Differential modulation of actin-severing activity of gelsolin by multiple isoforms of cultured rat cell tropomyosin. Potentiation of protective ability of tropomyosins by 83-kDa nonmuscle caldesmon

Multiple isoforms of tropomyosin (TM) of rat cultured cells show differential effects on actin-severing activity of gelsolin. Flow birefringence measurements have revealed that tropomyosin isoforms with high Mr values (high Mr TMs) partially protect actin filaments from fragmentation by gelsolin, while tropomyosins with low Mr values (low Mr TMs) have no significant protection even when the actin filaments have been fully saturated with low Mr TMs. We have also examined effect of nonmuscle caldesmon on the severing activity of gelsolin because 83-kDa nonmuscle caldesmon stimulates actin binding of rat cell TMs (Yamashiro-Matsumura, S., and Matsumura, F. (1988) J. Cell Biol. 106, 1973-1983). While nonmuscle caldesmon alone or low Mr TMs alone show no significant protection against fragmentation by gelsolin, the low Mr TMs coupled with 83-kDa protein are able to protect actin filaments. Further, high Mr TMs together with 83-kDa protein appear to block the severing activity completely. Electron microscopic analyses of length distribution of actin filaments have confirmed the results. The average length of control actin filaments is measured as 1.46 +/- microns, and gelsolin shortens the average length to 0.084 +/- 0.039 micron. Similar short average lengths are obtained when gelsolin severs actin complexed with low Mr TMs (0.080 +/- 0.045 micron) or with nonmuscle caldesmon (0.11 +/- 0.072 micron) while longer average length (0.22 +/- 0.18 micron) is measured in the presence of high Mr TMs. The simultaneous addition of nonmuscle caldesmon makes the average length considerably longer, i.e. 0.61 +/- 0.37 micron in the presence of low Mr TMs and 1.57 +/- 0.97 micron in the presence of high Mr TMs. Furthermore, the actin binding of gelsolin is strongly inhibited by co-addition of high Mr TMs and nonmuscle caldesmon. These results suggest that TM and gelsolin share the same binding site on actin molecules and that the differences in the actin affinities between TMs are related to their abilities of protection against gelsolin.     

The production of 53-55-kDa isoforms is not required for rat L-histidine decarboxylase activity

Post-translational processing of the histamine-producing enzyme, L-histidine decarboxylase (HDC), leads to the formation of multiple carboxyl-truncated isoforms. Nevertheless, it has been widely reported that the mature catalytically active dimer is dependent specifically on the production of carboxyl-truncated 53-55-kDa monomers. Here we use transiently transfected COS-7 cells to study the properties of carboxyl-truncated rat HDC isoforms in the 52-58-kDa size range. Amino acid sequences important for the production of a 55-kDa HDC isoform were identified by successive truncations through amino acids 502, 503, and 504. Mutating this sequence in the full-length protein prevented the production of 55-kDa HDC but did not affect enzymatic activity. Further truncations to amino acid 472 generated an inactive 53-kDa HDC isoform that was degraded by the proteasome pathway. These results suggested that processed isoforms, apart from 53-55-kDa ones, contribute toward histamine biosynthesis in vivo. This was confirmed in physiological studies where regulated increases in HDC activity were associated with the expression of isoforms that were greater than 55 kDa in size. We provide evidence to show that regulation of HDC expression can be achieved by the differential production or differential stabilization of multiple enzyme isoforms.     

Intracellular localization of the 55-kD actin-bundling protein in cultured cells: spatial relationships with actin, alpha-actinin, tropomyosin, and fimbrin

The 55-kD protein is a new actin-bundling protein purified from HeLa cells (Yamashiro-Matsumura, S., and F. Matsumura, 1985, J. Biol. Chem., 260:5087-5097). We have prepared monoclonal antibodies against the 55-kD protein and examined its intracellular localization, as well as its spatial relationships with other components of microfilaments in cultured cells by double-label immunofluorescence. The localization of the 55-kD protein is similar to that of actin. The antibody to the 55-kD protein stained strongly both microspikes and stress fibers. The 55-kD protein was found from the basal portions to the extremities of microspikes while alpha-actinin was localized only in the basal portions. In stress fibers, the 55-kD protein was found rather continuously in comparison to the periodic localizations of alpha-actinin and tropomyosin. Although fimbrin is located in microspikes and ruffling membranes, fimbrin is hardly found in stress fibers unlike the 55-kD protein. These observations coupled with the actin-bundling activity of the 55-kD protein imply that the 55-kD protein is involved in the formation of microfilament bundles in both microspikes and stress fibers.     

