Involvement of protein kinase A in fibroblast growth factor-2-activated transcription

Polypeptide growth factors activate common signal transduction pathways, yet they can induce transcription of different target genes. The mechanisms that control this specificity are not completely understood. Recently, we have described a fibroblast growth factor (FGF)-inducible response element, FiRE, on the syndecan-1 gene. In NIH 3T3 cells, the FiRE is activated by FGF-2 but not by several other growth factors, such as platelet-derived growth factor or epidermal growth factor, suggesting that FGF-2 activates signaling pathways that diverge from pathways activated by other growth factors. In this paper, we report that the activation of FiRE by FGF-2 requires protein kinase A (PKA) in NIH 3T3 cells. The PKA-specific inhibitor H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) blocked the FGF-2-induced activation of FiRE, the transcription of the syndecan-1 gene, and cell proliferation. Also, expression of a dominant-negative form of PKA inhibited the FGF-2-induced FiRE activation and the transcription of the syndecan-1 gene. The binding of activator protein-1 transcription-factor complexes, required for the activation of FiRE, was blocked by inhibition of PKA activity before FGF-2 treatment. In accordance with the growth factor specificity of FiRE, the activity of PKA was stimulated by FGF-2 but not by platelet-derived growth factor or epidermal growth factor. Furthermore, a portion of the PKA catalytic subunit pool was translocated to the nucleus by FGF-2. Noticeably, the total cellular cAMP concentration was not affected by FGF-2 stimulus. We propose that the FGF-2-selective transcriptional activation through FiRE is caused by the ability of FGF-2 to control PKA activity.

Guanidine hydrochloride blocks a critical step in the propagation of the prion-like determinant [PSI+] of Saccharomyces cerevisiae

The cytoplasmic heritable determinant [PSI+] of the yeast Saccharomyces cerevisiae reflects the prion-like properties of the chromosome-encoded protein Sup35p. This protein is known to be an essential eukaryote polypeptide release factor, namely eRF3. In a [PSI+] background, the prion conformer of Sup35p forms large oligomers, which results in the intracellular depletion of functional release factor and hence inefficient translation termination. We have investigated the process by which the [PSI+] determinant can be efficiently eliminated from strains, by growth in the presence of the protein denaturant guanidine hydrochloride (GuHCl). Strains are “cured” of [PSI+] by millimolar concentrations of GuHCl, well below that normally required for protein denaturation. Here we provide evidence indicating that the elimination of the [PSI+] determinant is not derived from the direct dissolution of self-replicating [PSI+] seeds by GuHCl. Although GuHCl does elicit a moderate stress response, the elimination of [PSI+] is not enhanced by stress, and furthermore, exhibits an absolute requirement for continued cell division. We propose that GuHCl inhibits a critical event in the propagation of the prion conformer and demonstrate that the kinetics of curing by GuHCl fit a random segregation model whereby the heritable [PSI+] element is diluted from a culture, after the total inhibition of prion replication by GuHCl.

Mutated epithelial cadherin is associated with increased tumorigenicity and loss of adhesion and of responsiveness to the motogenic trefoil factor 2 in colon carcinoma cells

Epithelial (E)-cadherin and its associated cytoplasmic proteins (α-, β-, and γ-catenins) are important mediators of epithelial cell–cell adhesion and intracellular signaling. Much evidence exists suggesting a tumor/invasion suppressor role for E-cadherin, and loss of expression, as well as mutations, has been described in a number of epithelial cancers. To investigate whether E-cadherin gene (CDH1) mutations occur in colorectal cancer, we screened 49 human colon carcinoma cell lines from 43 patients by single-strand conformation polymorphism (SSCP) analysis and direct sequencing. In addition to silent changes, polymorphisms, and intronic variants in a number of the cell lines, we detected frameshift single-base deletions in repeat regions of exon 3 (codons 120 and 126) causing premature truncations at codon 216 in four replication-error-positive (RER+) cell lines (LS174T, HCT116, GP2d, and GP5d) derived from 3 patients. In LS174T such a mutation inevitably contributes to its lack of E-cadherin protein expression and function. Transfection of full-length E-cadherin cDNA into LS174T cells enhanced intercellular adhesion, induced differentiation, retarded proliferation, inhibited tumorigenicity, and restored responsiveness to the migratory effects induced by the motogenic trefoil factor 2 (human spasmolytic polypeptide). These results indicate that, although inactivating E-cadherin mutations occur relatively infrequently in colorectal cancer cell lines overall (3/43 = 7%), they are more common in cells with an RER+ phenotype (3/10 = 30%) and may contribute to the dysfunction of the E-cadherin–catenin-mediated adhesion/signaling system commonly seen in these tumors. These results also indicate that normal E-cadherin-mediated cell adhesion can restore the ability of colonic tumor cells to respond to trefoil factor 2.

