PubMed Health⌕ Search

Biomedical subjects

P A Rubenstein

Publications and source records attributed to P A Rubenstein.

At least 19 recordsLinked to original sources
PubMed1992

Isolation and characterization of the rat liver actin N-acetylaminopeptidase.

Actins from most eukaryotes undergo a unique post-translational modification of the amino terminus called "processing." Processing consists of the removal of an amino-terminal Ac-Met or Ac-Cys to leave an acidic amino-terminal residue. We have previously demonstrated that this reaction is not catalyzed by the ribosomally associated methionine aminopeptidase or by other previously described acetylaminopeptidases. Here we present the isolation and characterization of the actin N-acetylaminopeptidase (ANAP) from rat liver. A five-step purification protocol achieves a 4100-fold purification of the enzyme with an overall 8% recovery of activity. ANAP is a 77-kDa monomer with a pI of 4.6. Using unprocessed yeast actin as a substrate, the Km of ANAP is 3.5 microM. Purified ANAP was used to generate a polyclonal antibody. The antibody has been used along with activity assays to demonstrate the presence of ANAP in a variety of rat tissues. Finally, evidence is presented that in mammals, ANAP may function with a second, as yet unpurified, component to process actin amino termini.

Actins↗
PubMed1992

Splicing of two alternative exon pairs in beta-tropomyosin pre-mRNA is independently controlled during myogenesis.

Two known tissue-specific tropomyosin (TM) isoforms are produced from the rodent beta-TM gene. Skeletal muscle beta-TM uses the alternative exons 6b and 9a and the exon 9a-associated poly(A) site. Fibroblast and smooth muscle TM-1 use exons 6a and 9b and the exon-9b associated poly(A) site. We have identified a new skeletal muscle beta-TM isoform, beta-TM2. beta-TM2 contains exon 6b (muscle) and exon 9b (nonmuscle). Full-length beta-TM2 cDNA clones were isolated from a cDNA library of mouse muscle BC3H1 cells. Its mRNA was also found in mouse skeletal muscle tissue but not in other tissues. beta-TM2 mRNA level and protein synthesis are differentiation-dependent, with a transient high level in the early stages of myogenesis both in BC3H1 cells and in mouse embryo limbs. Trace amounts of beta-TM3 mRNA, the other hybrid form (exons 6a + 9a), were found in less differentiated BC3H1 cells, mouse uterus, heart, and 3T3 fibroblasts but not skeletal muscle tissue. Thus, the selection of the two alternative exons appears to be controlled independently. Furthermore, during myogenesis, there is a sequential switch in the internal alternative exon, the terminal exon, and the poly(A) site from the nonmuscle to the muscle type.

Amino Acid Sequence↗
PubMed1992

Removal of the amino-terminal acidic residues of yeast actin. Studies in vitro and in vivo.

We have examined the role of the acidic residues Asp2 and Glu4 at the NH2 terminus of Saccharomyces cerevisiae actin through site-directed mutagenesis. In DNEQ actin, these residues have been changed to Asn2 and Gln4, whereas in delta DSE actin, the Asp2-Ser-Glu tripeptide has been deleted. Both mutant actins can replace wild type yeast actin. Peptide mapping studies reveal that DNEQ, like wild type actin, retains the initiator Met and is NH2 terminally acetylated, whereas delta DSE has a free NH2 terminus and has lost the initiator Met. Interestingly, microscopic examination of filaments of these two actins reveal the appearance of bundled filaments. The DNEQ bundles are smaller and more ordered, whereas the delta DSE bundles are larger and more loosely organized. Additionally, both mutant actins activate the ATPase activity of rabbit muscle myosin S1 fragment to a lesser extent than wild type. We have also developed a sensitive assay for actin function in vivo that enabled us to detect a slight defect in the ability of these mutant actins to support secretion, an important function in yeast. Thus, although the mutant actins resulted in no gross phenotypic changes, we were able to detect a defect in actin function through this assay. From these studies we can conclude that 1) although NH2-terminal negative charges are not essential to yeast life, the loss of such charges does result in a slight defect in the actins' ability to support secretion, 2) removal of the NH2-terminal negative charges promotes the bundling of actin filaments, and 3) actins lacking NH2-terminal negative charges are unable to activate the myosin S1 ATPase activity as well as wild type actin.

Acetylation↗
PubMed1992

Amino-terminal processing of actins mutagenized at the Cys-1 residue.

