Computational prediction of actin–actin interaction

Actin is one of the most abundant proteins in eukaryotic cells, where it plays key roles in cell shape, motility, and regulation. Actin is found in globular (G) and filamentous (F) structure in the cell. The helix of actin occurs as a result of polymerization of monomeric G-actin molecules through sequential rowing, is called F-actin. Recently, the crystal structure of an actin dimer has been reported, which details molecular interface in F-actin. In this study, the computational prediction model of actin and actin complex has been constructed base on the atomic model structure of G-actin. To this end, a docking simulation was carried out using predictive docking tools to obtain modeled structures of the actin–actin complex. Following molecular dynamics refinement, hot spots interactions at the protein interface were identified, that were predicted to contribute substantially to the free energy of binding. These provided a detailed prediction of key amino acid interactions at the protein–protein interface. The obtained model can be used for future experimental and computational studies to draw biological and functional conclusions. Also, the identified interactions will be used for designing next studies to understand the occurrence of F-actin structure.     

Adsorption of actin at the air-water interface: a monolayer study

The intrinsic surface activity of the contractile protein actin has been determined from surface tension measurements using the Wilhelmy hanging-plate method. Actin, a very soluble protein, moves from the subphase to the air-water interface to make a film. In the absence of magnesium, actin is monomeric and is known as G-actin. During the compression the monomers change their conformation or orientation at the interface and they are then pushed reversibly into the subphase upon further compression. No collapse occurs. Actin monomers in the presence of magnesium become activated; at concentrations greater than some critical value, actin polymerizes to form filaments of F-actin. The actin filaments have a higher surface activity than the actin monomers either because they are more hydrophobic or because F-actin, a rigid polymer, is much more efficient at creating excluded volume. The actin filaments then form a rigid film at the interface that collapses when the surface area is decreased. At less than the critical concentration, the actin monomers are present in the subphase in their activated form. However, their concentration increases at the interface during film compression until the critical concentration is reached. The surface pressure isotherm in this case has the characteristics of a G-actin film at the beginning of the compression and of an F-actin film at the end of the compression process.     

The cytotoxic effect of diphtheria toxin on the actin cytoskeleton

Diphtheria toxin (DT) and its N-terminal fragment A (FA) catalyse the transfer of the ADP-ribose moiety of nicotinamide adenine dinucleotide (NAD) into a covalent linkage with eukaryotic elongation factor 2 (eEF2). DT-induced cytotoxicity is versatile, and it includes DNA cleavage and the depolymerisation of actin filaments. The inhibition of the ADP-ribosyltransferase (ADPrT) activity of FA did not affect the deoxyribonuclease activity of FA or its interaction with actin. The toxin entry rate into cells (HUVEC) was determined by measuring the ADP-ribosyltransferase activity. DT uptake was nearly 80% after 30 min. The efficiency was determined as K_m = 2.2 nM; V_max = 0.25 pmol.min⁻¹. The nuclease activity was tested with hyperchromicity experiments, and it was concluded that G-actin has an inhibitory effect on DT nuclease activity. In thepresence of DT and mutant of diphtheria toxin (CRM197), F-actin depolymerisation was determined with gel filtration, WB and fluorescence techniques. In the presence of DT and CRM197, 60–65% F-actin depolymerisation was observed. An in vitro FA-actin interaction and F-actin depolymerisation were reported in our previous paper. The present study thus confirms the depolymerisation of actin cytoskeleton in vivo.     

The quantitation of G- and F-actin in cultured cells

An improved method to quantitate the amounts of filamentous (F-actin) and monomeric (globular) actin (G-actin) in cultured cells was developed. Cells are lysed into a myosin-containing buffer and F-actin is removed by centrifugation. The pelleted F-actin is then depolymerized to G-actin in a 1 mM ATP-containing buffer for 1 h before measuring the levels of G-actin using the DNase I inhibition assay. Partitioning of G-actin in the supernatant (greater than 95%) and recovery of actin in both fractions (greater than 85%) were measured by adding [3H]actin to cultured cells. Actin in the separated fractions is stable for at least 72 h at 0 degree C. Asynchronous monolayer cultures of Chinese hamster ovary (CHO) cells contain 2.5 +/- 0.2% of the total protein as actin with 72.4 +/- 5.7% as F-actin. About 10% of this F-actin is not associated with the readily sedimented Triton-cytoskeleton. CHO cells grown in suspension contain 55.8% of the actin as F-actin; following plating about 90 min is required for these cells to flatten and for the F-actin level to reach the monolayer value of about 70%.     

