relative to other anionic phospholipids in both conformational states using potential of mean force and free-energy-perturbation calculations. Our results identify state-dependent binding of PIP₂ to sites involving the proximal C-terminus, and we show that PIP₂ promotes a conformational transition from a down state toward an intermediate that resembles the up state. These results are consistent with functional data for PIP₂ regulation, and together provide evidence for a structural mechanism of TREK-1 channel activation by phosphoinositides.     

Stimulation of phosphoinositide degradation and phosphatidylinositol-4-phosphate phosphorylation by GTP exclusively in plasma membrane of rat brain

The effect of GTP on the hydrolysis of [³H]phosphatidyinositol (PI), [³H]phosphatidylinositol-4-phosphate (PIP) and [³H]phosphatidylinositol-4,5-bisphosphate (PIP₂) by phospholipase C of rat brain plasma membrane, microsomes and cytosol was determined. Moreover the regulation of PI and PIP phosphorylation by GTP in brain plasma membrane was investigated.In the presence of EGTA PIP₂ was actively degradted, opposite to PI and PIP which require Ca²⁺ for their hydrolysis. Addition of calcium ions in each case caused stimulation of inositide phosphodiesterase(s). GTP independently of calcium ions activates by about 3 times phospholipase C acting on PIP and PIP₂ exclusively in the plasma membrane. PI degradation was unaffected by GTP. In the presence of Ca²⁺ guanine nucleotides have synergistic stimulatory effect on plasma membrane bound phospholipase C acting on PIP₂. PIP kinase of brain plasma membrane was stimulated by GTP by about 20–100% in the presence of exogenous and endogenous substrate respectively. PI kinase was negligible activated by about 20% exclusively in the presence of endogenous substrate. These results indicated that guanine nucleotide modulates the level of second messengers as diacylglycerol and IP₃ through the activation of phospholipase C acting on PIP₂ exclusively in brain plasma membrane. The stimulation of phospholipase C by GTP may occur directly or through the enhancement of substrate level PIP₂ due to stimulation of PIP kinase.     

Pasteurella multocida toxin activates Gβγ dimers of heterotrimeric G proteins

The mitogenic Pasteurella multocida toxin (PMT) is a major virulence factor of P. multocida, which causes Pasteurellosis in man and animals. The toxin activates the small GTPase RhoA, the MAP kinase ERK and STAT proteins via the stimulation of members of two G protein families, G_q and G_12/13. PMT action also results in an increase in inositol phosphates, which is due to the stimulation of PLCβ via Gα_q. Recent studies indicate that PMT additionally activates Gα_i to inhibit adenylyl cyclase. Here we show that PMT acts not only via Gα but also through Gβγ signaling. Activation of Gβγ by PMT causes stimulation of phosphoinositide 3-kinase (PI3K) γ and formation of phosphatidylinositol-3,4,5-trisphosphate (PIP₃) as indicated by the recruitment of a PIP₃-binding pleckstrin homology (PH) domain-containing protein to the plasma membrane. Moreover, it is demonstrated that Gβγ is necessary for PMT-induced signaling via Gα. Mutants of Gα_q incapable of binding or releasing Gβγ are not activated by PMT. Similarly, sequestration of Gβγ inhibits PMT-induced Gα-signaling.     

Molecular and Enzymatic Characterization of Three Phosphoinositide-Specific Phospholipase C Isoforms from Potato