A deoxyribonucleotidase in mitochondria: Involvement in regulation of dNTP pools and possible link to genetic disease

Three cytosolic and one plasma membrane-bound 5′-nucleotidases have been cloned and characterized. Their various substrate specificities suggest widely different functions in nucleotide metabolism. We now describe a 5′-nucleotidase in mitochondria. The enzyme, named dNT-2, dephosphorylates specifically the 5′- and 2′(3′)-phosphates of uracil and thymine deoxyribonucleotides. The cDNA of human dNT-2 codes for a 25.9-kDa polypeptide with a typical mitochondrial leader peptide, providing the structural basis for two-step processing during import into the mitochondrial matrix. The deduced amino acid sequence is 52% identical to that of a recently described cytosolic deoxyribonucleotidase (dNT-1). The two enzymes share many catalytic properties, but dNT-2 shows a narrower substrate specificity. Mitochondrial localization of dNT-2 was demonstrated by the mitochondrial fluorescence of 293 cells expressing a dNT-2-green fluorescent protein (GFP) fusion protein. 293 cells expressing fusion proteins without leader peptide or with dNT-1 showed a cytosolic fluorescence. During in vitro import into mitochondria, the preprotein lost the leader peptide. We suggest that dNT-2 protects mitochondrial DNA replication from overproduction of dTTP, in particular in resting cells. Mitochondrial toxicity of dTTP can be inferred from a severe inborn error of metabolism in which the loss of thymidine phosphorylase led to dTTP accumulation and aberrant mitochondrial DNA replication. We localized the gene for dNT-2 on chromosome 17p11.2 in the Smith–Magenis syndrome-critical region, raising the possibility that dNT-2 is involved in the etiology of this genetic disease.

NMR structure of the bovine prion protein

The NMR structures of the recombinant 217-residue polypeptide chain of the mature bovine prion protein, bPrP(23–230), and a C-terminal fragment, bPrP(121–230), include a globular domain extending from residue 125 to residue 227, a short flexible chain end of residues 228–230, and an N-terminal flexibly disordered “tail” comprising 108 residues for the intact protein and 4 residues for bPrP(121–230), respectively. The globular domain contains three α-helices comprising the residues 144–154, 173–194, and 200–226, and a short antiparallel β-sheet comprising the residues 128–131 and 161–164. The best-defined parts of the globular domain are the central portions of the helices 2 and 3, which are linked by the only disulfide bond in bPrP. Significantly increased disorder and mobility is observed for helix 1, the loop 166–172 leading from the β-strand 2 to helix 2, the end of helix 2 and the following loop, and the last turn of helix 3. Although there are characteristic local differences relative to the conformations of the murine and Syrian hamster prion proteins, the bPrP structure is essentially identical to that of the human prion protein. On the other hand, there are differences between bovine and human PrP in the surface distribution of electrostatic charges, which then appears to be the principal structural feature of the “healthy” PrP form that might affect the stringency of the species barrier for transmission of prion diseases between humans and cattle.

An ethylene-induced cDNA encoding a lipase expressed at the onset of senescence

A cDNA clone encoding a lipase (lipolytic acyl hydrolase) expressed at the onset of petal senescence has been isolated by screening a cDNA expression library prepared from carnation flowers (Dianthus caryophyllus). The cDNA contains the lipase consensus sequence, ITFAGHSLGA, and encodes a 447-amino acid polypeptide with a calculated molecular mass of 50.2 kDa that appears to be a cytosolic protein. Over-expression of the clone in Escherichia coli yielded a protein of the expected molecular weight that proved capable of deesterifying fatty acids from p-nitrophenylpalmitate, tri-linolein, soybean phospholipid, and Tween in both in vitro and in situ assays of enzyme activity. The abundance of the lipase mRNA increases just as carnation flowers begin to senesce, and expression of the gene is also induced by treatment with ethylene. Southern blot analyses of carnation genomic DNA have indicated that the lipase is a single copy gene. The lipase gene is also expressed in carnation leaves and is up-regulated when the leaves are treated with ethylene. Deesterification of membrane lipids and ensuing loss of membrane structural integrity are well established early events of plant senescence, and the expression pattern of this lipase gene together with the lipolytic activity of its cognate protein indicate that it plays a fundamentally central role in mediating the onset of senescence.