Most actins examined to date undergo a unique posttranslational modification termed processing, catalyzed by the actin N-acetylaminopeptidase. Processing is the removal of acetylmethionine from the amino terminus in class I actins with Met-Asp(Glu) amino termini. For class II actins with Met-X-Asp(Glu) amino termini, processing is the removal of the second residue as an N-acetylamino acid. Other cytosolic proteins with these amino termini are not processed suggesting that the reaction may be specific for actins. In actin, X is usually cysteine. However, there are some class II actins in which this residue is other than cysteine, suggesting a broader substrate specificity for actin N-acetylaminopeptidase than acetylmethionine or acetylcysteine. We constructed mutant actins in which this cysteine was replaced with serine, asparagine, glycine, aspartic acid, histidine, phenylalanine, and tyrosine and used these to determine the substrate specificity of rat liver actin N-acetylaminopeptidase in vitro. Amino-terminal acetylmethinonine was cleaved from adjacent aspartic acid, asparagine, or histidine, but not serine, glycine, phenylalanine, or tyrosine. Of the acetylated actin amino termini tested, only acetylmethionine and acetylcysteine were cleaved. Histidine was never N-acetylated and was not cleaved. When phenylalanine and tyrosine were adjacent to the initiator methionine, no initiator methionine was cleaved even though it was acetylated. These results suggest a narrow substrate specificity for the rat liver actin N-acetylaminopeptidase. They also demonstrate that the adjacent residue can effect actin N-acetylaminopeptidase specificity.

Actins↗
PubMed1992

Choice of 3' cleavage/polyadenylation site in beta-tropomyosin RNA processing is differentiation-dependent in mouse BC3H1 muscle cells.

The rodent beta-tropomyosin (TM) gene produces either a 1.2-kilobase (kb) skeletal muscle beta-TM mRNA or a 1.1-kb fibroblast/smooth muscle TM-1 mRNA through tissue-specific alternative exon splicing and 3' cleavage/polyadenylation at two alternative poly(A) sites. beta-TM mRNA contains exon 6b, 9a, and the poly(A) site immediately following exon 9a, whereas TM-1 mRNA contains exon 6a, 9b, and the poly(A) site following exon 9b. We isolated a novel 2.1-kb beta-TM cDNA clone, pUTM, from a cDNA library of 2-day differentiated mouse BC3H1 muscle-like cells. This cDNA contains the entire sequence of mature beta-TM mRNA with a normal but unused poly(A) site associated with exon 9a. Instead, 3' cleavage/polyadenylation of this cDNA occurred at the exon 9b-associated distal poly(A) site, resulting in the retention of a 1-kb intron and the TM-1 exon 9b. We identified a 2.3-kb functional mRNA, UTM RNA, corresponding to pUTM. UTM RNA appeared early during BC3H1 cell differentiation and gradually decreased as the beta-TM mRNA increased. UTM RNA was also detected in mouse C2C12 muscle cells and in skeletal muscle tissue isolated from mouse leg. Thus, in the processing of beta-TM gene transcripts, selection of alternative terminal exons and alternative poly(A) sites are not necessarily linked as they appear to be in other gene systems.

Animals↗
PubMed1992

Effects of profilin and profilactin on actin structure and function in living cells.

Previous studies have yielded conflicting results concerning the physiological role of profilin, a 12-15-kD actin- and phosphoinositide-binding protein, as a regulator of actin polymerization. We have addressed this question by directly microinjecting mammalian profilins, prepared either from an E. coli expression system or from bovine brain, into living normal rat kidney (NRK) cells. The microinjection causes a dose-dependent decrease in F-actin content, as indicated by staining with fluorescent phalloidin, and a dramatic reduction of actin and alpha-actinin along stress fibers. In addition, it has a strong inhibitory effect toward the extension of lamellipodia. However, the injection of profilin causes no detectable perturbation to the cell-substrate focal contact and no apparent depolymerization of filaments in either the nonlamellipodial circumferential band or the contractile ring of dividing cells. Furthermore, cytokinesis of injected cells occurs normally as in control cells. In contrast to pure profilin, high-affinity profilin-actin complexes from brain induce an increase in total cellular F-actin content and an enhanced ruffling activity, suggesting that the complex may dissociate readily in the cell and that there may be multiple states of profilin that differ in their ability to bind or release actin molecules. Our results indicate that profilin and profilactin can function as effective regulators for at least a subset of actin filaments in living cells.

Actins↗
PubMed1991

Unusual metabolism of the yeast actin amino terminus.