Many cellular responses to stimulation of cell-surface receptors by extracellular signals are transmitted across the plasma membrane by hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP₂), which is cleaved into diacylglycerol and inositol-1,4,5-tris-phosphate by phosphoinositide-specific phospholipase C (PI-PLC). We present structural, biochemical, and RNA expression data for three distinct PI-PLC isoforms, StPLC1, StPLC2, and StPLC3, which were cloned from a guard cell-enriched tissue preparation of potato (Solanum tuberosum) leaves. All three enzymes contain the catalytic X and Y domains, as well as C₂-like domains also present in all PI-PLCs. Analysis of the reaction products obtained from PIP₂ hydrolysis unequivocally identified these enzymes as genuine PI-PLC isoforms. Recombinant StPLCs showed an optimal PIP₂-hydrolyzing activity at 10 μm Ca²⁺ and were inhibited by Al³⁺ in equimolar amounts. In contrast to PI-PLC activity in plant plasma membranes, however, recombinant enzymes could not be activated by Mg²⁺. All three stplc genes are expressed in various tissues of potato, including leaves, flowers, tubers, and roots, and are affected by drought stress in a gene-specific manner.     

Regulation of PI3K by PKC and MARCKS: Single-Molecule Analysis of a Reconstituted Signaling Pathway

In chemotaxing ameboid cells, a complex leading-edge signaling circuit forms on the cytoplasmic leaflet of the plasma membrane and directs both actin and membrane remodeling to propel the leading edge up an attractant gradient. This leading-edge circuit includes a putative amplification module in which Ca²⁺-protein kinase C (Ca²⁺-PKC) is hypothesized to phosphorylate myristoylated alanine-rich C kinase substrate (MARCKS) and release phosphatidylinositol-4,5-bisphosphate (PIP₂), thereby stimulating production of the signaling lipid phosphatidylinositol-3,4,5-trisphosphate (PIP₃) by the lipid kinase phosphoinositide-3-kinase (PI3K). We investigated this hypothesized Ca²⁺-PKC-MARCKS-PIP₂-PI3K-PIP₃ amplification module and tested its key predictions using single-molecule fluorescence to measure the surface densities and activities of its protein components. Our findings demonstrate that together Ca²⁺-PKC and the PIP₂-binding peptide of MARCKS modulate the level of free PIP₂, which serves as both a docking target and substrate lipid for PI3K. In the off state of the amplification module, the MARCKS peptide sequesters PIP₂ and thereby inhibits PI3K binding to the membrane. In the on state, Ca²⁺-PKC phosphorylation of the MARCKS peptide reverses the PIP₂ sequestration, thereby releasing multiple PIP₂ molecules that recruit multiple active PI3K molecules to the membrane surface. These findings 1) show that the Ca²⁺-PKC-MARCKS-PIP₂-PI3K-PIP₃ system functions as an activation module in vitro, 2) reveal the molecular mechanism of activation, 3) are consistent with available in vivo data, and 4) yield additional predictions that are testable in live cells. More broadly, the Ca²⁺-PKC-stimulated release of free PIP₂ may well regulate the membrane association of other PIP₂-binding proteins, and the findings illustrate the power of single-molecule analysis to elucidate key dynamic and mechanistic features of multiprotein signaling pathways on membrane surfaces.     

Human Sensory Neuron-specific Mas-related G Protein-coupled Receptors-X1 Sensitize and Directly Activate Transient Receptor Potential Cation Channel V1 via Distinct Signaling Pathways

Sensory neuron-specific Mas-related G protein-coupled receptors-X1 (MRGPR-X1) are primate-specific proteins that are exclusively expressed in primary sensory neurons and provoke pain in humans. Hence, MRGPR-X1 represent promising targets for future pain therapy, but signaling pathways activated by MRGPR-X1 are poorly understood. The transient receptor potential cation channel V1 (TRPV1) is also expressed in primary sensory neurons and detects painful stimuli such as protons and heat. G_q-promoted signaling has been shown to sensitize TRPV1 via protein kinase C (PKC)-dependent phosphorylation. In addition, recent studies suggested TRPV1 activation via a G_q-mediated mechanism involving diacylglycerol (DAG) or phosphatidylinositol-4,5-bisphosphate (PIP₂). However, it is not clear if DAG-promoted TRPV1 activation occurs independently from classic TRPV1 activation modes induced by heat and protons. Herein, we analyzed putative functional interactions between MRGPR-X1 and TRPV1 in a previously reported F11 cell line stably over-expressing MRGPR-X1. First, we found that MRGPR-X1 sensitized TRPV1 to heat and protons in a PKC-dependent manner. Second, we observed direct MRGPR-X1-mediated TRPV1 activation independent of MRGPR-X1-induced Ca²⁺-release and PKC activity or other TRPV1 affecting enzymes such as lipoxygenase, extracellular signal-regulated kinases-1/2, sarcoma, or phosphoinositide 3-kinase. Investigating several TRPV1 mutants, we observed that removal of the TRPV1 binding site for DAG and of the putative PIP₂ sensor decreased MRGPR-X1-induced TRPV1 activation by 71 and 43%, respectively. Therefore, we demonstrate dual functional interactions between MRGPR-X1 and TRPV1, resulting in PKC-dependent TRPV1 sensitization and DAG/PIP₂-mediated activation. The molecular discrimination between TRPV1 sensitization and activation may help improve the specificity of current pain therapies.     