Germinal center-specific protein human germinal center associated lymphoma directly interacts with both myosin and actin and increases the binding of myosin to actin

Human germinal center associated lymphoma (HGAL) is a germinal center-specific gene whose expression correlates with a favorable prognosis in patients with diffuse large B-cell and classic Hodgkin lymphomas. HGAL is involved in negative regulation of lymphocyte motility. The movement of lymphocytes is directly driven by actin polymerization and actin-myosin interactions. We demonstrate that HGAL interacts directly and independently with both actin and myosin and delineate the HGAL and myosin domains responsible for the interaction. Furthermore, we show that HGAL increases the binding of myosin to F-actin and inhibits the ability of myosin to translocate actin by reducing the maximal velocity of myosin head/actin movement. No effects of HGAL on actomyosin ATPase activity and the rate of actin polymerization from G-actin to F-actin were observed. These findings reveal a new mechanism underlying the inhibitory effects of germinal center-specific HGAL protein on lymphocyte and lymphoma cell motility.     

Distribution of G-actin is related to root hair growth of wheat

BACKGROUND AND AIMS: Actin distribution in root hair tips is a controversial topic. Although the relationship between Ca2+ gradient and actin dynamics in plant tip-growth has been a focus of study, there is still little direct evidence on the exact relationship in root hair tip-growth. METHODS: G-actin was labelled by fluorescein isothiocyanate-DNase I. F-actin was labelled by tetramethylrhodamine isothiocyanate-phalloidin. Actin in root hairs of Triticum aestivum (wheat) was investigated using confocal laser-scanning microscopy. KEY RESULTS: Thick F-actin bundles did not extend into a region of approx. 5-10 microm from the tip of the growing root hairs, although they gave off branches of fine actin filaments in the hair tips. A tip-focused G-actin gradient was shown at the extreme apex of growing root hairs. In full-grown wheat root hairs, the tip-focused G-actin gradient disappeared while the thick F-actin bundles extended into the tips. BAPTA-AM, a Ca2+ disruption agent, also caused the tip-focused G-actin gradient to disappear and the diffuse F-actin bundles to appear in the tips of wheat root hairs. CONCLUSIONS: These results suggest that the tip-focused gradient of intracellular G-actin concentration at the extreme apex may be essential for root hair growth, and that preserving the tip-focused gradient needs a high Ca2+ concentration in the root hair tips.     

Botulinum C2 toxin ADP-ribosylates actin and disorganizes the microfilament network in intact cells

Botulinum C2 toxin ADP-ribosylates actin in [32P]orthophosphate-labelled intact chick embryo cells (CEC). The toxin-induced rounding up of CEC is correlated with ADP-ribosylation of actin in intact cells in a time and concentration-dependent manner. Both, rounding up of cells and actin ADP-ribosylation, depend on the presence of both components of botulinum C2 toxin (components I and II) and are independent of the ability of CEC to divide. Treatment of CEC with botulinum C2 toxin induced a time-dependent disorganization of the typical architecture of the microfilament network as shown by fluorescein-phalloidin staining. Botulinum C2 toxin decreased the amount of Triton X-100 insoluble actin, while the fraction of Triton soluble actin was increased. Actin, which was 32P-labelled by botulinum C2 toxin in intact CEC, was recovered in the Triton soluble but not in the Triton insoluble actin fraction. It is suggested that in intact CEC botulinum C2 toxin causes ADP-ribosylation of G-actin but not of F-actin thereby leading to an accumulation in the pool of monomeric actin.     

Toxofilin, a novel actin-binding protein from Toxoplasma gondii, sequesters actin monomers and caps actin filaments

Toxoplasma gondii relies on its actin cytoskeleton to glide and enter its host cell. However, T. gondii tachyzoites are known to display a strikingly low amount of actin filaments, which suggests that sequestration of actin monomers could play a key role in parasite actin dynamics. We isolated a 27-kDa tachyzoite protein on the basis of its ability to bind muscle G-actin and demonstrated that it interacts with parasite G-actin. Cloning and sequence analysis of the gene coding for this protein, which we named Toxofilin, showed that it is a novel actin-binding protein. In in vitro assays, Toxofilin not only bound to G-actin and inhibited actin polymerization as an actin-sequestering protein but also slowed down F-actin disassembly through a filament end capping activity. In addition, when green fluorescent protein-tagged Toxofilin was overexpressed in mammalian nonmuscle cells, the dynamics of actin stress fibers was drastically impaired, whereas green fluorescent protein-Toxofilin copurified with G-actin. Finally, in motile parasites, during gliding or host cell entry, Toxofilin was localized in the entire cytoplasm, including the rear end of the parasite, whereas in intracellular tachyzoites, especially before they exit from the parasitophorous vacuole of their host cell, Toxofilin was found to be restricted to the apical end.     