In this paper we have examined the post-translational modifications of the NH2 terminus of actin from the yeast Saccharomyces cerevisiae. Like actins examined previously, this actin contains an acetylated NH2 terminus. Actins in other organisms undergo a unique post-translational processing event in which the initial amino acid(s) are removed by an actin-specific processing enzyme in an acetylation-dependent reaction. This is defined as actin processing. In yeast, actin retains its initiator Met in vivo and is thus not processed even though a rat liver actin processing enzyme can process yeast actin in vitro. This lack of actin processing appears to be a general property of fungi, as the actin from three other species, Aspergillus nidulans, Schizosaccharomyces pombe, and Candida albicans are not NH2 terminally processed either. Yeast actin is a class I actin; its initiator Met directly precedes an acidic residue. We converted yeast actin to a class II species by inserting a Cys codon between the Met-1 and Asp-2 codons. In normal class II actins the Cys residue is removed as acetyl-Cys during processing. Neither the mutant actin nor chick beta-actin (a class I actin) are processed when expressed in yeast. S. cerevisiae thus appears to be also incapable of processing exogenous actins. Further study of the mutant actin containing a Cys at position 2 shows that 30-40% of this actin is stably unacetylated. This unacetylated actin does not have a shorter half-life than the acetylated form. From these studies we conclude that 1) NH2-terminal actin-specific processing is not required for actin function in yeast and three other fungi, 2) yeast are apparently incapable of processing any type of actin precursor, and 3) the stability of a yeast pseudo-class II actin is not affected by the acetylation state of the NH2 terminus.

Acetylation↗
PubMed1990

The functional importance of multiple actin isoforms.

Actin is a protein that plays an important role in cell structure, cell motility, and the generation of contractile force in both muscle and nonmuscle cells. In many organisms, multiple forms of actin, or isoactins, are found. These are products of different genes and have different, although very similar, amino acid sequences. Furthermore, these isoactins are expressed in a tissue specific fashion that is conserved across species, suggesting that their presence is functionally important and their behavior can be distinguished quantitatively from one another in vitro. In muscle cells, they are differentially distributed within the cell and some are specifically associated with structures such as costameres, mitochondria, and neuromuscular junctions. There is also good evidence for specific isoactin function in microvascular pericytes and in the intestinal brush border. However, the necessity of specific isoactins for various functions has not yet been conclusively demonstrated.

Actins↗
PubMed1989

Identification of N-acetylmethionine as the product released during the NH2-terminal processing of a pseudo-class I actin.

Genes for the various isoactins define two classes of actin. Class I actin genes code for Met-Asp(Glu)-actin, and class II actin genes code for Met-X-Asp(Glu)-actin where X is usually cysteine. Amino termini of both are removed in an acetylation-dependent processing reaction yielding acetyl-Asp(Glu)-actin. Both classes are processed at approximately equal rate (t1/2 = 15 min) in vivo. In vitro, class II actins are 90% processed by endogenous enzymes after 60 min in a rabbit reticulocyte lysate system, whereas class I actins are only minimally processed during this period. Using site-directed mutagenesis of a human skeletal muscle isoactin coupled with in vitro transcription and translation methods, we have synthesized a pseudo-class I actin in which the penultimate cysteine has been changed to an aspartic acid, thus placing a class I amino terminus on an otherwise class II actin molecule. The pseudo-class I actin was less than 20% processed during the translation period as determined by peptide mapping. It was further processed by exogenous processing enzyme at a rate compatible with a class I actin. These results indicate that the major actin determinant controlling differential actin-processing rates is the amino-terminal residue being cleaved, not the remaining structure of the actin molecule. We have also demonstrated for the first time that N-acetylmethionine is the immediately released product from the amino terminus of a pseudo-class I actin during processing.

Acetylation↗
PubMed1989

Synthesis of mammalian profilin in Escherichia coli and its characterization.

Profilin is a G-actin binding protein that may have a role in controlling the ratio of G/F actin within the cell. To devise a way for obtaining large amounts of mammalian profilin in an active state, we transfected Escherichia coli with a plasmid containing a full-length rat spleen profilin cDNA adjacent to a promoter inducible by isopropyl thiogalactoside (IPTG). Upon induction, they synthesized a new protein of 15,000 MW constituting approximately 5% of the total cell protein. This protein bound to poly-L-proline Sepharose and could be eluted with 7 M urea, behavior similar to that exhibited by authentic profilin. The protein could be released from the bacteria in soluble form following sonication, and the profilin could then be purified to homogeneity following chromatography on Sephadex G-75 and DEAE A-50 Sephadex. The protein began with an unblocked Ala, indicating that the initiating formyl and methionine residues had been removed. The dissociation of the recombinant profilin from chicken skeletal muscle actin was characterized by a Kd of approximately 2 microM based on gel filtration analysis and actin polymerization assays. These results show that purified active mammalian profilin can be made conveniently in large quantities. This study also demonstrates the feasibility of using bacterially synthesized profilin in structure-function studies involving mutant profilins altered by site-directed mutagenesis.