Phosphatidylinositol 4,5-Bisphosphate Activates Slo3 Currents and Its Hydrolysis Underlies the Epidermal Growth Factor-induced Current Inhibition

The Slo3 gene encodes a high conductance potassium channel, which is activated by both voltage and intracellular alkalinization. Slo3 is specifically expressed in mammalian sperm cells, where it gives rise to pH-dependent outwardly rectifying K⁺ currents. Sperm Slo3 is the main current responsible for the capacitation-induced hyperpolarization, which is required for the ensuing acrosome reaction, an exocytotic process essential for fertilization. Here we show that in intact spermatozoa and in a heterologous expression system, the activation of Slo3 currents is regulated by phosphatidylinositol 4,5-bisphosphate (PIP₂). Depletion of endogenous PIP₂ in inside-out macropatches from Xenopus oocytes inhibited heterologously expressed Slo3 currents. Whole-cell recordings of sperm Slo3 currents or of Slo3 channels co-expressed in Xenopus oocytes with epidermal growth factor receptor, demonstrated that stimulation by epidermal growth factor (EGF) could inhibit channel activity in a PIP₂-dependent manner. High concentrations of PIP₂ in the patch pipette not only resulted in a strong increase in sperm Slo3 current density but also prevented the EGF-induced inhibition of this current. Mutation of positively charged residues involved in channel-PIP₂ interactions enhanced the EGF-induced inhibition of Slo3 currents. Overall, our results suggest that PIP₂ is an important regulator for Slo3 activation and that receptor-mediated hydrolysis of PIP₂ leads to inhibition of Slo3 currents both in native and heterologous expression systems.     

Effects of Ca2+, Mg2+, and depolarizing agents,on the32Pi-labeling and degradation of phosphatidylinositols in rat brain synaptosomes

In isolated synaptosomes from rat brain, 100 M antimycin A and 10 M oxamic acid inhibit the³²Pi-labeling of phosphatidylinositol-4,5-bisphosphate (PIP₂) and phosphatidylinositol-4-phosphate (PIP) by 90% and 95–99% respectively. 10 mM sodium fluoride inhibits the labeling by 50–60% and 10 mM A23187 inhibits the labeling by 63–70%. Phospholipase A₂ inhibits the labeling of PIP₂ and PIP by 93–94% and stimulates their degradation by 84–92%. Depolarization of synaptosomes with 75 mM K⁺ or 100 M veratrine decreases the labeling of PIP₂ and PIP by 66–74%. The decreased labeling results in large part from the Ca²⁺-dependent degradation of³²P-labeled PIP₂ and PIP as shown by pulse-chase experiments in which PIP₂ and PIP were prelabeled with³²Pi. Depolarization of synaptosomes results in the stimulation of⁴⁵Ca²⁺ uptake with the concomitant hydrolysis of PIP and PIP₂. Addition of 1 mM Ca²⁺ accounts for 25% of the enhanced degradation whereas depolarization with 75 mM K⁺ accounts for 75% of the enhanced degradation of PIP₂ and PIP. Depolarization with 100 mM veratrine results in a 223% increase in inositol trisphosphate as evidenced by stimulation of⁴⁵Ca²⁺ uptake. EGTA (10mM) and Mg²⁺ (5–10 mM) inhibit the degradation of PIP and PIP₂ and counteract the action of 1 mM Ca²⁺. Our data demonstrate that⁴⁵Ca²⁺, Mg²⁺, and membrane depolarization play an important role in the turnover of membrane phosphatidylinositols.Abbreviations ATP adenosine triphosphate - Pi inorganic orthophosphate - PIP phosphatidylinositol-4-phosphate - PIP₂ phosphatidylinositol-4,5,-bisphosphate - IP₃ inositol-1,4,5-trisphosphate     

