Chemotactic peptide-induced changes in neutrophil actin conformation

The effect of the chemotatic peptide, N- formylmethionylleucylphenylalanine (FMLP), on actin conformation in human neutrophils (PMN) was studied by flow cytometry using fluorescent 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin to quantitate cellular F-actin content. Uptake of NBD-phallacidin by fixed PMN was saturable and inhibited by fluid phase F-actin but not G-actin. Stimulation of PMN by greater than 1 nM FMLP resulted in a dose-dependent and reversible increase in F-actin in 70-95% of PMN by 30 s. The induced increase in F-actin was blocked by 30 microM cytochalasin B or by a t- BOC peptide that competitively inhibits FMLP binding. Under fluorescence microscopy, NBD-phallacidin stained, unstimulated PMN had faint homogeneous cytoplasmic fluorescence while cells exposed to FMLP for 30 s prior to NBD-phallacidin staining had accentuated subcortical fluorescence. In the continued presence of an initial stimulatory dose of FMLP, PMN could respond with increased F-actin content to the addition of an increased concentration of FMLP. Thus, FMLP binding to PMN induces a rapid transient conversion of unpolymerized actin to subcortical F-actin and repetitive stimulation of F-actin formation can be induced by increasing chemoattractant concentration. The directed movement of PMN in response to chemoattractant gradients may require similar rapid reversible changes in actin conformation.     

The regulation of actin polymerization and the inhibition of monomeric actin ATPase activity by Acanthamoeba profilin

Profilin inhibits the rate of nucleation of actin polymerization and the rate of filament elongation and also reduces the concentration of F-actin at steady state. Addition of profilin to solutions of F-actin causes depolymerization. The same steady state concentrations of polymerized and nonpolymerized actin are reached whether profilin is added before initiation of polymerization or after polymerization is complete. The KD for formation of the 1:1 complex between Acanthamoeba profilin and Acanthamoeba actin is in the range of 4 to 11 microM; the KD for the reaction between Acanthamoeba profilin and rabbit skeletal muscle actin is about 60 to 80 microM, irrespective of the concentrations of KCl or MgCl2. The critical concentration of actin for polymerization and the KD for the actin-profilin interaction are independent of each other; therefore, a change in the critical concentration of actin alters the amount of actin bound to profilin at steady state. As a consequence, the presence of profilin greatly amplifies the effects of small changes in the actin critical concentration on the concentration of F-actin. Profilin also inhibits the ATPase activity of monomeric actin, the profilin-actin complex being entirely inactive.     

Polymorphonuclear leukocyte adherence induces actin polymerization by a transduction pathway which differs from that used by chemoattractants

Nitrobenzoxadiazole-phallacidin in combination with quantitative fluorescent microscopy have been used to measure F-actin concentrations in human polymorphonuclear leukocytes (PMN) as they adhere to a plastic surface. Like stimulation with chemoattractants, adherence is associated with a twofold rise in F-actin content. However unlike the rapid rise in F-actin induced by chemoattractants which peaks within 30 s, actin assembly induced by adherence is slower, maximum F-actin values not being observed until 10 min. Furthermore the rise in F-actin induced by adherence is persistent, remaining constant over 60 min while F-actin returns to near basal levels after 20 min exposure to chemoattractant. The combination of adherence (5 min) followed by chemoattractant (FMLP 5 x 10(-8) M for 40 s) resulted in an additive rise in F-actin content to greater than threefold over unstimulated values. Unlike chemoattractant induced actin assembly, adherence- associated PMN actin polymerization was not inhibited by pertussis toxin, but was markedly reduced by lowering extracellular Ca2+. Fluorescent micrographs of adherent PMN stained with nitrobenzoxadiazole-phallacidin revealed F-actin in the lamellipodia and in small foci on the adherent surface. These findings suggest that the transduction mechanisms by which adherence induces PMN actin polymerization differ from those used by chemoattractant receptors.     