Polymerization of nonfilamentous actin into microfilaments is an important process for porcine oocyte maturation and early embryo development

Actin is one of the major proteins in mammalian oocytes. Most developmental events are dependent on the normal distribution of filamentous (F-) actin. Polymerization of nonfilamentous (G-) actin into F-actin is important for both meiosis and mitosis. This study examined G- and F-actin distribution in pig oocytes and embryos by immunocytochemical staining and confocal microscopy. Actin protein was quantified by electrophoresis and immunoblotting. G-Actin was distributed in the whole cytoplasm of oocytes and embryos irrespective of their stages. F-Actin was distributed at the cortex of oocytes and embryos at all stages, at the joint of blastomeres in the embryos, in the cytoplasm around the germinal vesicle (GV), and in the perinuclear area of 2- to 4-cell-stage embryos. No differences in the amount of actin protein were found among oocytes and embryos. Oocytes cultured in medium with cytochalasin D (CD), an inhibitor of microfilament polymerization, underwent GV breakdown and reached metaphase I but did not proceed to metaphase II. Two- to 4-cell-stage embryos cultured in medium with CD did not develop to blastocysts. When GV-stage oocytes or 2- to 4-cell-stage embryos treated with CD for 6 h were re-cultured in media without CD, oocytes or embryos re-assembled actin filaments and underwent a meiotic maturation or blastocyst formation similar to that of controls. These results indicate that it is the polymerization of G-actin into F-actin, not actin protein synthesis, that is important for both meiosis and mitosis in pig oocytes and embryos.     

Identification of actin as a 15-deoxy-Delta12,14-prostaglandin J2 target in neuroblastoma cells: mass spectrometric, computational, and functional approaches to investigate the effect on cytoskeletal derangement

A proteomic approach was used to identify 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) protein targets in human neuroblastoma SH-SY5Y cells. By using biotinylated 15d-PGJ2, beta-actin was found as the major adducted protein; at least 12 proteins were also identified as minor biotin-positive spots, falling in different functional classes, including glycolytic enzymes (enolase and lactate dehydrogenase), redox enzymes (biliverdin reductase), and a eukaryotic regulatory protein (14-3-3gamma). 15d-PGJ2 induced marked morphological changes in the actin filament network and in particular promoted F-actin depolymerization as confirmed by Western blot analysis. By using a mass spectrometric approach, we found that 15d-PGJ2 reacts with isolated G-actin in a 1:1 stoichiometric ratio and selectively binds the Cys374 site through a Michael adduction mechanism. Computational studies showed that the covalent binding of 15d-PGJ2 induces a significant unfolding of actin structure and in particular that 15d-PGJ2 distorts the actin subdomains 2 and 4, which define the nucleotide binding sites impeding the nucleotide exchange. The functional effect of 15d-PGJ2 on G-actin was studied by polymerization measurement: in the presence of 15d-PGJ2, a lower amount of F-actin forms, as followed by the increase in pyrenyl-actin fluorescence intensity, as the major effect of increasing 15d-PGJ2 concentrations occurs on the maximum extent of actin polymerization, whereas it is negligible on the initial rate of reaction. In summary, the results here reported give an insight into the role of 15d-PGJ2 as a cytotoxic compound in neuronal cell dysfunction. Actin is the main protein cellular target of 15d-PGJ2, which specifically binds through a Michael adduction to Cys374, leading to a protein conformational change that can explain the disruption of the actin cytoskeleton, F-actin depolymerization, and impairment of G-actin polymerization.     