Purification and characterization of 55-kDa protein with 3,5,3'-triiodo-L-thyronine-binding activity and protein disulfide-isomerase activity from beef liver membrane

Beef liver membranes were shown to have different kinds of 3,5,3'-triiodo-L-thyronine binding proteins including the 55-kDa protein which had been reported to have this activity in many cells by affinity labelling with N-bromoacetyl-3,5,3'-[125I]triiodo-L-thyronine. In order to characterize the molecular features of these binding proteins, the 55-kDa protein was purified from a beef liver membrane fraction abundant in the plasma membrane. The protein was solubilized with 0.5% Chaps and purified by chromatography on gel filtration, hydroxyapatite, and Mono Q anion-exchange columns. The purity was confirmed with reversed-phase HPLC and SDS/PAGE. Consequently, 0.4% of the total proteins in the membrane fraction was recovered as the 55-kDa protein. One fourth of the amino acid composition of this protein was Glx (14.6%) plus Asx (11.7%) and the pI of this protein was 4.5. The purified protein has triiodothyronine-binding activity with a Kd of 57 nM which is similar to the high-affinity binding site of the membranes. The anti-(55-kDa protein) sera specifically recognized the 55-kDa protein of beef, rat and human cells. The immunoglobulin G fraction of the anti-(55-kDa protein) sera inhibited triiodothyronine binding to the beef liver membrane fraction. The purified protein also showed the activity of protein disulfide-isomerase (EC 5.3.4.1) as determined by reactivating scrambled ribonuclease. These data strongly suggested that the multi-functional 55-kDa protein which has triiodothyronine-binding activity and the activity of protein disulfide-isomerase, which is also reported to be the beta subunit of prolyl-4-hydroxylase, glycosylation-site-binding protein of oligosaccharyl transferase and iodothyronine 5'-monodeionidase, could be significant in the action of triiodothyronine towards the target cells.     

Purification and characterization of multiple isoforms of tropomyosin from rat cultured cells

We have previously shown that rat cultured cells contain five isoforms of tropomyosin (Matsumura, F., Yamashiro-Matsumura, S., and Lin, J. J.-C. (1983) J. Biol. Chem. 258, 6636-6644) and that these tropomyosins are differentially expressed upon cell transformation (Matsumura, F., Lin, J. J.-C., Yamashiro-Matsumura, S., Thomas, G. P., and Topp, W. C. (1983) J. Biol. Chem. 258, 13954-13964). To examine functions of tropomyosin in microfilament organization, we have purified and partially separated the multiple isoforms of tropomyosin by chromatography on hydroxylapatite. Analyses of cross-linked dimers produced by air oxidation have revealed that all isoforms except the tropomyosin isoform with apparent Mr of 35,000 form homodimers. Although these tropomyosins share many properties characteristic of tropomyosin, structural analyses at a peptide level and immunological analyses have shown that the five isoforms can be classified into two groups, i.e. tropomyosins with higher apparent Mr (Mr = 40,000, 36,500, and 35,000) and tropomyosins with lower apparent Mr (Mr = 32,400 and 32,000). The low Mr tropomyosins show less ability for head-to-tail polymerization and lower affinity to actin than the high Mr tropomyosins. We suggest that these differences in properties may be related to the changes in microfilament organization observed in transformed cells.     

