Heterodimeric capping protein from Arabidopsis is regulated by phosphatidic acid

The cytoskeleton is a key regulator of morphogenesis, sexual reproduction, and cellular responses to extracellular stimuli. Changes in the cellular architecture are often assumed to require actin-binding proteins as stimulus-response modulators, because many of these proteins are regulated directly by binding to intracellular second messengers or signaling phospholipids. Phosphatidic acid (PA) is gaining widespread acceptance as a major, abundant phospholipid in plants that is required for pollen tube tip growth and mediates responses to osmotic stress, wounding, and phytohormones; however, the number of identified effectors of PA is rather limited. Here we demonstrate that exogenous PA application leads to significant increases in filamentous actin levels in Arabidopsis suspension cells and poppy pollen grains. To investigate further these lipid-induced changes in polymer levels, we analyzed the properties of a key regulator of actin filament polymerization, the heterodimeric capping protein from Arabidopsis thaliana (AtCP). AtCP binds to PA with a K(d) value of 17 muM and stoichiometry of approximately 1:2. It also binds well to PtdIns(4,5)P(2), but not to several other phosphoinositide or acidic phospholipids. The interaction with PA inhibited the actin-binding activity of CP. In the presence of PA, CP is unable to block the barbed or rapidly growing and shrinking end of actin filaments. Precapped filament barbed ends can also be uncapped by addition of PA, allowing rapid filament assembly from an actin monomer pool that is buffered with profilin. The findings support a model in which the inhibition of CP activity in cells by elevated PA results in the stimulation of actin polymerization from a large pool of profilin-actin. Such regulation may be important for the response of plant cells to extracellular stimuli as well as for the normal process of pollen tube tip growth.     

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.     

Arabidopsis capping protein senses cellular phosphatidic acid levels and transduces these into changes in actin cytoskeleton dynamics

Plants respond rapidly and precisely to a broad spectrum of developmental, biotic and abiotic cues. In many instances, signaling cascades involved in transducing this information result in changes to the cellular architecture and cytoskeletal rearrangements. Based originally on paradigms for animal cell signaling, phospholipids have received increased scrutiny as key intermediates for transmitting information to the actin cytoskeleton. Significantly, a wealth of biochemical data for plant actin-binding proteins (ABPs) demonstrates that many of these interact with phosphoinositide lipids in vitro. Moreover, phosphatidic acid (PA) has been identified not only as an abundant structural lipid in plants, but also as an intermediary in developmental and stress signaling pathways that lead to altered actin organization. Several years ago, the heterodimeric capping protein (CP) from Arabidopsis was demonstrated to bind PA and is negatively regulated by this lipid in vitro. Whether this form of regulation occurs in cells, however, remained a mystery. A new study, that combines live-cell imaging of cytoskeletal dynamics with reverse-genetic analyses in Arabidopsis, provides compelling new evidence that CP is inhibited from binding filament ends in the presence of PA in vivo. This allows rapid actin polymerization and increases in filament abundance following stimulation and could be one key factor in the physiological responses of plant cells to environmental stimuli.     