Role of the Phosphatase PTEN in Early Vascular Remodeling

Background

The phosphatase PTEN represents an important physiological inhibitor of phosphatidylinositol-3 kinase (PI3-K)/protein kinase B (Akt) signalling, however, the functional role of PTEN in the initial phase of angioplasty-induced vascular injury remains elusive. In the present study we sought to determine PTEN''s effect on vascular smooth muscle cell (VSMC) apoptosis following acute injury in vivo and in vitro.

Methods and Results

Immunohistochemistry indicated a faint basal expression and equal distribution of PTEN in uninjured rat carotid arteries. 12 h following balloon-injury, PTEN expression was strongly increased in apoptotic (TUNEL+) VSMC. In vitro, stimulation with serum or different growth factors or subjecting VSMC to cyclic stretch had no effect on PTEN expression, whereas stimulation with H₂O₂ robustly increased PTEN expression in a time- and dose-dependent manner. To evaluate the functional role of PTEN expression, human VSMC were transduced with WT-PTEN. Overexpression of PTEN increased the number of apoptotic VSMC (19.8%±4.4 vs. 5.6%±2.3; P<0.001) as determined by TUNEL assay. In contrast, siRNA-mediated knock-down of PTEN attenuated the basal as well as H₂O₂-induced apoptosis of VSMC. Mechanistically, overexpression of PTEN prevented serum-induced Akt-phosphorylation, whereas siRNA-mediated knock down of PTEN augmented Akt-activation. Moreover, co-transfection of PTEN and a constitutive active Akt mutant prevented PTEN-dependent augmentation of VSMC apoptosis, indicating, that PTEN regulates VSMC apoptosis by inhibition of Akt phosphorylation/activation.

Conclusion

By interfering with the PI3-K/Akt-dependent survival signalling, the oxidative stress-induced up regulation of PTEN in VSMC of injured arteries augments the sensitivity of VSMC to apoptotic stimuli in the early phase following vascular injury, augmenting the initial injury and cell loss of the injured vessel wall. Thus, these data add to our understanding of PTEN''s role during vascular remodelling.     

Ca2+-Calmodulin and PIP2 interactions at the proximal C-terminus of Kv7 channels

In the heart, co-assembly of Kv7.1 with KCNE1 produces the slow I_KS potassium current, which repolarizes the cardiac action potential and mutations in human Kv7.1 and KCNE1 genes cause cardiac arrhythmias. The proximal Kv7.1 C-terminus binds calmodulin (CaM) and phosphatidylinositol-4,5-bisphosphate (PIP₂) and recently we revealed the competition of PIP₂ with the calcified CaM N-lobe to a previously unidentified site in Kv7.1 helix B, also known to harbor a LQT mutation. Data indicated that PIP₂ and Ca²⁺-CaM perform the same function on I_KS channel gating to stabilize the channel open state. Here we show that similar features were observed for Kv7.1 currents expressed alone. We also find that conservation of homologous residues in helix B of other Kv7 subtypes confer similar competition of Ca²⁺-CaM with PIP2 binding to their proximal C-termini and suggest that PIP2-CaM interactions converge to Kv7 helix B to modulates channel activity in a Kv7 subtype-dependent manner.     

