Molecular cloning of cDNAs for the nerve-cell specific phosphoprotein, synapsin I.

To provide access to synapsin I-specific DNA sequences, we have constructed cDNA clones complementary to synapsin I mRNA isolated from rat brain. Synapsin I mRNA was specifically enriched by immunoadsorption of polysomes prepared from the brains of 10-14 day old rats. Employing this enriched mRNA, a cDNA library was constructed in pBR322 and screened by differential colony hybridization with single-stranded cDNA probes made from synapsin I mRNA and total polysomal poly(A)+ RNA. This screening procedure proved to be highly selective. Five independent recombinant plasmids which exhibited distinctly stronger hybridization with the synapsin I probe were characterized further by restriction mapping. All of the cDNA inserts gave restriction enzyme digestion patterns which could be aligned. In addition, some of the cDNA inserts were shown to contain poly(dA) sequences. Final identification of synapsin I cDNA clones relied on the ability of the cDNA inserts to hybridize specifically to synapsin I mRNA. Several plasmids were tested by positive hybridization selection. They specifically selected synapsin I mRNA which was identified by in vitro translation and immunoprecipitation of the translation products. The established cDNA clones were used for a blot-hybridization analysis of synapsin I mRNA. A fragment (1600 bases) from the longest cDNA clone hybridized with two discrete RNA species 5800 and 4500 bases long, in polyadenylated RNA from rat brain and PC12 cells. No hybridization was detected to RNA from rat liver, skeletal muscle or cardiac muscle.     

Molecular cloning of mammalian Spred-3 which suppresses tyrosine kinase-mediated Erk activation

We have reported on Spred-1 and Spred-2, which inhibit MAP kinase activation by interacting with c-kit and ras/raf. Here, we report the cloning of a third member in this family, Spred-3. Spred-3 is expressed exclusively in the brain and its gene locates in chromosome 19q13.13 in human. Like Spred-1 and -2, Spred-3 contains an EVH1 domain in the N-terminus and a Sprouty-related cysteine-rich region (SPR domain) in the C-terminus that is necessary for membrane localization. However, Spred-3 does not possess a functional c-kit binding domain (KBD), since the critical amino acid Arg residue in this region was replaced with Gly in Spred-3. Although Spred-3 suppressed growth factor-induced MAP kinase (Erk) activation, inhibitory activity of Spred-3 was lower than that of Spred-1 or Spred-2. By the analysis of chimeric molecules between Spred-3 and Spred-1, we found that the SPR domain, rather than KBD, is responsible for efficient Erk suppression. The finding of Spred-3 revealed the presence of a novel family of regulators for the Ras/MAP kinase pathway, each member of which may have different specificities for extracellular signals.     

Molecular cloning, expression, and uses of mammalian glycosyltransferases.

Lineage specific expression patterns of mammalian oligosaccharides are regulated largely by glycosyltransferases. Several distinct glycosyltransferase genes have recently been isolated by gene transfer and traditional cloning approaches. These each maintain a type II transmembrane topology, that yields a COOH-terminal, Golgi-resident catalytic domain. However, significant primary sequence similarities between cloned glycosyltransferases have been found only between enzymes with substantial functional similarity. These cloned DNA segments, and their cognate enzymes, have been used to determine glycosyltransferase expression patterns, characterize the molecular basis for mutant glycosyltransferase alleles, and identify oligosaccharide ligands for members of the Selectin family of cell adhesion molecules. They have also shown promise for use in the preparative synthesis of defined oligosaccharide molecules, and for transgenic animal approaches designed to explore the function(s) of oligosaccharides in mammalian organisms.     

cDNA cloning of the HMGI-C phosphoprotein, a nuclear protein associated with neoplastic and undifferentiated phenotypes.

The HMGI-C protein is a nuclear phosphoprotein expressed at high levels in transformed cells. The cDNA encoding the mouse protein has been isolated and the sequence of the encoded protein shows that it is related to the HMGY and I proteins, proteins which bind in the minor groove of DNA containing stretches of A and T. The HMGI-C protein has three short highly basic domains, an acidic C-terminal domain, and potential CDC2/p34 and casein kinase II phosphorylation sites. Analysis of mRNA levels demonstrate that the HMGI-C gene is not expressed in a variety of mouse tissues but is expressed in Lewis lung carcinoma cells.     

Nickel serves as a substrate recognition motif for the endopeptidase involved in hydrogenase maturation.

The interaction of the hydrogenase maturation endopeptidase HycI with its substrate, the precursor of the large subunit, was studied. Replacement of conserved amino-acid residues in HycI, which have been shown to bind a cadmium ion from the crystallization buffer in crystals of HybD (endopeptidase for hydrogenase 2), abolished or strongly reduced processing activity. Atomic absorption spectroscopy of purified HycI and HybD proteins showed the absence of nickel. In vitro processing assays showed that the reaction requires nickel to be bound to the precursor and the protease does not have a function in nickel delivery to the substrate. Radioactive labelling of cells with 63Ni, devoid of endopeptidase, resolved several forms of the precursor which are possibly intermediates in the maturation pathway. It is concluded that the endopeptidase uses the metal in the large subunit of [NiFe]-hydrogenases as a recognition motif.     