Isolation and sequencing of cDNAs for splice variants of growth hormone-releasing hormone receptors from human cancers

The proliferation of various tumors is inhibited by the antagonists of growth hormone-releasing hormone (GHRH) in vitro and in vivo, but the receptors mediating the effects of GHRH antagonists have not been identified so far. Using an approach based on PCR, we detected two major splice variants (SVs) of mRNA for human GHRH receptor (GHRH-R) in human cancer cell lines, including LNCaP prostatic, MiaPaCa-2 pancreatic, MDA-MB-468 breast, OV-1063 ovarian, and H-69 small-cell lung carcinomas. In addition, high-affinity, low-capacity binding sites for GHRH antagonists were found on the membranes of cancer cell lines such as MiaPaCa-2 that are negative for the vasoactive intestinal peptide/pituitary adenylate cyclase-activating polypeptide receptor (VPAC-R) or lines such as LNCaP that are positive for VPAC-R. Sequence analysis of cDNAs revealed that the first three exons in SV1 and SV2 are replaced by a fragment of retained intron 3 having a new putative in-frame start codon. The rest of the coding region of SV1 is identical to that of human pituitary GHRH-R, whereas in SV2 exon 7 is spliced out, resulting in a 1-nt upstream frameshift, which leads to a premature stop codon in exon 8. The intronic sequence may encode a distinct 25-aa fragment of the N-terminal extracellular domain, which could serve as a proposed signal peptide. The continuation of the deduced protein sequence coded by exons 4–13 in SV1 is identical to that of pituitary GHRH-R. SV2 may encode a GHRH-R isoform truncated after the second transmembrane domain. Thus SVs of GHRH-Rs have now been identified in human extrapituitary cells. The findings support the view that distinct receptors are expressed on human cancer cells, which may mediate the antiproliferative effect of GHRH antagonists.

The role of cdc2 in the expression of herpes simplex virus genes

Earlier reports have shown that cdc2 kinase is activated in cells infected with herpes simplex virus 1 and that the activation is mediated principally by two viral proteins, the infected cell protein 22 (ICP22) and the protein kinase encoded by UL13. The same proteins are required for optimal expression of a subset of late (γ2) genes exemplified by US11. In this study, we used a dominant-negative cdc2 protein to determine the role of cdc2 in viral gene expression. We report the following. (i) The cdc2 dominant-negative protein had no effect in the synthesis and accumulation of at least two α-regulatory proteins (ICP4 and ICP0), two β-proteins (ribonucleotide reductase major subunit and single-stranded DNA-binding protein), and two γ1-proteins (glycoprotein D and viral protease). US11, a γ2-protein, accumulated only in cells in which cdc2 dominant-negative protein could not be detected or was made in very small amounts. (ii) The sequence of amino acids predicted to be phosphorylated by cdc2 is present in at least 27 viral proteins inclusive of the regulatory proteins ICP4, ICP0, and ICP22. In in vitro assays, we demonstrated that cdc2 specifically phosphorylated a polypeptide consisting of the second exon of ICP0 but not a polypeptide containing the sequence of the third exon as would be predicted from the sequence analysis. We conclude that cdc2 is required for optimal expression of a subset of γ2-proteins whose expression is also regulated by the viral proteins (ICP22 and UL13) that mediate the activation of cdc2 kinase.