Gelsolin Modulates Phospholipase C Activity In Vivo through Phospholipid Binding

Gelsolin and CapG are actin regulatory proteins that remodel the cytoskeleton in response to phosphatidylinositol 4,5-bisphosphate (PIP₂) and Ca²⁺ during agonist stimulation. A physiologically relevant rise in Ca²⁺ increases their affinity for PIP₂ and can promote significant interactions with PIP₂ in activated cells. This may impact divergent PIP₂- dependent signaling processes at the level of substrate availability. We found that CapG overexpression enhances PDGF-stimulated phospholipase C_γ (PLC_γ) activity (Sun, H.-q., K. Kwiatkowska, D.C. Wooten, and H.L. Yin. 1995. J. Cell Biol. 129:147–156). In this paper, we examined the ability of gelsolin and CapG to compete with another PLC for PIP₂ in live cells, in semiintact cells, and in vitro. We found that CapG and gelsolin overexpression profoundly inhibited bradykinin-stimulated PLC_β. Inhibition occurred at or after the G protein activation step because overexpression also reduced the response to direct G protein activation with NaF. Bradykinin responsiveness was restored after cytosolic proteins, including gelsolin, leaked out of the overexpressing cells. Conversely, exogenous gelsolin added to permeabilized cells inhibited response in a dose-dependent manner. The washout and addback experiments clearly establish that excess gelsolin is the primary cause of PLC inhibition in cells. In vitro experiments showed that gelsolin and CapG stimulated as well as inhibited PLC_β, and only gelsolin domains containing PIP₂-binding sites were effective. Inhibition was mitigated by increasing PIP₂ concentration in a manner consistent with competition between gelsolin and PLC_β for PIP₂. Gelsolin and CapG also had biphasic effects on tyrosine kinase– phosphorylated PLC_γ, although they inhibited PLC_γ less than PLC_β. Our findings indicate that as PIP₂ level and availability change during signaling, cross talk between PIP₂-regulated proteins provides a selective mechanism for positive as well as negative regulation of the signal transduction cascade.     

Regional Distribution of Phospholipids and Polyphosphatidyl Inositides in the Rabbit's Spinal Cord

The plasticity of the membrane phospholipids in general and stimulated phosphoinositides turnover in particular are the subjects in a variety of neural paradigms studying the molecular mechanisms of neuronal changes under normal and pathological conditions. The regional modifiability of phospholipids (SM, PC, PS, PI, PA + DG, PE), polyphosphatidylinositides (PI, PIP, PIP₂) and diacylglycerol-dependent incorporation of CDP-choline into phosphatidylcholine in the gray matter, white matter, dorsal horns, intermediate zone and ventral horns of the rabbit's spinal cord was studied. We have found 1. a significant increase in the concentration of SM, PC, PS, DG + PA and PE in the white matter in comparison to the gray one, 2. the highest concentration of the outer membrane leaflet-bound phospholipids in the dorsal horns and the inner membrane phospholipids in the intermediate zone in comparison to the gray matter, 3. a substantial amount of labeled polyphosphatidylinositides (poly-PI_s) in the spinal cord white matter with descending order PIP > PI > PIP₂, 4. similar incorporation of myo-2-[³H]inositol into all poly-PI_s in ventral horns and intermediate zone, but a different, lower incorporation into PI and PIP and higher into PIP₂ in the dorsal horns, 5. higher diacylglycerol-dependent incorporation of CDP-choline into PC in the regionally undivided gray matter than in the white matter taken as a whole, 6. the high proportion of diacylglycerol-dependent incorporation of CDP-choline into PC in both the ventral and dorsal horns, whereas that in the intermediate zone remained low.     

Mutual regulation of plant phospholipase D and the actin cytoskeleton

Membrane lipids and cytoskeleton dynamics are intimately inter‐connected in the eukaryotic cell; however, only recently have the molecular mechanisms operating at this interface in plant cells been addressed experimentally. Phospholipase D (PLD) and its product phosphatidic acid (PA) were discovered to be important regulators in the membrane–cytoskeleton interface in eukaryotes. Here we report the mechanistic details of plant PLD–actin interactions. Inhibition of PLD by n‐butanol compromises pollen tube actin, and PA rescues the detrimental effect of n‐butanol on F‐actin, showing clearly the importance of the PLD–PA interaction for pollen tube F‐actin dynamics. From various candidate tobacco PLDs isoforms, we identified NtPLDβ1 as a regulatory partner of actin, by both activity and in vitro interaction assays. Similarly to published data, the activity of tobacco PIP₂‐dependent PLD (PLDβ) is specifically enhanced by F‐actin and inhibited by G‐actin. We then identified the NtPLDβ1 domain responsible for actin interactions. Using sequence‐ and structure‐based analysis, together with site‐directed mutagenesis, we identified Asn323 and Thr382 of NtPLDβ1 as the crucial amino acids in the actin‐interacting fold. The effect of antisense‐mediated suppression of NtPLDβ1 or NtPLDδ on pollen tube F‐actin dynamics shows that NtPLDβ1 is the active partner in PLD–actin interplay. The positive feedback loop created by activation of PLDβ by F‐actin and of F‐actin by PA provides an important mechanism to locally increase membrane–F‐actin dynamics in the cortex of plant cells.     