Identification of tyrosine hydroxylase as a physiological substrate for Cdk5

Cyclin-dependent kinase 5 (Cdk5) is emerging as a neuronal protein kinase involved in multiple aspects of neurotransmission in both post- and presynaptic compartments. Within the reward/motor circuitry of the basal ganglia, Cdk5 regulates dopamine neurotransmission via phosphorylation of the postsynaptic signal transduction pathway integrator, DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, M(r) 32,000). Cdk5 has also been implicated in regulating various steps in the presynaptic vesicle cycle. Here we report that Cdk5 phosphorylates tyrosine hydroxylase (TH), the key enzyme for synthesis of dopamine. Using phosphopeptide mapping, site-directed mutagenesis, and phosphorylation state-specific antibodies, the site was identified as Ser31, a previously defined extracellular signal-regulated kinases 1/2 (ERK1/2) site. The phosphorylation of Ser31 by Cdk5 versus ERK1/2 was investigated in intact mouse striatal tissue using a pharmacological approach. The results indicated that Cdk5 phosphorylates TH directly and also regulates ERK1/2-dependent phosphorylation of TH through the phosphorylation of mitogen-activated protein kinase kinase 1 (MEK1). Finally, phospho-Ser31 TH levels were increased in dopaminergic neurons of rats trained to chronically self-administer cocaine. These results demonstrate direct and indirect regulation of the phosphorylation state of a Cdk5/ERK1/2 site on TH and suggest a role for these pathways in the neuroadaptive changes associated with chronic cocaine exposure.     

An involucrin-like protein in hepatocytes serves as a substrate for tissue transglutaminase during apoptosis.

Cornified envelopes and apoptotic bodies are transglutaminase-cross-linked end-products of physiological cell death pathways. The two structures have similar amino acid composition. Involucrin has been considered as a cornified envelope precursor protein expressed specifically in terminally differentiating keratinocytes and squamous epithelia. We report the presence in hepatocytes of an involucrin-like protein which could be purified from dog liver with procedures characteristic to involucrins. When compared to purified dog esophagus involucrin, the liver protein also reacts with anti-involucrin antibodies, has the same relative molecular mass, possesses similar amino acid composition, and shows almost identical peptide mapping pattern. The involucrin-like protein is detectable by immunohistochemistry in normal and apoptotic hepatocytes, is a substrate of tissue transglutaminase, and is incorporated into cross-linked apoptotic bodies. These results suggest that there are overlapping molecular components in the two characteristic forms (cornification and apoptosis) of naturally occurring cell death.     

The product of the UL31 gene of herpes simplex virus 1 is a nuclear phosphoprotein which partitions with the nuclear matrix.

The nucleotide sequence of the UL31 open reading frame is predicted to encode a basic protein with a hydrophilic amino terminus and a nuclear localization signal. To identify its gene product, we constructed a viral genome in which the thymidine kinase gene was inserted between the UL31 and UL32 open reading frames. The thymidine kinase gene was then deleted, and in the process, the 5' terminus of the UL31 open reading frame was replaced with a 64-bp sequence in frame with the complete, authentic sequence of the UL31 open reading frame. The inserted sequence encoded a hydrophilic epitope derived from glycoprotein B of human cytomegalovirus and for which a monoclonal antibody is available. We report that in infected cells, the tagged protein localized in and was dispersed throughout the nucleus. Nuclear fractionation studies revealed that the UL31 protein partitions with the nuclear matrix. The protein is phosphorylated in infected cells maintained in medium containing 32Pi.     

Molecular cloning and characterization of a novel mammalian endo-apyrase (LALP1)

Here we describe the cloning, localization, and characterization of a novel mammalian endo-apyrase (LALP1) in human and mouse. The predicted human LALP1 gene encodes a 604-amino acid protein, whereas the mouse Lalp1 gene encodes a 606-amino acid protein. The human and mouse genes have 88% amino acid sequence identity. These genes share considerable homologies with hLALP70, a recently discovered mammalian lysosomal endo-apyrase. The human LALP1 gene resides on chromosome 10q23-q24 and contains 12 exons and 11 introns covering a genomic region of approximately 46 kilobase pairs. The subcellular localization and enzymatic activity of LALP1 indicated that LALP1 is indeed an endo-apyrase with substrate preference for nucleoside triphosphates UTP, GTP, and CTP.     

Molecular cloning of a novel retinoic acid-responsive gene, HA1R-62, which is also up-regulated in Hoxa-1-overexpressing cells.

Using a PCR-based cDNA subtractive hybridization method (L. Diatchenko et al., Proc. Natl. Acad. Sci. USA, 93: 6025-6030, 1996), we cloned a cDNA fragment of a novel gene that is highly expressed in F9-10; F9-10 is an F9 teratocarcinoma stem cell line that expresses high levels of exogenous Hoxa-1 mRNA and protein in comparison to F9 wild-type stem cells, which do not express endogenous Hoxa-1 mRNA in the absence of retinoic acid (RA). Rapid amplification of cDNA ends was used to clone the full-length cDNA of this gene, designated HA1R-62 (Hoxa1 regulated-62). We have shown that HA1R-62 is also a RA-responsive gene and that it is expressed (mRNA size, approximately 4.3 kb) in adult mouse thymus, lung, kidney, and ovary as well as in 12.5-day mouse embryos. DNA sequence analysis and in vitro translation experiments have shown that HA1R-62 encodes a protein with a molecular mass of approximately 26 kDa. Elucidation of the function of the HA1R-62 gene product will provide new insights into the functions of RA and homeobox genes.     