Alternative splicing of the rat sodium/bile acid transporter changes its cellular localization and transport properties

Bile secretion involves the structural and functional interplay of hepatocytes and cholangiocytes, the cells lining the intrahepatic bile ducts. Hepatocytes actively secrete bile acids into the canalicular space and cholangiocytes then transport bile acids in a vectorial manner across their apical and basolateral plasma membranes. The initial step in the transepithelial transport of bile acids across rat cholangiocytes is apical uptake by a Na+-dependent bile acid transporter (ASBT). To date, the molecular basis of the obligate efflux mechanism for extrusion of bile acids across the cholangiocyte basolateral membrane remains unknown. We have identified an exon-2 skipped, alternatively spliced form of ASBT, designated t-ASBT, expressed in rat cholangiocytes, ileum, and kidney. Alternative splicing causes a frameshift that produces a 154-aa protein. Antipeptide antibodies detected the ≈19 kDa t-ASBT polypeptide in rat cholangiocytes, ileum, and kidney. The t-ASBT was specifically localized to the basolateral domain of cholangiocytes. Transport studies in Xenopus oocytes revealed that t-ASBT can function as a bile acid efflux protein. Thus, alternative splicing changes the cellular targeting of ASBT, alters its functional properties, and provides a mechanism for rat cholangiocytes and other bile acid-transporting epithelia to extrude bile acids. Our work represents an example in which a single gene appears to encode via alternative splicing both uptake and obligate efflux carriers in a bile acid-transporting epithelial cell.

Coordinate control of translation and localization of Vg1 mRNA in Xenopus oocytes

Vg1, a member of the transforming growth factor-β family involved in mesoderm induction, is translated subsequent to the localization of its mRNA to the vegetal pole of Xenopus oocytes. Whereas the localization of Vg1 mRNA is known to be directed by the 3′ untranslated region (UTR), the basis of its translational regulation is unknown. We show here that the 3′ UTR of Vg1 causes translational repression of two different reporter mRNAs in Xenopus oocytes. A 350-nucleotide region of the 3′ UTR, which is distinct from the localization element, is necessary and sufficient for mediating translational repression and specifically binds to a 38-kDa polypeptide. The translational repression activity is found throughout the oocyte and at all stages of oogenesis. These results suggest that factors colocalized with Vg1 mRNA at the vegetal pole relieve translational repression to allow expression of Vg1 protein.

Nonneuronal isoforms of STOP protein are responsible for microtubule cold stability in mammalian fibroblasts

A number of cycling mammalian cells, such as NIH 3T3, contain abundant subsets of cold-stable microtubules. The origin of such microtubule stabilization in nonneuronal cells is unknown. We have previously described a neuronal protein, stable tubule-only polypeptide (STOP), that binds to microtubules and induces cold stability. We find that NIH 3T3 fibroblasts contain a major 42-kDa isoform of STOP (fibroblastic STOP, F-STOP). F-STOP contains the central repeats characteristic of brain STOP but shows extensive deletions of N- and C-terminal protein domains that are present in brain STOP. These deletions arise from differences in STOP RNA splicing. Despite such deletions, F-STOP has full microtubule stabilizing activity. F-STOP accumulates on cold-stable microtubules of interphase arrays and is present on stable microtubules within the mitotic spindle of NIH 3T3 cells. STOP inhibition by microinjection of affinity-purified STOP central repeat antibodies into NIH 3T3 cells abolishes both interphase and spindle microtubule cold stability. Similar results were obtained with Rat2 cells. These results show that STOP proteins have nonneuronal isoforms that are responsible for the microtubule cold stability observed in mammalian fibroblasts.

Inactivation of the mouse sperm receptor, mZP3, by site-directed mutagenesis of individual serine residues located at the combining site for sperm

To initiate fertilization, mouse sperm bind to Ser- (O-) linked oligosaccharides located at the sperm combining site of zona pellucida glycoprotein mZP3. Apparently, the oligosaccharides are present on one or more of five Ser residues clustered in the carboxyl-terminal region of the mZP3 polypeptide. Here, each of the Ser residues, as well as an intervening Asn residue, was converted to a small, nonhydroxy amino acid by site-directed mutagenesis. Mouse embryonal carcinoma (EC) cells were then stably transfected with the wild-type and mutated mZP3 genes. In each case, transfected cells synthesized and secreted recombinant EC-mZP3 into the culture medium. The glycoproteins were partially purified and assayed for their ability to inhibit binding of sperm to ovulated eggs in vitro. As compared with wild-type EC-mZP3, mutations of Ser-329, Ser-331, or Ser-333 had no effect on sperm receptor activity. Mutation of Asn-330, a potential N-linked glycosylation site, also had no effect on sperm receptor activity. On the other hand, mutation of either Ser-332 or Ser-334, or mutation of Ser-332, Ser-333, and Ser-334, resulted in complete inactivation of EC-mZP3 as a sperm receptor. These results suggest that Ser-332 and Ser-334, residues conserved in mouse, hamster, and human ZP3, are essential for sperm receptor activity.