Arp2/3 Complex from Acanthamoeba Binds Profilin and Cross-links Actin Filaments

The Arp2/3 complex was first purified from Acanthamoeba castellanii by profilin affinity chromatography. The mechanism of interaction with profilin was unknown but was hypothesized to be mediated by either Arp2 or Arp3. Here we show that the Arp2 subunit of the complex can be chemically cross-linked to the actin-binding site of profilin. By analytical ultracentrifugation, rhodamine-labeled profilin binds Arp2/3 complex with a K_d of 7 μM, an affinity intermediate between the low affinity of profilin for barbed ends of actin filaments and its high affinity for actin monomers. These data suggest the barbed end of Arp2 is exposed, but Arp2 and Arp3 are not packed together in the complex exactly like two actin monomers in a filament. Arp2/3 complex also cross-links actin filaments into small bundles and isotropic networks, which are mechanically stiffer than solutions of actin filaments alone. Arp2/3 complex is concentrated at the leading edge of motile Acanthamoeba, and its localization is distinct from that of α-actinin, another filament cross-linking protein. Based on localization and actin filament nucleation and cross-linking activities, we propose a role for Arp2/3 in determining the structure of the actin filament network at the leading edge of motile cells.     

Phospholipid Mediated Activation of Calcium Dependent Protein Kinase 1 (CaCDPK1) from Chickpea: A New Paradigm of Regulation

Phospholipids, the major structural components of membranes, can also have functions in regulating signaling pathways in plants under biotic and abiotic stress. The effects of adding phospholipids on the activity of stress-induced calcium dependent protein kinase (CaCDPK1) from chickpea are reported here. Both autophosphorylation as well as phosphorylation of the added substrate were enhanced specifically by phosphatidylcholine and to a lesser extent by phosphatidic acid, but not by phosphatidylethanolamine. Diacylgylerol, the neutral lipid known to activate mammalian PKC, stimulated CaCDPK1 but at higher concentrations. Increase in V_max of the enzyme activity by these phospholipids significantly decreased the K_m indicating that phospholipids enhance the affinity towards its substrate. In the absence of calcium, addition of phospholipids had no effect on the negligible activity of the enzyme. Intrinsic fluorescence intensity of the CaCDPK1 protein was quenched on adding PA and PC. Higher binding affinity was found with PC (K_½ = 114 nM) compared to PA (K_½ = 335 nM). We also found that the concentration of PA increased in chickpea plants under salt stress. The stimulation by PA and PC suggests regulation of CaCDPK1 by these phospholipids during stress response.     

Regulation of high molecular weight bovine brain neutral protease by phospholipids in vitro

The activity of the heat stable, glycosylated high molecular weight bovine brain neutral protease (HMW protease) is differentially regulated by phospholipids. While phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidic acid (PA) had only marginal stimulatory effect (40–75%) on the activity of HMW protease, lysophoshatidylcholine (lysoPC) and lysophosphatidic acid (lysoPA) activated the enzyme by more than two-fold. Both lysoPC and lysoPA exhibited concentration-dependent saturation kinetics for the activation of HMW protease. Surprisingly, phosphoinositides (phosphatidylinositol, PI; phosphatidylinositol 4-phosphate, PIP; and phosphatidylinositol 4,5-bisphosphate, PIP₂) modulated the activity of protease differently: activation of the enzyme was higher with PIP (90%) as compared to PI (21%), whereas PIP₂ inhibited the enzyme (16%). The inhibition of the protease by PIP₂ was concentration-dependent. During receptor-coupled cell activation, phospholipase A₂ (PLA₂) converts PC and PA to lysoPC and lysoPA, respectively; PI is converted to PIP₂ by successive enzymatic phosphorylation by PI 4-kinase and PIP 5-kinase; and phospholipase C (PLC) degrades PIP₂ to diacylglycerol and inositol 1,4,5-trisphosphate. Therefore, the data suggest that HMW protease may be coupled to cell signal transduction where PLA₂, PI 4-kinase, PIP 5-kinase and PLC are involved.     