How ATP hydrolysis controls filament assembly from profilin-actin: implication for formin processivity

Formins catalyze rapid filament growth from profilin-actin, by remaining processively bound to the elongating barbed end. The sequence of elementary reactions that describe filament assembly from profilin-actin at either free or formin-bound barbed ends is not fully understood. Specifically, the identity of the transitory complexes between profilin and actin terminal subunits is not known; and whether ATP hydrolysis is directly or indirectly coupled to profilin-actin assembly is not clear. We have analyzed the effect of profilin on actin assembly at free and FH1-FH2-bound barbed ends in the presence of ADP and non-hydrolyzable CrATP. Profilin blocked filament growth by capping the barbed ends in ADP and CrATP/ADP-Pi states, with a higher affinity when formin is bound. We confirm that, in contrast, profilin accelerates depolymerization of ADP-F-actin, more efficiently when FH1-FH2 is bound to barbed ends. To reconcile these data with effective barbed end assembly from profilin-MgATP-actin, the nature of nucleotide bound to both terminal and subterminal subunits must be considered. All data are accounted for quantitatively by a model in which a barbed end whose two terminal subunits consist of profilin-ATP-actin cannot grow until ATP has been hydrolyzed and Pi released from the penultimate subunit, thus promoting the release of profilin and allowing further elongation. Formin does not change the activity of profilin but simply uses it for its processive walk at barbed ends. Finally, if profilin release from actin is prevented by a chemical cross-link, formin processivity is abolished.     

Actin from Thyone sperm assembles on only one end of an actin filament: a behavior regulated by profilin

Thyone sperm were demembranated with Triton X-100 and, after washing, extracted with 30 mM Tris at pH 8.0 and 1 mM MgCl2. After the insoluble contaminants were removed by centrifugation, the sperm extract was warmed to 22 degrees C. Actin filaments rapidly assembled and aggregated into bundles when KCl was added to the extract. When we added preformed actin filaments, i.e., the acrosomal filament bundles of Limulus sperm, to the extract, the actin monomers rapidly assembled on these filaments. What was unexpected was that assembly took place on only one end of the bundle--the end corresponding to the preferred end for monomer addition. We showed that the absence of growth on the nonpreferred end was not due to the presence of a capper because exogenously added actin readily assembled on both ends. We also analyzed the sperm extract by SDS gel electrophoresis. Two major proteins were present in a 1:1 molar ratio: actin and a 12,500-dalton protein whose apparent isoelectric point was 8.4. The 12,500-dalton protein was purified by DEAE chromatography. We concluded that it is profilin because of its size, isoelectric point, molar ratio to actin, inability to bind to DEAE, and its effect on actin assembly. When profilin was added to actin in the presence of Limulus bundles, addition of monomers on the nonpreferred end of the bundle was inhibited, even though actin by itself assembled on both ends. Using the Limulus bundles as nuclei, we determined the critical concentration for assembly off each end of the filament and estimated the Kd for the profilin-actin complex (approximately 10 microM). We present a model to explain how profilin may regulate the extension of the Thyone acrosomal process in vivo: The profilin-actin complex can add to only the preferred end of the filament bundle. Once the actin monomer is bound to the filament, the profilin is released, and is available to bind to additional actin monomers. This mechanism accounts for the rapid rate of filament elongation in the acrosomal process in vivo.     

Filament assembly from profilin-actin

Profilin plays a major role in the assembly of actin filament at the barbed ends. The thermodynamic and kinetic parameters for barbed end assembly from profilin-actin have been measured turbidimetrically. Filament growth from profilin-actin requires MgATP to be bound to actin. No assembly is observed from profilin-CaATP-actin. The rate constant for association of profilin-actin to barbed ends is 30% lower than that of actin, and the critical concentration for F-actin assembly from profilin-actin units is 0.3 microM under physiological ionic conditions. Barbed ends grow from profilin-actin with an ADP-Pi cap. Profilin does not cap the barbed ends and is not detectably incorporated into filaments. The EDC-cross-linked profilin-actin complex (PAcov) both copolymerizes with F-actin and undergoes spontaneous self-assembly, following a nucleation-growth process characterized by a critical concentration of 0.2 microM under physiological conditions. The PAcov polymer is a helical filament that displays the same diffraction pattern as F-actin, with layer lines at 6 and 36 nm. The PAcov filaments bound phalloidin with the same kinetics as F-actin, bound myosin subfragment-1, and supported actin-activated ATPase of myosin subfragment-1, but they did not translocate in vitro along myosin-coated glass surfaces. These results are discussed in light of the current models of actin structure.     