Molecular basis for dissimilar nuclear trafficking of the actin-bundling protein isoforms T- and L-plastin

T- and L-plastin are highly similar actin-bundling proteins implicated in the regulation of cell morphology, lamellipodium protrusion, bacterial invasion and tumor progression. We show that T-plastin localizes predominantly to the cytoplasm, whereas L-plastin distributes between nucleus and cytoplasm in HeLa or Cos cells. T-plastin shows nuclear accumulation upon incubation of cells with the CRM1 antagonist leptomycin B (LMB). We identified a Rev-like nuclear export sequence (NES) in T-plastin that is able to export an otherwise nuclear protein in an LMB-dependent manner. Deletion of the NES promotes nuclear accumulation of T-plastin. Mutation of residues L17, F21 or L26 in the T-plastin NES inhibits nuclear efflux. L-plastin harbors a less conserved NES and lacks the F21 T-plastin residue. Insertion of a Phe residue in the L-plastin NES specifically enhances its export activity. These findings explain why both isoforms exhibit specific distribution patterns in eukaryotic cells.     

55-kD actin-bundling protein (p55) is a specific marker for identifying vascular dendritic cells.

Compared with other members of the dendritic cell family, the antigen profile of the recently recognized vascular dendritic cells has received limited attention. This study demonstrates that vascular dendritic cells in the human aorta and carotid arteries express 55-kD actin-bundling protein (p55), a specific marker for blood dendritic cells and Langerhans cells. This finding will facilitate screening of dendritic cells during their isolation from the arterial wall, as well as other investigations.(J Histochem Cytochem 47:1481-1486, 1999)     

Modulation of a major 30-kDa skeletal muscle protein by thyroid hormone

Thyroidectomy results in the transformation of type II fibres to type I in rat soleus muscle. In vitro translations containing polyribosomes indicate that the template activity of mRNA coding for a 30-kDa protein is increased in hypothyroid (6 months) rats. The cellular content of this protein is also increased in hypothyroid rats. The in vitro synthesis of the 30-kDa protein is not observed in thyroidectomized (10 weeks) rats that have been treated with triiodothyronine. The synthesis and accumulation of this protein are directly related to the proportion of type I fibres in rat skeletal muscle and appear to be modulated by thyroid hormone.     

Modulation of actin mechanics by caldesmon and tropomyosin

The ability of cells to sense and respond to physiological forces relies on the actin cytoskeleton, a dynamic structure that can directly convert forces into biochemical signals. Because of the association of muscle actin-binding proteins (ABPs) may affect F-actin and hence cytoskeleton mechanics, we investigated the effects of several ABPs on the mechanical properties of the actin filaments. The structural interactions between ABPs and helical actin filaments can vary between interstrand interactions that bridge azimuthally adjacent actin monomers between filament strands (i.e. by molecular stapling as proposed for caldesmon) or, intrastrand interactions that reinforce axially adjacent actin monomers along strands (i.e. as in the interaction of tropomyosin with actin). Here, we analyzed thermally driven fluctuations in actin's shape to measure the flexural rigidity of actin filaments with different ABPs bound. We show that the binding of phalloidin increases the persistence length of actin by 1.9-fold. Similarly, the intrastrand reinforcement by smooth and skeletal muscle tropomyosins increases the persistence length 1.5- and 2- fold respectively. We also show that the interstrand crosslinking by the C-terminal actin-binding fragment of caldesmon, H32K, increases persistence length by 1.6-fold. While still remaining bound to actin, phosphorylation of H32K by ERK abolishes the molecular staple (Foster et al. 2004. J Biol Chem 279;53387-53394) and reduces filament rigidity to that of actin with no ABPs bound. Lastly, we show that the effect of binding both smooth muscle tropomyosin and H32K is not additive. The combination of structural and mechanical studies on ABP-actin interactions will help provide information about the biophysical mechanism of force transduction in cells.     

The 135-kDa actin-bundling protein from lily pollen tubes arranges F-actin into bundles with uniform polarity

 A plant 135-kDa actin-bundling protein (P-135-ABP) isolated from pollen tubes of Lilium longiflorum (Thunb.) binds stoichiometrically to F-actin filaments and bundles them in vitro (E. Yokota et al., 1998, Plant Physiol. 116: 1421–1429). To further understand the mechanism of actin-filament bundle formation by P-135-ABP, the polarity of each F-actin filament in bundles was examined using myosin subfragment 1 (S-1). Dissociation of F-actin filaments from bundles organized by P-135-ABP was induced by S-1. However, F-actin filaments that remained in a bundle and decorated by S-1 showed uniform polarity. These results indicate that P-135-ABP arranges F-actin filaments into bundles with uniform polarity and consequently plays a key role in the orientation of cytoplasmic streaming in pollen tubes. Received: 23 February 1999 / Accepted: 22 April 1999     