Interaction of actin with the capping protein, CapZ from sea bass (Dicentrarchus labrax) white skeletal muscle

We have compared the functional properties of CapZ from fish white skeletal muscle with those of CapZ from chicken muscle. CapZ is a heterodimer, which enhances actin nucleation and inhibits the depolymerization process by binding to the barbed ends of microfilaments. Here, we report the interaction of CapZ not only with F-actin, but also with monomeric actin. The affinity of sea bass CapZ for G-actin estimated by enzyme-linked immunosorbent assay (ELISA) was in the μM range. This association was PIP₂ dependent. Binding contacts with the barbed end of actin were delimited by both ELISA and fluorescence approaches. One site (actin sequence 338–348) was located in a helical region of the subdomain 1, region already implicated in the interaction with other actin binding proteins such as gelsolin. Another site implicates the C-terminal region (sequence 360–372) of actin. Finally, the partial competition of antibodies directed against CapZ α or β-subunits towards CapZ interaction with actin filaments suggests both subunits participate in the complex with actin.     

Molecular Basis of the Membrane Interaction of the β2e Subunit of Voltage-Gated Ca2+ Channels

The auxiliary β subunit plays an important role in the regulation of voltage-gated calcium (Ca_V) channels. Recently, it was revealed that β2e associates with the plasma membrane through an electrostatic interaction between N-terminal basic residues and anionic phospholipids. However, a molecular-level understanding of β-subunit membrane recruitment in structural detail has remained elusive. In this study, using a combination of site-directed mutagenesis, liposome-binding assays, and multiscale molecular-dynamics (MD) simulation, we developed a physical model of how the β2e subunit is recruited electrostatically to the plasma membrane. In a fluorescence resonance energy transfer assay with liposomes, binding of the N-terminal peptide (23 residues) to liposome was significantly increased in the presence of phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP₂). A mutagenesis analysis suggested that two basic residues proximal to Met-1, Lys-2 (K2) and Trp-5 (W5), are more important for membrane binding of the β2e subunit than distal residues from the N-terminus. Our MD simulations revealed that a stretched binding mode of the N-terminus to PS is required for stable membrane attachment through polar and nonpolar interactions. This mode obtained from MD simulations is consistent with experimental results showing that K2A, W5A, and K2A/W5A mutants failed to be targeted to the plasma membrane. We also investigated the effects of a mutated β2e subunit on inactivation kinetics and regulation of Ca_V channels by PIP₂. In experiments with voltage-sensing phosphatase (VSP), a double mutation in the N-terminus of β2e (K2A/W5A) increased the PIP₂ sensitivity of Ca_V2.2 and Ca_V1.3 channels by ∼3-fold compared with wild-type β2e subunit. Together, our results suggest that membrane targeting of the β2e subunit is initiated from the nonspecific electrostatic insertion of N-terminal K2 and W5 residues into the membrane. The PS-β2e interaction observed here provides a molecular insight into general principles for protein binding to the plasma membrane, as well as the regulatory roles of phospholipids in transporters and ion channels.     