TOM1, an Arabidopsis gene required for efficient multiplication of a tobamovirus, encodes a putative transmembrane protein

Host-encoded factors play an important role in virus multiplication, acting in concert with virus-encoded factors. However, information regarding the host factors involved in this process is limited. Here we report the map-based cloning of an Arabidopsis thaliana gene, TOM1, which is necessary for the efficient multiplication of tobamoviruses, positive-strand RNA viruses infecting a wide variety of plants. The TOM1 mRNA is suggested to encode a 291-aa polypeptide that is predicted to be a multipass transmembrane protein. The Sos recruitment assay supported the hypothesis that TOM1 is associated with membranes, and in addition, that TOM1 interacts with the helicase domain of tobamovirus-encoded replication proteins. Taken into account that the tobamovirus replication complex is associated with membranes, we propose that TOM1 participates in the in vivo formation of the replication complex by serving as a membrane anchor.

A double-stranded RNA element from a hypovirulent strain of Rhizoctonia solani occurs in DNA form and is genetically related to the pentafunctional AROM protein of the shikimate pathway

M2 is a double-stranded RNA (dsRNA) element occurring in the hypovirulent isolate Rhs 1A1 of the plant pathogenic basidiomycete Rhizoctonia solani. Rhs 1A1 originated as a sector of the virulent field isolate Rhs 1AP, which contains no detectable amount of the M2 dsRNA. The complete sequence (3,570 bp) of the M2 dsRNA has been determined. A 6.9-kbp segment of total DNA from either Rhs 1A1 or Rhs 1AP hybridizes with an M2-specific cDNA probe. The sequences of M2 dsRNA and of PCR products generated from Rhs 1A1 total DNA were found to be identical. Thus this report describes a fungal host containing full-length DNA copies of a dsRNA element. A major portion of the M2 dsRNA is located in the cytoplasm, whereas a smaller amount is found in mitochondria. Based on either the universal or the mitochondrial genetic code of filamentous fungi, one strand of M2 encodes a putative protein of 754 amino acids. The resulting polypeptide has all four motifs of a dsRNA viral RNA-dependent RNA polymerase (RDRP) and is phylogenetically related to the RDRP of a mitochondrial dsRNA associated with hypovirulence in strain NB631 of Cryphonectria parasitica, incitant of chestnut blight. This polypeptide also has significant sequence similarity with two domains of a pentafunctional polypeptide, which catalyzes the five central steps of the shikimate pathway in yeast and filamentous fungi.

Cloning and characterization of a third human lysyl hydroxylase isoform

Lysyl hydroxylase (EC 1.14.11.4), a homodimer, catalyzes the formation of hydroxylysine in collagens. Recently, an isoenzyme termed lysyl hydroxylase 2 has been cloned from human sources [M. Valtavaara, H. Papponen, A.-M. Pirttilä, K. Hiltunen, H. Helander and R. Myllylä (1997) J. Biol. Chem. 272, 6831–6834]. We report here on the cloning of a third human lysyl hydroxylase isoenzyme, termed lysyl hydroxylase 3. The cDNA clones encode a 738 amino acid polypeptide, including a signal peptide of 24 residues. The overall amino acid sequence identity between the processed human lysyl hydroxylase 3 and 1 polypeptides is 59%, and that between the processed lysyl hydroxylase 3 and 2 polypeptides is 57%, whereas the identity to the processed Caenorhabditis elegans polypeptide is only 45%. All four recently identified critical residues at the catalytic site, two histidines, one aspartate, and one arginine, are conserved in all these polypeptides. The mRNA for lysyl hydroxylase 3 was found to be expressed in a variety of tissues, but distinct differences appear to exist in the expression patterns of the three isoenzyme mRNAs. Recombinant lysyl hydroxylase 3 expressed in insect cells by means of a baculovirus vector was found to be more soluble than lysyl hydroxylase 1 expressed in the same cell type. No differences in catalytic properties were found between the recombinant lysyl hydroxylase 3 and 1 isoenzymes. Deficiency in lysyl hydroxylase 1 activity is known to cause the type VI variant of the Ehlers–Danlos syndrome, and it is therefore possible that deficiency in lysyl hydroxylase 3 activity may lead to some other variant of this syndrome or to some other heritable connective tissue disorder.