Identification of a 55-kDa high-affinity calmodulin-binding protein from Electrophorus electricus

A high-affinity calcium-dependent calmodulin-binding protein (CaMBP) has been isolated from Electrophorus electricus main electric organ. This 55-kDa CaMBP has been purified to homogeneity by ion exchange and calmodulin-Sepharose affinity chromatography and electrophoretic elution from preparative sodium dodecyl sulfate-polyacrylamide gels. Antibodies against the 55-kDa CaMBP were raised in sheep and were affinity purified. A 47-kDa high-affinity CaMBP species was demonstrated by limited protease digestion and immunoblot analysis to be derived from the 55-kDa CaMBP. The 55-kDa CaMBP has also been isolated from skeletal muscle. It is not detectable by immunoblot analysis in nonexcitable tissues. Characterization of the 55-kDa high-affinity calmodulin-acceptor protein may further elucidate the role of calcium-calmodulin in the regulation of bioelectricity.     

Modulation of protein kinase C isoforms by PAF in cerebral cortex.

The effect of platelet activating factor (PAF) on subcellular distribution of protein kinase C isoforms in rat cerebral cortex was investigated. PAF induced an increase in levels of protein kinase C epsilon and gamma in membrane fraction. Results also indicate that PAF induced an increase in protein kinase C delta levels in both cytosolic and membrane fraction. This effect is possibly due to an increase in enzyme synthesis, as indicated by the results obtained from the experiments performed in the presence of cycloheximide and actinomycin. All the effects induced by PAF were time- and dose-dependent, and were mediated through the activation of PAF receptor. These findings indicate that the three isoforms may be involved in signal transduction of PAF in the brain.     

Phosphoenolpyruvate-dependent phosphorylation of a 55-kDa protein of Streptococcus faecalis catalyzed by the phosphotransferase system

Abstract A protein with an M _r of 55000 was isolated from glucose-grown Streptococcus faecalis cells. The protein becomes phosphorylated in a phosphoenolpyruvate-dependent reaction catalyzed by enzyme I and HPr of the bacterial phosphotransferase system. It did not stimulate phosphoenolpyruvate-dependent glucose phosphorylation. Several sugars were tested for their ability to dephosphorylate the phosphorylated protein in the presence of membrane fragments. Even though some of the sugars were able to dephosphorylate phospho-HPr quickly, the factor III-like 55-kDa protein remained phosphorylated. We therefore assumed that this protein is not involved in any sugar uptake reaction but that it exerts a regulatory function in Gram-positive bacteria comparable to the function of factor III specific for glucose in Escherichia coli .     

Epidermal keratinocytes: regulation of multiple cell phenotypes by multiple protein kinase C isoforms

Squamous cells form the outermost layers of the epidermis, and though they are readily discarded from the tissue, they serve a vital water barrier function while in the stratum corneum. The generation of cornified or squamous keratinocytes involves a complex, multi-step differentiation process that insures the proper physical and immunological barrier functions of the epidermis are maintained. The regulation of keratinocyte terminal differentiation is influenced by a large number of signaling pathways. This article will review some recent findings regarding the roles of the protein kinase C (PKC) family in normal keratinocyte differentiation, as well as their involvement in skin diseases, especially skin cancer.     