Actins↗
PubMed1989

Nonuniform behavior of multiple isoactins in the same cell is a cell-dependent phenomenon.

The functional significance of multiple isoactins in the same cell is still not understood. To address this question, we examined the response of smooth muscle and cardiac muscle alpha-isoactins to a serial extraction procedure applied to both muscle and nonmuscle cell types. We compared these extraction results with results obtained with the beta- and gamma-nonmuscle actin isoforms from the same cells. In differentiated BC3H1 nonfusing muscle cells (smooth muscle alpha-isoactin), in human rhabdomyosarcoma cells (cardiac alpha-isoactin), and in chick skeletal muscle cells (cardiac alpha-isoactin), different fractions were found selectively enriched in either the nonmuscle or the muscle-specific actin isoforms compared with their relative abundance in whole cell extracts. Conversely, when these same isoactins were examined either in undifferentiated BC3H1 cells or in mouse nonmuscle cells stably transfected with a cardiac alpha-isoactin gene, no enrichment of these isoforms above their relative abundance in whole cell extracts was observed. These results indicate that within the muscle or muscle-like cells examined, the different actin isoforms were either selectively utilized or localized. These results further show that isoactin-specific responses observed were apparently related to the cell type in which they were found and not to differences in inherent physical properties such as solubility of the different isoactins examined.

Actins↗
PubMed1988

Studies on the role of actin's aspartic acid 3 and aspartic acid 11 using oligodeoxynucleotide-directed site-specific mutagenesis.

One or more of the five acidic amino-terminal residues of skeletal muscle actin have been implicated as being important in a number of actin-related processes. We have constructed a series of actins containing mutations at Asp3 and Asp11 and tested these mutant proteins for their ability to bind to DNase I-agarose, polymerize with rabbit skeletal muscle actin, undergo amino-terminal processing, and bind to the myosin-S1 subfragment. The mutant actins were expressed in vitro using a coupled transcription/translation system which involves the synthesis of mutant RNAs with SP6 RNA polymerase followed by their translation in a rabbit reticulocyte lysate. When Asp3 was changed to Ala, His, or Asn there was no difference in the tested properties as compared to wild type actin. These results suggest that an acidic residue at position 3 is not critical for the actin functions measured. When Asp11 was changed to Glu, Asn, or His or if the conserved Asp-Asn sequence at positions 11 and 12 was reversed, the mutants were able to copolymerize with rabbit skeletal muscle actin and be cross-linked to myosin-S1 to nearly the same extent as wild type actin. However, the amount of in vitro-synthesized actin capable of binding to DNase I-agarose with high affinity or undergoing amino-terminal processing was reduced significantly relative to the wild type actin synthesized in vitro. The Asp11 mutants ran anomalously on native polyacrylamide gels suggestive of a conformational change induced in the actin. Together, these results suggest that Asp11 may be important in proper actin folding and function.

Actins↗
PubMed1988

Epidermal growth factor controls smooth muscle alpha-isoactin expression in BC3H1 cells.

We have examined the effects of epidermal growth factor (EGF), platelet-derived growth factor, and insulin on the differentiation of a mouse vascular smooth muscle-like cell line, the BC3H1 cells. On the basis of cell morphology and smooth muscle alpha-isoactin synthesis, we demonstrate that EGF at physiological concentrations prevents the differentiation of these cells, whereas platelet-derived growth factor has no apparent effect. The induction of alpha-isoactin synthesis by serum deprivation is inhibited by EGF in a dose-dependent manner with a half-maximal effect at 3-5 ng/ml and a maximal inhibition at approximately 30 ng/ml. Northern analysis also shows that EGF blocks the accumulation of alpha-isoactin mRNA normally observed during cell differentiation. Addition of EGF to differentiated cells results in a repression of alpha-isoactin synthesis, a stimulation of beta- and gamma-isoactin synthesis, and the stabilization of the nonmuscle isoactins. The synthesis of creatine phosphokinase, a muscle-specific noncontractile protein, is also regulated by EGF in a similar fashion. Modulation by EGF of alpha-isoactin expression is not affected by aphidicolin and is therefore independent of its mitogenic effect on these cells. Insulin is not required for observation of the EGF-dependent effects but instead seems to promote differentiation. Our results show that EGF can replace serum in controlling the differentiation of BC3H1 cells.