Molecular cloning of MINK, a novel member of mammalian GCK family kinases, which is up-regulated during postnatal mouse cerebral development

A new germinal center kinase (GCK) family kinase, Misshapen/NIKs-related kinase (MINK), has been cloned and its expression has been characterized in several tissues and various developmental stages of the mouse brain. MINK encodes a 1300 amino acid polypeptide, consisting of an N-terminal kinase domain, a proline-rich intermediate region, and a C-terminal GCK homology region. The expression of MINK is up-regulated during the postnatal development of the mouse brain. MINK activates the cJun N-terminal kinase and the p38 pathways.     

Nucleophosmin serves as a rate-limiting nuclear export chaperone for the Mammalian ribosome

Nucleophosmin (NPM) (B23) is an essential protein in mouse development and cell growth; however, it has been assigned numerous roles in very diverse cellular processes. Here, we present a unified mechanism for NPM's role in cell growth; NPM directs the nuclear export of both 40S and 60S ribosomal subunits. NPM interacts with rRNA and large and small ribosomal subunit proteins and also colocalizes with large and small ribosomal subunit proteins in the nucleolus, nucleus, and cytoplasm. The transduction of NPM shuttling-defective mutants or the loss of Npm1 inhibited the nuclear export of both the 40S and 60S ribosomal subunits, reduced the available pool of cytoplasmic polysomes, and diminished overall protein synthesis without affecting rRNA processing or ribosome assembly. While the inhibition of NPM shuttling can block cellular proliferation, the dramatic effects on ribosome export occur prior to cell cycle inhibition. Modest increases in NPM expression amplified the export of newly synthesized rRNAs, resulting in increased rates of protein synthesis and indicating that NPM is rate limiting in this pathway. These results support the idea that NPM-regulated ribosome export is a fundamental process in cell growth.     

Purification, characterization, and molecular cloning of a 60-kDa phosphoprotein in rabbit skeletal sarcoplasmic reticulum which is an isoform of phosphoglucomutase.

A 60-kDa substrate of calmodulin-dependent protein kinase in rabbit "heavy" skeletal sarcoplasmic reticulum (SR) was characterized by purification and cDNA cloning. Purification was achieved by column chromatography using DEAE-Sephacel, heparin-agarose, and hydroxylapatite in 0.5% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid (CHAPS). Analyses of amino acid sequence and composition indicated that the CHAPS-soluble 60-kDa protein is an isoform of phosphoglucomutase (PGM). cDNAs encoding two isoforms of PGM were isolated from rabbit skeletal muscles. The translated amino acid sequences show that the isoforms, PGM1 and PGM2, differ in the N-terminal 77 amino acids and that PGM2 is identical to the 60-kDa protein in the SR. Northern blot analysis showed that the size of the mRNA encoding PGM2 is 2.4 kilobases. The PGM enzyme activity was markedly inhibited in SR membranes, while perturbation of the membranes with CHAPS or guanidine-HCl recovered the enzyme activity. KCl (0.15-1 M) led to a partial recovery of the enzyme activity suggesting that the charge interaction is not the primary force for PGM-SR interaction. PGM is localized in the heavy fraction of SR, where calsequestrin and Ca2+ release channel are enriched. Our results demonstrate that an isoform of PGM localized in junctional skeletal SR is the 60-kDa substrate of calmodulin-dependent protein kinase.     

IL-1 serves as a secondary signal for IL-9 expression.

IL-9 is produced in vitro by activated CD4+ T cell lines of the Th2 subtype and by naive CD4+ T cells. Here we show that T cell lines stimulated with Con A in the presence of accessory cells (AC) such as irradiated spleen cells or bone marrow-derived macrophages produced substantially more IL-9 than T cells stimulated with Con A alone. These data suggest that AC influence the production of IL-9 through accessory signals that result in an at least 10-fold increase of IL-9 secretion by the respective T cells. Addition of IL-1 to T cells activated by Con A, PHA, or anti-CD3 antibodies revealed that this monokine was responsible for the potentiation of IL-9 production. This finding was confirmed by applying anti-IL-1 antibodies. The production of other lymphokines, namely, IL-3, IL-4, and IL-6, by activated T cells was not or only marginally enhanced in the presence of AC or IL-1, thus indicating that the synthesis of IL-9 is regulated differently from that of other Th2-derived lymphokines. Furthermore, it was demonstrated by Northern blot analysis that IL-1 increases IL-9 expression at the pretranslational level. Because IL-1 alone failed to induce the production of IL-9 by T cells, this monokine acts as a costimulator in combination with a T cell receptor-mediated signal.