Dynamic phospholipid interaction of β2e subunit regulates the gating of voltage-gated Ca2+ channels

High voltage-activated Ca²⁺ (Ca_V) channels are protein complexes containing pore-forming α1 and auxiliary β and α2δ subunits. The subcellular localization and membrane interactions of the β subunits play a crucial role in regulating Ca_V channel inactivation and its lipid sensitivity. Here, we investigated the effects of membrane phosphoinositide (PI) turnover on Ca_V2.2 channel function. The β2 isoform β2e associates with the membrane through electrostatic and hydrophobic interactions. Using chimeric β subunits and liposome-binding assays, we determined that interaction between the N-terminal 23 amino acids of β2e and anionic phospholipids was sufficient for β2e membrane targeting. Binding of the β2e subunit N terminus to liposomes was significantly increased by inclusion of 1% phosphatidylinositol 4,5-bisphosphate (PIP₂) in the liposomes, suggesting that, in addition to phosphatidylserine, PIs are responsible for β2e targeting to the plasma membrane. Membrane binding of the β2e subunit slowed Ca_V2.2 current inactivation. When membrane phosphatidylinositol 4-phosphate and PIP₂ were depleted by rapamycin-induced translocation of pseudojanin to the membrane, however, channel opening was decreased and fast inactivation of Ca_V2.2(β2e) currents was enhanced. Activation of the M₁ muscarinic receptor elicited transient and reversible translocation of β2e subunits from membrane to cytosol, but not that of β2a or β3, resulting in fast inactivation of Ca_V2.2 channels with β2e. These results suggest that membrane targeting of the β2e subunit, which is mediated by nonspecific electrostatic insertion, is dynamically regulated by receptor stimulation, and that the reversible association of β2e with membrane PIs results in functional changes in Ca_V channel gating. The phospholipid–protein interaction observed here provides structural insight into mechanisms of membrane–protein association and the role of phospholipids in ion channel regulation.     

Homer3 regulates the establishment of neutrophil polarity

Most chemoattractants rely on activation of the heterotrimeric G-protein Gαi to regulate directional cell migration, but few links from Gαi to chemotactic effectors are known. Through affinity chromatography using primary neutrophil lysate, we identify Homer3 as a novel Gαi2-binding protein. RNA interference–mediated knockdown of Homer3 in neutrophil-like HL-60 cells impairs chemotaxis and the establishment of polarity of phosphatidylinositol 3,4,5-triphosphate (PIP₃) and the actin cytoskeleton, as well as the persistence of the WAVE2 complex. Most previously characterized proteins that are required for cell polarity are needed for actin assembly or activation of core chemotactic effectors such as the Rac GTPase. In contrast, Homer3-knockdown cells show normal magnitude and kinetics of chemoattractant-induced activation of phosphoinositide 3-kinase and Rac effectors. Chemoattractant-stimulated Homer3-knockdown cells also exhibit a normal initial magnitude of actin polymerization but fail to polarize actin assembly and intracellular PIP₃ and are defective in the initiation of cell polarity and motility. Our data suggest that Homer3 acts as a scaffold that spatially organizes actin assembly to support neutrophil polarity and motility downstream of GPCR activation.     

Phosphatidylinositol 4,5-Biphosphate (PIP2) Modulates Interaction of Syntaxin-1A with Sulfonylurea Receptor 1 to Regulate Pancreatic β-Cell ATP-sensitive Potassium Channels