Non-muscle actin filament elongation from complexes of profilin with nucleotide-free actin and divalent cation-free ATP-actin

Using vertebrate cytoplasmic actin consisting of a mixture of beta and gamma isoforms, we previously characterized profilin and nucleotide binding to monomeric actin (Kinosian, H. J., et al. (2000) Biochemistry 39, 13176-13188) and F-actin barbed end elongation from profilin-actin (PA) (Kinosian, H. J., et al. (2002) Biochemistry 41, 6734-6743). Our initial calculations indicated that elongation of F-actin from PA was more energetically favorable than elongation of F-actin from monomeric actin; therefore, the overall actin elongation reaction scheme described by these two linked reactions appeared to be thermodynamically unbalanced. However, we hypothesized that the profilin-induced weakening of MgATP binding by actin reduces the negative free energy change for the formation of profilin-MgATP-actin from MgATP-actin. When this was taken into account, the overall reaction scheme was calculated to be thermodynamically balanced. In our present work, we test this hypothesis by measuring actin filament barbed end elongation of nucleotide-free actin (NF-A) and nucleotide-free profilin-actin (NF-PA). We find that the free energy change for elongation of F-actin by NF-PA is equal to that for elongation of F-actin from NF-A, indicating energetic balance of the linked reactions. In the absence of actin-bound divalent cation, profilin has very little effect on ATP binding to actin; analysis of elongation experiments with divalent cation-free ATP-actin and profilin yielded an approximately energetically balanced reaction scheme. Thus, the data in this present report support our earlier hypothesis.     

Structural basis for the recruitment of profilin-actin complexes during filament elongation by Ena/VASP

Cells sustain high rates of actin filament elongation by maintaining a large pool of actin monomers above the critical concentration for polymerization. Profilin-actin complexes constitute the largest fraction of polymerization-competent actin monomers. Filament elongation factors such as Ena/VASP and formin catalyze the transition of profilin-actin from the cellular pool onto the barbed end of growing filaments. The molecular bases of this process are poorly understood. Here we present structural and energetic evidence for two consecutive steps of the elongation mechanism: the recruitment of profilin-actin by the last poly-Pro segment of vasodilator-stimulated phosphoprotein (VASP) and the binding of profilin-actin simultaneously to this poly-Pro and to the G-actin-binding (GAB) domain of VASP. The actin monomer bound at the GAB domain is proposed to be in position to join the barbed end of the growing filament concurrently with the release of profilin.     

Compartmentalization and actin binding properties of ABP-50: the elongation factor-1 alpha of Dictyostelium.

ABP-50 is the elongation factor-1 alpha (EF-1 alpha) of Dictyostelium discoideum (Yang et al.: Nature 347:494-496, 1990). ABP-50 is also an actin filament binding and bundling protein (Demma et al.: J. Biol. Chem. 265:2286-2291, 1990). In the present study we have investigated the compartmentalization of ABP-50 in both resting and stimulated cells. Immunofluorescence microscopy shows that in addition to being colocalized with F-actin in surface extensions in unstimulated cells, ABP-50 exhibits a diffuse distribution throughout the cytosol. Upon addition of cAMP, a chemoattractant, ABP-50 becomes localized in the filopodia that are extended as a response to stimulation. Quantification of ABP-50 in Triton-insoluble and -soluble fractions of resting cells indicates that 10% of the total ABP-50 is recovered in the Triton cytoskeleton, while the remainder is in the soluble cytosolic fraction. Stimulation with cAMP increases the incorporation of ABP-50 into the Triton cytoskeleton. The peak of incorporation of ABP-50 at 90 sec is concomitant with filopod extension. Immunoprecipitation of the cytosolic ABP-50 from unstimulated cells using affinity-purified polyclonal anti ABP-50 results in the coprecipitation of non-filamentous actin with ABP-50. Purified ABP-50 binds to G-actin with a Kd of approximately 0.09 microM. The interaction between ABP-50 and G-actin is inhibited by GTP but not by GDP, while the bundling of F-actin by ABP-50 is unaffected by guanine nucleotides. We conclude that a significant amount of ABP-50 is bound to either G- or F-actin in vivo and that the interaction between ABP-50 and F-actin in the cytoskeleton is regulated by chemotactic stimulation.     