Influence of botulinum C2 toxin on F-actin and N-formyl peptide receptor dynamics in human neutrophils

Stimulation of human neutrophils with the chemotactic N-formyl peptide causes production of oxygen radicals and conversion of monomeric actin (G-actin) to polymeric actin (F-actin). The effects of the binary botulinum C2 toxin on the amount of F-actin and on neutrophil cell responses were studied. Two different methods for analyzing the actin response were used in formyl peptide-stimulated cells: staining of F-actin with rhodamine-phalloidin and a transient right angle light scatter. Preincubation of neutrophils with 400 ng/ml component I and 1,600 ng/ml component II of botulinum C2 toxin for 30 min almost completely inhibited the formyl peptide-stimulated polymerization of G-actin and at the same time decreased the amount of F-actin in unstimulated neutrophils by an average of approximately 30%. Botulinum C2 toxin preincubation for 60 min destroyed approximately 75% of the F-actin in unstimulated neutrophils. Right angle light scatter analysis showed that control neutrophils exhibited the transient response characteristic of actin polymerization; however, after botulinum C2 toxin treatment, degranulation was detected. Single components of the binary botulinum C2 toxin were without effect on the actin polymerization response. Fluorescence flow cytometry and fluorospectrometric binding studies showed little alteration in N-formyl peptide binding or dissociation dynamics in the toxin-treated cells. However, endocytosis of the fluorescent N-formyl peptide ligand-receptor complex was slower but still possible in degranulating neutrophils treated with botulinum C2 toxin for 60 min. The half-time of endocytosis, estimated from initial rates, was 4 and 8 min in control and botulinum C2 toxin-treated neutrophils, respectively.     

Inhibition of sliding movement of F-actin by crosslinking emphasizes the role of actin structure in the mechanism of motility

The effects of crosslinking of monomeric and polymeric actin with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), disuccinimidyl suberate (DSS) and glutaraldehyde on the interaction with heavy meromyosin (HMM) in solution and on the sliding movement on glass-attached HMM were examined. The Vmax values of actin-activated HMM ATPase decreased in the following order: intact actin = EDC F-actin greater than DSS actin greater than glutaraldehyde F-actin = glutaraldehyde G-actin greater than EDC G-actin. The affinity of actin for HMM in the presence of ATP decreased in the following order: DSS actin greater than glutaraldehyde F-actin = glutaraldehyde G-actin greater than intact actin greater than EDC F-actin greater than EDC G-actin. However, sliding movement was inhibited only in the case of glutaraldehyde-crosslinked F and G-actin and EDC-crosslinked G-actin. Interestingly, after copolymerization of "non-motile" glutaraldehyde or EDC-crosslinked monomers with "motile" monomers of intact actin sliding of the copolymers was observed and its rate was independent of the type of crosslinked monomer, i.e. of the manner of their interaction with HMM. These data strongly indicate that inhibition of the sliding of actin by crosslinking cannot be explained entirely by changes in the Vmax value or affinity for myosin heads. We conclude that movement is generated by interaction of myosin with segments of F-actin containing a number of intact monomers, and the mechanism of inhibition involves an effect of the crosslinkers on the structure of F-actin itself.     

Tracking down the different forms of nuclear actin

Actin is a rather uncommitted protein with a high degree of structural plasticity: it can adopt a variety of structural states, depending on the specific ionic conditions or the interaction with ligand proteins. These interactions lock actin into a distinct conformation, which specifies the oligomeric or polymeric form it can assume. The interplay between monomeric, oligomeric and polymeric forms is used by the cell to execute an enormous variety of motility processes, such as lamellipodium formation during locomotion or intracellular transport of vesicles. In these cytoplasmic events, monomeric G-actin and filamentous F-actin are the prevalent forms. However, there might be other structural states of actin in cells that have so far not received the attention they deserve. Here, we propose that specific, "unconventional" actin conformations might contribute especially to the multitude of functions executed by actin in the nucleus. We present evidence for the existence of different forms of nuclear actin, taken from studies with selected antibodies.     

Binding of human angiogenin inhibits actin polymerization

Angiogenin is a potent inducer of angiogenesis, a process of blood vessel formation. It interacts with endothelial and other cells and elicits a wide range of cellular responses including migration, proliferation, and tube formation. One important target of angiogenin is endothelial cell-surface actin and their interaction might be one of essential steps in angiogenin-induced neovascularization. Based on earlier indications that angiogenin promotes actin polymerization, we studied the binding interactions between angiogenin and actin in a wide range of conditions. We showed that at subphysiological KCl concentrations, angiogenin does not promote, but instead inhibits polymerization by sequestering G-actin. At low KCl concentrations angiogenin induces formation of unstructured aggregates, which, as shown by NMR, may be caused by angiogenin’s propensity to form oligomers. Binding of angiogenin to preformed F-actin does not cause depolymerization of actin filaments though it causes their stiffening. Binding of tropomyosin and angiogenin to F-actin is not competitive at concentrations sufficient for saturation of actin filaments. These observations suggest that angiogenin may cause changes in the cell cytoskeleton by inhibiting polymerization of G-actin and changing the physical properties of F-actin.     