Differential Effects of Lysophosphatidic Acid and Phosphatidylinositol 4,5-Bisphosphate on Actin Dynamics by Direct Association with the Actin-binding Protein Villin

We have previously reported that the epithelial cell-specific actin-binding protein villin directly associates with phosphatidylinositol 4,5-bisphosphate (PIP₂) through three binding sites that overlap with actin-binding sites in villin. As a result, association of villin with PIP₂ in hibits actin depolymerization and enhances actin cross-linking by villin. In this study, we demonstrate that these three PIP₂-binding sites also bind the more hydrophilic phospholipid, lysophosphatidic acid (LPA) but with a higher affinity than PIP₂ (dissociation constant (K_d) of 22 μm versus 39.5 μm for PIP₂). More interestingly, unlike PIP₂, the association of villin with LPA inhibits all actin regulatory functions of villin. In addition, unlike PIP₂, LPA dramatically stimulates the tyrosine phosphorylation of villin by c-Src kinase. These studies suggest that in cells, selective interaction of villin with either PIP₂ or LPA could have dramatically different outcomes on actin reorganization as well as phospholipid-regulated cell signaling. These studies provide a novel regulatory mechanism for phospholipid-induced changes in the microfilament structure and cell function and suggest that LPA could be an intracellular regulator of the actin cytoskeleton.     

Phosphatidylinositol 3-kinase activation is required to form the NKG2D immunological synapse

The receptor NKG2D allows natural killer (NK) cells to detect virally infected, stressed, and tumor cells. In human cells, NKG2D signaling is mediated through the associated DAP10 adapter. Here we show that engagement of NKG2D by itself is sufficient to stimulate the formation of the NK immunological synapse (NKIS), with recruitment of NKG2D to the center synapse. Mutagenesis studies of DAP10 revealed that the phosphatidylinositol 3-kinase binding site, but not the Grb2 binding site, was required and sufficient for recruitment of DAP10 to the NKIS. Surprisingly, we found that in the absence of the Grb2 binding site, Grb2 was still recruited to the NKIS. Since the recruitment of Grb2 was dependent on phosphatidylinositol-(3,4,5)-trisphosphate (PIP₃), we explored the possibility that recruitment to the NKIS is mediated by a pleckstrin homology (PH) domain-containing binding partner for Grb2. We found that the PH domain of SOS1, but not that of Vav1, was able to be recruited by PIP₃. These results provide new insights into the mechanism of immunological synapse formation and also demonstrate how multiple mechanisms can be used to recruit the same signaling proteins to the plasma membrane.     

Structural Determinants of Phosphatidylinositol 4,5-Bisphosphate (PIP2) Regulation of BK Channel Activity through the RCK1 Ca2+ Coordination Site

Big or high conductance potassium (BK) channels are activated by voltage and intracellular calcium (Ca²⁺). Phosphatidylinositol 4,5-bisphosphate (PIP₂), a ubiquitous modulator of ion channel activity, has been reported to enhance Ca²⁺-driven gating of BK channels, but a molecular understanding of this interplay or even of the PIP₂ regulation of this channel''s activity remains elusive. Here, we identify structural determinants in the KDRDD loop (which follows the αA helix in the RCK1 domain) to be responsible for the coupling between Ca²⁺ and PIP₂ in regulating BK channel activity. In the absence of Ca²⁺, RCK1 structural elements limit channel activation through a decrease in the channel''s PIP₂ apparent affinity. This inhibitory influence of BK channel activation can be relieved by mutation of residues that (a) connect either the RCK1 Ca²⁺ coordination site (Asp³⁶⁷ or its flanking basic residues in the KDRDD loop) to the PIP₂-interacting residues (Lys³⁹² and Arg³⁹³) found in the αB helix or (b) are involved in hydrophobic interactions between the αA and αB helix of the RCK1 domain. In the presence of Ca²⁺, the RCK1-inhibitory influence of channel-PIP₂ interactions and channel activity is relieved by Ca²⁺ engaging Asp³⁶⁷. Our results demonstrate that, along with Ca²⁺ and voltage, PIP₂ is a third factor critical to the integral control of BK channel activity.     