Actins↗
PubMed1987

Studies on the role of actin's N tau-methylhistidine using oligodeoxynucleotide-directed site-specific mutagenesis.

The primary structure of all actins except that isolated from Naegleria gruberi contains a unique N tau-methylhistidine (MeHis) at position 73. This modified residue has been implicated as possibly being important for the post-translational processing of actin's amino terminus, the binding of actin to DNase I, and in the polymerization of G-actin. We have investigated the potential role of MeHis in each of these processes by utilizing site-directed mutagenesis to change His-73 of skeletal muscle actin to Arg and Tyr. Wild type and mutant actins were synthesized in vivo, using non-muscle cells transfected with mutant cDNAs, and in vitro by translating mutant RNAs synthesized using SP6 RNA polymerase in a rabbit reticulocyte lysate. We have found that actins containing Arg or Tyr at position 73 undergo amino-terminal processing, bind to DNase I-agarose, and become incorporated into the cytoskeleton of a nonmuscle cell as efficiently as wild type actin. Furthermore, using an in vitro copolymerization assay we have found that although there is no difference between the Arg mutant and the wild type actins, the Tyr mutant has a slightly greater critical concentration for polymerization. These results show that MeHis is not absolutely required for any of these processes.

Actins↗
PubMed1987

Alternate pathways for removal of the class II actin initiator methionine.

Class II actin genes usually specify a polypeptide with a Met-Cys-Asp NH2 terminus, whereas the actin itself begins with an acetyl (Ac)-Asp(Glu). Previous studies with Drosophila actin showed that the first detectable intermediate is one with an Ac-Cys NH2 terminus which is subsequently cleaved in a novel reaction to expose the Asp. The initiator Met was probably removed early in translation as a free amino acid. To determine whether the class II actin initiating Met could also be removed in an acetylation-dependent manner, we translated Drosophila mRNA in a rabbit reticulocyte lysate in which protein acetylation was inhibited. After 60 min, three actin intermediates were detected, NH2-Met-Cys-Asp-actin, Ac-Met-Cys-Asp-actin, and NH2-Cys-Asp-actin. During processing in the presence of acetyl-CoA, three additional species were observed with NH2-terminal Ac-Cys-Asp, NH2-Asp, and Ac-Asp segments. In a time- and acetyl-CoA-dependent fashion, Met-Cys-Asp-actin was processed to the mature actin, presumably through an Ac-Met-Cys-Asp intermediate. Thus, two different pathways for removal of the initiator Met of class II actins, acetylation-dependent and independent, are possible. Since no class II actin intermediate containing the initiator Met is seen in vivo, although in class I actins this intermediate is observed, the most probable pathway for class II actins in vivo is the cotranslational removal of the initiator Met as a free amino acid.

Acetyl Coenzyme A↗
PubMed1986

Characterization of actin mRNA levels during BC3H1 cell differentiation.

The expression of a vascular smooth muscle specific alpha-actin isoform can be induced in mouse BC3H1 smooth muscle cells by treating confluent monolayers with serum-free medium (Strauch, A. R., and Rubenstein, P. A. (1984) J. Biol. Chem. 259, 3152-3159; 7224-7229). Using blot hybridization techniques, two size classes of actin RNA were identified in BC3H1 cells with the relative amount of RNA in each size class varying according to the developmental state of the cells; a 2100-nucleotide actin RNA was most abundant in myoblasts, whereas a smaller 1500-nucleotide actin RNA was found predominantly in fully differentiated myocytes. Results of in vitro translation experiments suggested that the 2100-nucleotide actin RNA on blots of myoblast total RNA corresponded to a mixture of similar size transcripts encoding both beta- and gamma-actin, while the 1500-nucleotide actin RNA in myocytes was an alpha-actin mRNA. Cell-cell contact and serum withdrawal initiated a 6-fold increase in the level of alpha-actin mRNA in BC3H1 cells that was followed by a 3-fold decrease in the amount of beta- and gamma-actin mRNA when confluent cells were exposed to serum-free medium for prolonged periods. Vascular smooth muscle alpha-actin was the major alpha-actin isoform synthesized in L-[35S]cysteine-labeled BC3H1 myocytes, indicating that the 1500-nucleotide actin mRNA size class in these cells may be enriched for vascular smooth muscle alpha-actin transcripts.

Actins↗