Activation of neutrophils: measurement of actin conformational changes by flow cytometry

Actin, which comprises approximately 10% of the weight of cytoplasmic protein of neutrophils, is the principal component of the cytoplasmic microfilament lattice. It can exist in either of two physical states, G-actin, which is monomeric, or F-actin, which is polymeric or filamentous. Actin microfilaments support many forms of cell movement. Continuous remodeling of the microfilament lattice, which seems integral to sustained movement, is possible in part because of the ability of actin to change rapidly between its monomeric G-state and its filamentous F-state. Changes in the G- and F-actin equilibrium may be studied by flow analysis using a fluorescent probe which is specific for F-actin, 7-nitrobenz-2-oxa-1,3-diazole-(NBD)-phallacidin. Alterations in neutrophil F-actin have been measured in response to chemotactic agents (e.g., formyl peptides and leukotriene B4), inhibitors of cell movement (e.g., N-ethylmaleimide and cytochalasin B), agents that promote the oxidative burst (e.g., formyl peptides and phorbol esters), and priming agents [e.g., tumor necrosis factor (TNF)]. Measurements may be taken at intervals of a few seconds, allowing comparison of rapid changes in the F-actin content to other rapidly occurring changes, such as altered membrane ion permeability and activation of cellular enzymes. The use of metabolic inhibitors has allowed dissection of some of the biochemical pathways involved in actin assembly in living cells. Although clinical studies are few thus far, the technique has also been used to study basal and stimulated F-actin levels in circulating neutrophils in neonates and in family members of patients with neutrophil-actin dysfunction.     

Differences in the ionic interaction of actin with the motor domains of nonmuscle and muscle myosin II.

Changes in the actin-myosin interface are thought to play an important role in microfilament-linked cellular movements. In this study, we compared the actin binding properties of the motor domain of Dictyostelium discoideum (M765) and rabbit skeletal muscle myosin subfragment-1 (S1). The Dictyostelium motor domain resembles S1(A2) (S1 carrying the A2 light chain) in its interaction with G-actin. Similar to S1(A2), none of the Dictyostelium motor domain constructs induced G-actin polymerization. The affinity of monomeric actin (G-actin) was 20-fold lower for M765 than for S1(A2) but increasing the number of positive charges in the loop 2 region of the D. discoideum motor domain (residues 613-623) resulted in equivalent affinities of G-actin for M765 and for S1. Proteolytic cleavage and cross-linking approaches were used to show that M765, like S1, interacts via the loop 2 region with filamentous actin (F-actin). For both types of myosin, F-actin prevents trypsin cleavage in the loop 2 region and F-actin segment 1-28 can be cross-linked to loop 2 residues by a carbodiimide-induced reaction. In contrast with the S1, loop residues 559-565 of D. discoideum myosin was not cross-linked to F-actin, probably due to the lower number of positive charges. These results confirm the importance of the loop 2 region of myosin for the interaction with both G-actin and F-actin, regardless of the source of myosin. The differences observed in the way in which M765 and S1 interact with actin may be linked to more general differences in the structure of the actomyosin interface of muscle and nonmuscle myosins.     

Effect of Hypothyroidism on Different Forms of Actin in Rat Cerebral Neuronal Cultures Studied by an Improved DNase I Inhibition Assay

An improved DNase I inhibition assay for the filamentous actin (F-actin) and monomeric actin (G-actin) in brain cells has been developed. Unlike other methods, the cell lysis conditions and postlysis treatments, established by us, inhibited the temporal inactivation of actin in the cell lysate and maintained a stable F-actin/G-actin ratio for at least 4-5 h after lysis. The new procedure allowed separate quantitation of the noncytoskeletal F-actin in the Triton-soluble fraction (12,000 g, 10 min supernatant) that did not readily sediment with the Triton-insoluble cytoskeletal F-actin (12,000 g, 10 min pellet). We have applied this modified assay system to study the effect of hypothyroidism on different forms of actin using primary cultures of neurons derived from cerebra of neonatal normal and hypothyroid rats. Our results showed a 20% increase in the Triton-insoluble cytoskeletal F-actin in cultures from hypothyroid brain relative to normal controls. In the Triton-soluble fraction, containing the G-actin and the noncytoskeletal F-actin, cultures from hypothyroid brain showed a 15% increase in G-actin, whereas the F-actin remained unaltered. The 10% increase in total actin observed in this fraction from hypothyroid brain could be totally accounted for by the enhancement of G-actin. The mean F-actin/G-actin ratio in this fraction was about 30% higher in the cultures from normal brain compared to that of the hypothyroid system, which indicates that hypothyroidism tends to decrease the proportion of noncytoskeletal F-actin relative to G-actin.     