Actin filament barbed end elongation with nonmuscle MgATP-actin and MgADP-actin in the presence of profilin

We have quantitated the in vitro interactions of profilin and the profilin-actin complex (PA) with the actin filament barbed end using profilin and nonmuscle beta,gamma-actin prepared from bovine spleen. Actin filament barbed end elongation was initiated from spectrin seeds in the presence of varying profilin concentrations and followed by light scattering. We find that profilin inhibits actin polymerization and that this effect is much more pronounced for beta,gamma-actin than for alpha-skeletal muscle actin. Profilin binds to beta,gamma-actin filament barbed ends with an equilibrium constant of 20 microM, decreases the filament elongation rate by blocking addition of actin monomers, and increases the dissociation rate of actin monomers from the filament end. PA containing bound MgADP supports elongation of the actin filament barbed end, indicating that ATP hydrolysis is not necessary for PA elongation of filaments. Initial analysis of the energetics for these reactions suggested an apparent greater negative free energy change for actin filament elongation from PA than elongation from monomeric actin. However, we calculate that the free energy changes for the two elongation pathways are equal if the profilin-induced weakening of nucleotide binding to actin is taken into consideration.     

Formin differentially utilizes profilin isoforms to rapidly assemble actin filaments

Cells contain multiple formin isoforms that drive the assembly of profilin-actin for diverse processes. Given that many organisms also contain several profilin isoforms, specific formin/profilin pairs might be matched to optimally stimulate actin polymerization. We utilized a combination of bulk actin polymerization and single filament total internal reflection fluorescence microscopy assays to measure the effect of different profilin isoforms on the actin assembly properties of the cytokinesis formins from fission yeast (Cdc12p) and the nematode worm (CYK-1). We discovered that Cdc12p only effectively utilizes the single fission yeast profilin isoform SpPRF. Conversely, CYK-1 prefers the essential worm cytokinesis profilin CePFN-1 to the two non-essential worm profilin isoforms (SpPRF = CePFN-1 > CePFN-2 > CePFN-3). Chimeras containing the profilin-binding formin homology 1 (FH1) domain from one formin and the barbed-end associated FH2 domain from the other formin, revealed that both the FH1 and FH2 domains help confer profilin isoform specialization. Although the Cdc12p and CYK-1 FH1 domains cannot differentiate between profilin isoforms in the absence of actin, formin FH1 domains appear to preferentially select specific isoforms of profilin-actin. Surprisingly, analysis of profilin point mutants revealed that differences in highly conserved residues in both the poly-L-proline and actin binding regions of profilin do not explain their differential utilization by formin. Therefore, rapid formin-mediated elongation of profilin-actin depends upon favorable interactions of profilin-actin with the FH1 domain as well as the barbed-end associated FH2 domain. Specific formin FH1FH2 domains are tailored to optimally utilize actin bound to particular profilin isoforms.     

A cross-linked profilin-actin heterodimer interferes with elongation at the fast-growing end of F-actin

Profilin and beta/gamma-actin from calf thymus were covalently linked using the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide in combination with N-hydroxysuccinimide, yielding a single product with an apparent molecular mass of 60 kDa. Sequence analysis and x-ray crystallographic investigations showed that the cross-linked residues were glutamic acid 82 of profilin and lysine 113 of actin. The cross-linked complex was shown to bind with high affinity to deoxyribonuclease I and poly(l-proline). It also bound and exchanged ATP with kinetics close to that of unmodified profilin-actin and inhibited the intrinsic ATPase activity of actin. This inhibition occurred even in conditions where actin normally forms filaments. By these criteria the cross-linked profilin-actin complex retains the characteristics of unmodified profilin-actin. However, the cross-linked complex did not form filaments nor copolymerized with unmodified actin, but did interfere with elongation of actin filaments in a concentration-dependent manner. These results support a polymerization mechanism where the profilin-actin heterodimer binds to the (+)-end of actin filaments, followed by dissociation of profilin, and ATP hydrolysis and P(i) release from the actin subunit as it assumes its stable conformation in the helical filament.     

Analysis of the function of Spire in actin assembly and its synergy with formin and profilin

The Spire protein, together with the formin Cappuccino and profilin, plays an important role in actin-based processes that establish oocyte polarity. Spire contains a cluster of four actin-binding WH2 domains. It has been shown to nucleate actin filaments and was proposed to remain bound to their pointed ends. Here we show that the multifunctional character of the WH2 domains allows Spire to sequester four G-actin subunits binding cooperatively in a tight SA(4) complex and to nucleate, sever, and cap filaments at their barbed ends. Binding of Spire to barbed ends does not affect the thermodynamics of actin assembly at barbed ends but blocks barbed end growth from profilin-actin. The resulting Spire-induced increase in profilin-actin concentration enhances processive filament assembly by formin. The synergy between Spire and formin is reconstituted in an in vitro motility assay, which provides a functional basis for the genetic interplay between Spire, formin, and profilin in oogenesis.     