In β-cells, syntaxin (Syn)-1A interacts with SUR1 to inhibit ATP-sensitive potassium channels (K_ATP channels). PIP₂ binds the Kir6.2 subunit to open K_ATP channels. PIP₂ also modifies Syn-1A clustering in plasma membrane (PM) that may alter Syn-1A actions on PM proteins like SUR1. Here, we assessed whether the actions of PIP₂ on activating K_ATP channels is contributed by sequestering Syn-1A from binding SUR1. In vitro binding showed that PIP₂ dose-dependently disrupted Syn-1A·SUR1 complexes, corroborated by an in vivo Forster resonance energy transfer assay showing disruption of SUR1(-EGFP)/Syn-1A(-mCherry) interaction along with increased Syn-1A cluster formation. Electrophysiological studies of rat β-cells, INS-1, and SUR1/Kir6.2-expressing HEK293 cells showed that PIP₂ dose-dependent activation of K_ATP currents was uniformly reduced by Syn-1A. To unequivocally distinguish between PIP₂ actions on Syn-1A and Kir6.2, we employed several strategies. First, we showed that PIP₂-insensitive Syn-1A-5RK/A mutant complex with SUR1 could not be disrupted by PIP₂, consequently reducing PIP₂ activation of K_ATP channels. Next, Syn-1A·SUR1 complex modulation of K_ATP channels could be observed at a physiologically low PIP₂ concentration that did not disrupt the Syn-1A·SUR1 complex, compared with higher PIP₂ concentrations acting directly on Kir6.2. These effects were specific to PIP₂ and not observed with physiologic concentrations of other phospholipids. Finally, depleting endogenous PIP₂ with polyphosphoinositide phosphatase synaptojanin-1, known to disperse Syn-1A clusters, freed Syn-1A from Syn-1A clusters to bind SUR1, causing inhibition of K_ATP channels that could no longer be further inhibited by exogenous Syn-1A. These results taken together indicate that PIP₂ affects islet β-cell K_ATP channels not only by its actions on Kir6.2 but also by sequestering Syn-1A to modulate Syn-1A availability and its interactions with SUR1 on PM.     

Two structural components in CNGA3 support regulation of cone CNG channels by phosphoinositides

Cyclic nucleotide-gated (CNG) channels in retinal photoreceptors play a crucial role in vertebrate phototransduction. The ligand sensitivity of photoreceptor CNG channels is adjusted during adaptation and in response to paracrine signals, but the mechanisms involved in channel regulation are only partly understood. Heteromeric cone CNGA3 (A3) + CNGB3 (B3) channels are inhibited by membrane phosphoinositides (PIP_n), including phosphatidylinositol 3,4,5-triphosphate (PIP₃) and phosphatidylinositol 4,5-bisphosphate (PIP₂), demonstrating a decrease in apparent affinity for cyclic guanosine monophosphate (cGMP).Unlike homomeric A1 or A2 channels, A3-only channels paradoxically did not show a decrease in apparent affinity for cGMP after PIP_n application. However, PIP_n induced an ∼2.5-fold increase in cAMP efficacy for A3 channels. The PIP_n-dependent change in cAMP efficacy was abolished by mutations in the C-terminal region (R643Q/R646Q) or by truncation distal to the cyclic nucleotide-binding domain (613X). In addition, A3-613X unmasked a threefold decrease in apparent cGMP affinity with PIP_n application to homomeric channels, and this effect was dependent on conserved arginines within the N-terminal region of A3. Together, these results indicate that regulation of A3 subunits by phosphoinositides exhibits two separable components, which depend on structural elements within the N- and C-terminal regions, respectively. Furthermore, both N and C regulatory modules in A3 supported PIP_n regulation of heteromeric A3+B3 channels. B3 subunits were not sufficient to confer PIP_n sensitivity to heteromeric channels formed with PIP_n-insensitive A subunits. Finally, channels formed by mixtures of PIP_n-insensitive A3 subunits, having complementary mutations in N- and/or C-terminal regions, restored PIP_n regulation, implying that intersubunit N–C interactions help control the phosphoinositide sensitivity of cone CNG channels.     