HIV-1 Gag specificity for PIP2 is regulated by macromolecular electric properties of both protein and membrane local environments

HIV-1 assembly occurs at the plasma membrane, with the Gag polyprotein playing a crucial role. Gag association with the membrane is directed by the matrix domain (MA), which is myristoylated and has a highly basic region that interacts with anionic lipids. Several pieces of evidence suggest that the presence of phosphatidylinositol-(4,5)-bisphosphate (PIP₂) highly influences this binding. Furthermore, MA also interacts with nucleic acids, which is proposed to be important for the specificity of GAG for PIP₂-containing membranes. It is hypothesized that RNA has a chaperone function by interacting with the MA domain, preventing Gag from associating with unspecific lipid interfaces. Here, we study the interaction of MA with monolayer and bilayer membrane systems, focusing on the specificity for PIP₂ and on the possible effects of a Gag N-terminal peptide on impairing the binding for either RNA or membrane. We found that RNA decreases the kinetics of the protein association with lipid monolayers but has no effect on the selectivity for PIP₂. Interestingly, for bilayer systems, this selectivity increases in presence of both the peptide and RNA, even for highly negatively charged compositions, where MA alone does not discriminate between membranes with or without PIP₂. Therefore, we propose that the specificity of MA for PIP₂-containing membranes might be related to the electrostatic properties of both membrane and protein local environments, rather than a simple difference in molecular affinities. This scenario provides a new understanding of the regulation mechanism, with a macromolecular view, rather than considering molecular interactions within a ligand-receptor model.     

Mutations in Nature Conferred a High Affinity Phosphatidylinositol 4,5-Bisphosphate-binding Site in Vertebrate Inwardly Rectifying Potassium Channels

All vertebrate inwardly rectifying potassium (Kir) channels are activated by phosphatidylinositol 4,5-bisphosphate (PIP₂) (Logothetis, D. E., Petrou, V. I., Zhang, M., Mahajan, R., Meng, X. Y., Adney, S. K., Cui, M., and Baki, L. (2015) Annu. Rev. Physiol. 77, 81–104; Fürst, O., Mondou, B., and D''Avanzo, N. (2014) Front. Physiol. 4, 404–404). Structural components of a PIP₂-binding site are conserved in vertebrate Kir channels but not in distantly related animals such as sponges and sea anemones. To expand our understanding of the structure-function relationships of PIP₂ regulation of Kir channels, we studied AqKir, which was cloned from the marine sponge Amphimedon queenslandica, an animal that represents the phylogenetically oldest metazoans. A requirement for PIP₂ in the maintenance of AqKir activity was examined in intact oocytes by activation of a co-expressed voltage-sensing phosphatase, application of wortmannin (at micromolar concentrations), and activation of a co-expressed muscarinic acetylcholine receptor. All three mechanisms to reduce the availability of PIP₂ resulted in inhibition of AqKir current. However, time-dependent rundown of AqKir currents in inside-out patches could not be re-activated by direct application to the inside membrane surface of water-soluble dioctanoyl PIP₂, and the current was incompletely re-activated by the more hydrophobic arachidonyl stearyl PIP₂. When we introduced mutations to AqKir to restore two positive charges within the vertebrate PIP₂-binding site, both forms of PIP₂ strongly re-activated the mutant sponge channels in inside-out patches. Molecular dynamics simulations validate the additional hydrogen bonding potential of the sponge channel mutants. Thus, nature''s mutations conferred a high affinity activation of vertebrate Kir channels by PIP₂, and this is a more recent evolutionary development than the structures that explain ion channel selectivity and inward rectification.