Ena/VASP proteins capture actin filament barbed ends

Ena/VASP (vasodialator-stimulated protein) proteins regulate many actin-dependent events, including formation of protrusive structures, fibroblast migration, neurite extension, cell-cell adhesion, and Listeria pathogenesis. In vitro, Ena/VASP activities on actin are complex and varied. They promote actin assembly, protect filaments from cappers, bundle filaments, and inhibit filament branching. To determine the mechanisms by which Ena/VASP proteins regulate actin dynamics at barbed ends, we monitored individual actin filaments growing in the presence of VASP and profilin using total internal reflection fluorescence microscopy. Filament growth was unchanged by VASP, but filaments grew faster in profilin-actin and VASP than with profilin-actin alone. Actin filaments were captured directly by VASP-coated surfaces via interactions with growing barbed ends. End-attached filaments transiently paused but resumed growth after becoming bound to the surface via a filament side attachment. Thus, Ena/VASP proteins promote actin assembly by interacting directly with actin filament barbed ends, recruiting profilin-actin, and blocking capping.     

Reinvestigation of the inhibition of actin polymerization by profilin

In buffer containing 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 5 mM imidazole, pH 7.5, 0.1 mM CaCl2, 0.2 mM dithiothreitol, 0.01% NaN3, and 0.2 mM ATP, the KD for the formation of the 1:1 complex between Acanthamoeba actin and Acanthamoeba profilin was about 5 microM. When the actin was modified by addition of a pyrenyl group to cysteine 374, the KD increased to about 40 microM but the critical concentration (0.16 microM) was unchanged. The very much lower affinity of profilin for modified actin explains the anomalous critical concentrations curves obtained for 5-10% pyrenyl-labeled actin in the presence of profilin and the apparently weak inhibition by profilin of the rate of filament elongation when polymerization is quantified by the increase in fluorescence of pyrenyl-labeled actin. Light-scattering assays of the polymerization of unmodified actin in the absence and presence of profilin gave a similar value for the KD (about 5-10 microM) when determined by the increase in the apparent critical concentration of F-actin at steady state at all concentrations of actin up to 20 microM and by the inhibition of the initial rates of polymerization of actin nucleated by either F-actin or covalently cross-linked actin dimer. In the same buffer, but with ADP instead of ATP, the critical concentration of actin was higher (4.9 microM) and the KD of the profilin-actin complex was lower for both unmodified (1-2 microM) and 100% pyrenyl-labeled actin (4.9 microM).     

Model of formin-associated actin filament elongation

Formin FH2 domains associate processively with actin-filament barbed ends and modify their rate of growth. We modeled how the elongation rate depends on the concentrations of profilin and actin for four different formins. We assume that (1) FH2 domains are in rapid equilibrium among conformations that block or allow actin addition and that (2) profilin-actin is transferred rapidly to the barbed end from multiple profilin binding sites in formin FH1 domains. In agreement with previous experiments discussed below, we find an optimal profilin concentration with a maximal elongation rate that can exceed the rate of actin alone. High profilin concentrations suppress elongation, largely because free profilin displaces profilin-actin from FH1. The model supports a common polymerization mechanism for the four formin FH1FH2 constructs with differences attributed to varying parameter values. The mechanism does not require ATP hydrolysis by polymerized actin, but we cannot exclude that formins accelerate hydrolysis.     

Effects of macrophage profilin on actin in the presence and absence of acumentin and gelsolin

We purified profilin from rabbit alveolar macrophages and documented its structural and functional similarity to profilins isolated from other cells. The KD for formation of the macrophage profilin-actin complex was 3.0 +/- 0.8 microM (mean +/- S.D.). The affinity of this protein for actin did not change significantly in the presence of various concentrations of KCl and MgCl2, profilin-actin complex concentration being strictly dependent on the critical actin monomer concentration and free profilin concentration. We also examined profilin's interactions with actin in the presence of acumentin, a macrophage protein which inhibits actin monomer exchange at the "pointed" ends of actin filaments. Low concentrations of this protein caused substantial decreases in estimated profilin-actin complex concentration. The macrophage gelsolincalcium ion complex which blocks exchange at the "barbed" end of actin filaments, when added to profilin and actin solutions in substoichiometric concentrations, caused large increases in estimated profilin-actin complex concentration. The changes in calculated profilin-actin complex concentration induced by these two actin-modulating proteins were too large to be explained solely by their effects on critical actin monomer concentration.     