Partially Purified RhoA-Stimulated Phospholipase D Activity Specifically Binds to Phosphatidylinositol 4,5-Bisphosphate

Abstract: Phosphatidylinositol 4,5-bisphosphate (PIP₂) is absolutely required for the ADP-ribosylation factor-stimulated phospholipase D (PLD) activity. In the present study, partially purified rat brain PLD was found to be activated by another PLD activator, RhoA, when PIP₂, but not other acidic phospholipids, was included in vesicles comprising phosphatidylethanolamine (PE) and the PLD substrate phosphatidylcholine (PC) (PE/PC vesicles), demonstrating the absolute requirement of PIP₂ for the RhoA-stimulated PLD activation, too. It is interesting that the RhoA-dependent PLD activity in the partially purified preparation was drastically decreased after the preparation was incubated with and separated from PE/PC vesicles containing PIP₂. The PLD activity was extracted by higher concentrations of NaCl from the vesicles containing PIP₂ that were incubated with and then separated from the partially purified PLD preparation. These results demonstrate that RhoA-dependent PLD binds to PE/PC vesicles with PIP₂. The degree of binding of the RhoA-dependent PLD activity to the vesicles was totally dependent on the amount of PIP₂ in the vesicles and correlated well with the extent of the enzyme activation. Furthermore, it was found that a recombinant peptide of the pleckstrin homology domain of β-adrenergic receptor kinase fused to glutathione S-transferase, which specifically binds to PIP₂, inhibited the PIP₂-stimulated, RhoA-dependent PLD activity in a concentration-dependent manner. From these results, it is concluded that in vitro rat brain PLD translocates to the vesicles containing PIP₂, owing to its specific interaction with PIP₂, to access its substrate PC, thereby catalyzing the hydrolysis of PC. PLD appears to localize exclusively on plasma membranes of cells and tissues. An aminoglycoside, neomycin, that has high affinity for PIP₂ effectively extracted the RhoA-dependent PLD activity from rat brain membranes. This indicates that PIP₂ serves as an anchor to localize PLD on plasma membranes in vivo.     

How capping protein binds the barbed end of the actin filament

Cytoskeletal filaments are often capped at one end, regulating assembly and cellular location. The actin filament is a right-handed, two-strand long-pitch helix. The ends of the two protofilaments are staggered in relation to each other, suggesting that capping could result from one protein binding simultaneously to the ends of both protofilaments. Capping protein (CP), a ubiquitous alpha/beta heterodimer in eukaryotes, tightly caps (K(d) approximately 0.1-1 nM) the barbed end of the actin filament (the end favored for polymerization), preventing actin subunit addition and loss. CP is critical for actin assembly and actin-based motility in vivo and is an essential component of the dendritic nucleation model for actin polymerization at the leading edge of cells. However, the mechanism by which CP caps actin filaments is not well understood. The X-ray crystal structure of CP has inspired a model where the C termini ( approximately 30 amino acids) of the alpha and beta subunits of CP are mobile extensions ("tentacles"), and these regions are responsible for high-affinity binding to, and functional capping of, the barbed end. We tested the tentacle model in vitro with recombinant mutant CPs. Loss of both tentacles causes a complete loss of capping activity. The alpha tentacle contributes more to capping affinity and kinetics; its removal reduces capping affinity by 5000-fold and the on-rate of capping by 20-fold. In contrast, removal of the beta tentacle reduced the affinity by only 300-fold and did not affect the on-rate. These two regions are not close to each other in the three-dimensional structure, suggesting CP uses two independent actin binding tentacles to cap the barbed end. CP with either tentacle alone can cap, as can the isolated beta tentacle alone, suggesting that the individual tentacles interact with more than one actin subunit at a subunit interface at the barbed end.     

CARMIL is a potent capping protein antagonist: identification of a conserved CARMIL domain that inhibits the activity of capping protein and uncaps capped actin filaments