Visualization of F-actin and G-actin equilibrium using fluorescence resonance energy transfer (FRET) in cultured cells and neurons in slices

The plasticity of excitatory synapses has conventionally been studied from a functional perspective. Recent advances in neuronal imaging techniques have made it possible to study another aspect, the plasticity of the synaptic structure. This takes place at the dendritic spines, where most excitatory synapses are located. Actin is the most abundant cytoskeletal component in dendritic spines, and thus the most plausible site of regulation. The mechanism by which actin is regulated has not been characterized because of the lack of a specific method for detection of the polymerization status of actin in such a small subcellular structure. Here we describe an optical approach that allows us to monitor F-actin and G-actin equilibrium in living cells through the use of two-photon microscopy to observe fluorescence resonance energy transfer (FRET) between actin monomers. Our protocol provides the first direct method for looking at the dynamic equilibrium between F-actin and G-actin in intact cells.     

The role of the cytosolic free Ca2+ transient for fMet-Leu-Phe induced actin polymerization in human neutrophils

We have addressed the important question as to if and how the cytosolic free Ca2+ concentration, [Ca2+]i, is involved in fMet-Leu-Phe induced actin polymerization in human neutrophils. Stimulation of human neutrophils with the chemotactic peptide (10(-7) M), known to result in a prompt rise of the [Ca2+]i to above 500 nM, also induced a rapid decrease of monomeric actin, G-actin, content (to 35% of basal) and increase of filamentous actin, F-actin, content (to 320% of basal). A reduction of the fMet-Leu-Phe induced [Ca2+]i transient to about 250 nM, resulted in a less pronounced decrease of G-actin content (to 80% of basal) and increase of F-actin content (to 235% of basal). A total abolishment of the chemotactic peptide induced [Ca2+]i rise, still led to a decrease of the G-actin content (to 85% of basal) and increase of F-actin (to 200% of basal). These results indicate that the [Ca2+]i rise is not an absolute requirement, but has a modulating role for the fMet-Leu-Phe induced actin polymerization. Another possible intracellular candidate for fMet-Leu-Phe induced actin polymerization is protein kinase C. However, direct activation of protein kinase C by phorbol 12-myristate 13-acetate (PMA) only resulted in a minor increase of F-actin content. The recent hypothesis that a metabolite of the polyphosphoinositide cycle, independently of [Ca2+]i and protein kinase C, is responsible for actin polymerization agrees well with these results and by the fact that preexposure to pertussis toxin totally abolished a subsequent increase of F-actin content induced by fMet-Leu-Phe.     

Phosphorylation of Amoeba G-actin and its effect on actin polymerization

Mass culture of Amoeba proteus enabled us to do biochemical studies on this organism. Actin and profilin were purified from Amoeba to examine actin phosphorylation and polymerization. The apparent molecular weight of Amoeba actin was 44,000, and its isoelectric point was 5.8. The apparent molecular weight of Amoeba profilin was 12,000, and its isoelectric point was 4.9. It reduced the rate of actin polymerization as reported in the cases of profilins from other organisms. A protein of Mr = 44,000 (44 K protein) was phosphorylated in a Ca2+-dependent manner in cell homogenate of Amoeba without being inhibited by calmodulin antagonists. Using the homogenate as a kinase, purified Amoeba G-actin could be phosphorylated in proportion to the amount of actin. However, neither Amoeba F-actin nor rabbit skeletal muscle G-actin was phosphorylated. The phosphorylation of Amoeba actin with a kinase partially purified from A. proteus increased with dilution of the actin concentration. When Amoeba profilin was added, more than 80% of the actin was phosphorylated. By viscometry, electron microscopy, and ultracentrifugation analysis it was demonstrated that Amoeba G-actin phosphorylated in the presence of profilin and kinase did not polymerize in this solution. High-performance liquid chromatography analysis showed that phosphorylated Amoeba actin remained in a monomeric state even under conditions favorable for actin polymerization.