Quantitative analysis of the effect of Acanthamoeba profilin on actin filament nucleation and elongation

The current view of the mechanism of action of Acanthamoeba profilin is that it binds to actin monomers, forming a complex that cannot polymerize [Tobacman, L. S., & Korn, E. D. (1982) J. Biol. Chem. 257, 4166-4170; Tseng, P., & Pollard, T. D. (1982) J. Cell Biol. 94, 213-218; Tobacman, L. S., Brenner, S. L., & Korn, E. D. (1983) J. Biol. Chem. 258, 8806-8812]. This simple model fails to predict two new experimental observations made with Acanthamoeba actin in 50 mM KC1, 1 mM MgCl2, and 1 mM EGTA. First, Acanthamoeba profilin inhibits elongation of actin filaments far more at the pointed end than at the barbed end. According, to the simple model, the Kd for the profilin-actin complex is less than 5 microM on the basis of observations at the pointed end and greater than 50 microM for the barbed end. Second, profilin inhibits nucleation more strongly than elongation. According to the simple model, the Kd for the profilin-actin complex is 60-140 microM on the basis of two assays of elongation but 2-10 microM on the basis of polymerization kinetics that reflect nucleation. These new findings can be explained by a new and more complex model for the mechanism of action that is related to a proposal of Tilney and co-workers [Tilney, L. G., Bonder, E. M., Coluccio, L. M., & Mooseker, M. S. (1983) J. Cell Biol. 97, 113-124]. In this model, profilin can bind both to actin monomers with a Kd of about 5 microM and to the barbed end of actin filaments with a Kd of about 50-100 microM. An actin monomer bound to profilin cannot participate in nucleation or add to the pointed end of an actin filament. It can add to the barbed end of a filament. When profilin is bound to the barbed end of a filament, actin monomers cannot bind to that end, but the terminal actin protomer can dissociate at the usual rate. This model includes two different Kd's--one for profilin bound to actin monomers and one for profilin bound to an actin molecule at the barbed end of a filament. The affinity for the end of the filament is lower by a factor of 10 than the affinity for the monomer, presumably due to the difference in the conformation of the two forms of actin or to steric constraints at the end of the filament.     

Stimulation of human polymorphonuclear leukocyte phosphatidylinositol-4-phosphate kinase by concanavalin A and formyl-methionyl-leucyl-phenylalanine is calcium-independent. Correlation with maintenance of actin assembly.

Chemoattractants directly stimulate the enzyme activity that synthesizes phosphatidylinositol-4,5-bisphosphate (PIP2), phosphoinositol-4-monophosphate (PIP) kinase. The present study determined whether stimulation of this enzyme correlates with actin assembly by assessing the calcium dependence of this reaction. Incubation of neutrophils with 5 to 100 micrograms/ml Con A caused a concentration-dependent increase in PIP kinase activity ranging from 1.38- to 3.4-fold. The effective concentration which stimulated PIP kinase by 50% (17 micrograms/ml, EC50) corresponded with the EC50 for Con A-induced superoxide production (32 micrograms/ml). Like chemoattractants, the increase in PIP kinase by Con A was characterized by a 2.6-fold increase in the maximum velocity (Vmax) of the enzyme, and no change in the Km for ATP. The kinetics of FMLP- and Con A-induced filamentous actin formation preceded stimulation of PIP kinase and was sustained over the same time period that this increased enzyme activity was noted. Although transmembrane signaling by FMLP and Con A requires an increase in intracellular calcium for some polymorphonuclear leukocyte (PMN) functional responses, calcium depletion of PMN by incubation with 100 microM Quin 2 A/M and 5 mM EGTA did not prevent the stimulation of PIP kinase by FMLP or Con A. In addition, calcium depletion did not prevent the increase in filamentous actin formation by FMLP and Con A in PMN. These findings demonstrate that Con A increases PIP kinase activity in human PMN and that PIP kinase stimulation and maintenance of actin assembly are independent of calcium fluxes in these cells. Because PIP2 controls the function of the actin-regulatory proteins, profilin and gelsolin, changes in the synthetic rate of PIP2 through regulation of PIP kinase may provide a molecular basis for the prolonged stimulation of actin assembly in human PMN by agonists such as Con A and FMLP.