Acanthamoeba CARMIL was previously shown to co-purify with capping protein (CP) and to bind pure CP. Here we show that this interaction inhibits the barbed end-capping activity of CP. Even more strikingly, this interaction drives the uncapping of actin filaments previously capped with CP. These activities are CP-specific; CARMIL does not inhibit the capping activities of either gelsolin or CapG and does not uncap gelsolin-capped filaments. Although full-length (FL) CARMIL (residues 1-1121) possesses both anti-CP activities, C-terminal fragments like glutathione S-transferase (GST)-P (940-1121) that contain the CARMIL CP binding site are at least 10 times more active. We localized the full activities of GST-P to its C-terminal 51 residues (1071-1121). This sequence contains a stretch of 25 residues that is highly conserved in CARMIL proteins from protozoa, flies, worms, and vertebrates (CARMIL Homology domain 3; CAH3). Point mutations showed that the majority of the most highly conserved residues within CAH3 are critical for the anti-CP activity of GST-AP (862-1121). Finally, we found that GST-AP binds CP approximately 20-fold more tightly than does FL-CARMIL. This observation together with the elevated activities of C-terminal fragments relative to FL-CARMIL suggests that FL-CARMIL might exist primarily in an autoinhibited state. Consistent with this idea, proteolytic cleavage of FL-CARMIL with thrombin generated an approximately 14-kDa C-terminal fragment that expresses full anti-CP activities. We propose that, after some type of physiological activation event, FL-CARMIL could function in vivo as a potent CP antagonist. Given the pivotal role that CP plays in determining the global actin phenotype of cells, our results suggest that CARMIL may play an important role in the physiological regulation of actin assembly.     

Biological role and structural mechanism of twinfilin-capping protein interaction

Twinfilin and capping protein (CP) are highly conserved actin-binding proteins that regulate cytoskeletal dynamics in organisms from yeast to mammals. Twinfilin binds actin monomer, while CP binds the barbed end of the actin filament. Remarkably, twinfilin and CP also bind directly to each other, but the mechanism and role of this interaction in actin dynamics are not defined. Here, we found that the binding of twinfilin to CP does not affect the binding of either protein to actin. Furthermore, site-directed mutagenesis studies revealed that the CP-binding site resides in the conserved C-terminal tail region of twinfilin. The solution structure of the twinfilin-CP complex supports these conclusions. In vivo, twinfilin's binding to both CP and actin monomer was found to be necessary for twinfilin's role in actin assembly dynamics, based on genetic studies with mutants that have defined biochemical functions. Our results support a novel model for how sequential interactions between actin monomers, twinfilin, CP, and actin filaments promote cytoskeletal dynamics.     

Electron Microscopy and 3D Reconstruction Reveals Filamin Ig Domain Binding to F-Actin

Filamin A (FLNa) is an actin-binding protein that cross-links F-actin into networks of orthogonally branched filaments. FLNa also directs the networks to integrins while responding to mechanochemical signaling pathways. Flexible, 160-nm-long FLNa molecules are tail-to-tail dimers, each subunit of which contains an N-terminal calponin homology (CH)/actin-binding domain connected by a series of 24 immunoglobulin (Ig) repeats to a dimerization site at their C-terminal end. Whereas the contribution of the CH domains to F-actin affinity is weak (apparent K_a ~ 10⁵), the binding of the intact protein to F-actin is strong (apparent K_a ~ 10⁸), suggesting involvement of additional parts of the molecule in this association. Indeed, previous results indicate that Ig repeats along FLNa contribute significantly to the strength of the actin filament interaction. In the current study, we used electron microscopy and three-dimensional reconstruction to elucidate the structural basis of the Ig repeat–F-actin binding. We find that FLNa density is clearly delineated in reconstructions of F-actin complexed either with a four-Ig-repeat segment of FLNa containing Ig repeat 10 or with immunoglobulin-like filamin A repeat (IgFLNa)10 alone. The mass attributable to IgFLNa10 lies peripherally along the actin helix over the N-terminus of actin subdomain 1. The IgFLNa10 interaction appears to be specific, since no other individual Ig repeat or fragment of the FLNa molecule examined, besides ones with IgFLNa10 or CH domains, decorated F-actin filaments or were detected in reconstructions. We conclude that the combined interactions of CH domains and the IgFLNa10 repeat provide the binding strength of the whole FLNa molecule and propose a model for the association of IgFLNa10 on actin filaments.