Modulation of the actin-activated adenosinetriphosphatase activity of myosin by tropomyosin from vascular and gizzard smooth muscles

Tropomyosins from bovine aorta and pulmonary artery exhibit identical electrophoretic patterns in sodium dodecyl sulfate but differ from tropomyosins of either chicken gizzard or rabbit skeletal muscle. Each of the four tropomyosins binds readily to skeletal muscle F-actin as indicated by their sedimentation with actin and by their ability to maximally stimulate or inhibit actin-activated ATPase activity at a molar ratio of one tropomyosin per seven actin monomers. Smooth and skeletal muscle tropomyosins differ in their effects on activity of skeletal myosin or heavy meromyosin (HMM); the former can enhance activity under conditions in which the latter inhibits. Gizzard and arterial tropomyosins are usually equally effective in stimulating ATPase activity of skeletal acto-HMM, but at high concentrations of Mg2+ gizzard tropomyosin is more effective, a result that cannot be attributed to differences in the binding of the two tropomyosins to F-actin. The effects of tropomyosin also depend on the type of myosin; tropomyosin enhances activity of gizzard myosin under conditions in which it inhibits that of skeletal myosin. Increasing the pH or the Mg2+ concentration can reverse the effect of tropomyosin on actin-stimulated ATPase activity of skeletal HMM from activation to inhibition, but this reversal is not found with gizzard myosin. Activity in the absence of tropomyosin is independent of pH, and the loss of activation with increasing pH is not accompanied by loss of binding of tropomyosin to actin.     

Specification of actin filament function and molecular composition by tropomyosin isoforms

The specific functions of greater than 40 vertebrate nonmuscle tropomyosins (Tms) are poorly understood. In this article we have tested the ability of two Tm isoforms, TmBr3 and the human homologue of Tm5 (hTM5(NM1)), to regulate actin filament function. We found that these Tms can differentially alter actin filament organization, cell size, and shape. hTm5(NM1) was able to recruit myosin II into stress fibers, which resulted in decreased lamellipodia and cellular migration. In contrast, TmBr3 transfection induced lamellipodial formation, increased cellular migration, and reduced stress fibers. Based on coimmunoprecipitation and colocalization studies, TmBr3 appeared to be associated with actin-depolymerizing factor/cofilin (ADF)-bound actin filaments. Additionally, the Tms can specifically regulate the incorporation of other Tms into actin filaments, suggesting that selective dimerization may also be involved in the control of actin filament organization. We conclude that Tm isoforms can be used to specify the functional properties and molecular composition of actin filaments and that spatial segregation of isoforms may lead to localized specialization of actin filament function.     

Specific features of neuronal size and shape are regulated by tropomyosin isoforms

Spatially distinct populations of microfilaments, characterized by different tropomyosin (Tm) isoforms, are present within a neuron. To investigate the impact of altered tropomyosin isoform expression on neuronal morphogenesis, embryonic cortical neurons from transgenic mice expressing the isoforms Tm3 and Tm5NM1, under the control of the beta-actin promoter, were cultured in vitro. Exogenously expressed Tm isoforms sorted to different subcellular compartments with Tm5NM1 enriched in filopodia and growth cones, whereas the Tm3 was more broadly localized. The Tm5NM1 neurons displayed significantly enlarged growth cones accompanied by an increase in the number of dendrites and axonal branching. In contrast, Tm3 neurons displayed inhibition of neurite outgrowth. Recruitment of Tm5a and myosin IIB was observed in the peripheral region of a significant number of Tm5NM1 growth cones. We propose that enrichment of myosin IIB increases filament stability, leading to the enlarged growth cones. Our observations support a role for different tropomyosin isoforms in regulating interactions with myosin and thereby regulating morphology in specific intracellular compartments.     

Modulation of glucan-binding protein activity in streptococci by fluoride

Glucan-binding lectin (GBL) activity of Streptococcus sobrinus was significantly reduced by fluoride in the growth medium. Approximately 1.5 mM fluoride was required for a 50% reduction in GBL activity. In addition to the GBL, several other glucan-binding proteins were reduced when the bacteria were grown in subinhibitory fluoride. Fluoride had no effect on glucosyltransferases (GTFs), enzymes capable of converting sucrose into alpha-1,6-glucans. All the proteins were detected by use of enhanced chemiluminescence (ECL of fluorescein-labeled dextran) and Western blotting of renatured SDS-PAGE gels. The effects of fluoride on the bacteria were abrogated when the manganous ion was included in the growth medium. It thus appears that one mechanism of action of fluoridated water is its effects on glucan-binding proteins. The fluoride may be reducing metabolism of the mangano aquo ion, essential for expression of the glucan-binding proteins.