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J Corbin

Publications and source records attributed to J Corbin.

At least 19 recordsLinked to original sources
PubMed2000

Autoinhibition of a calmodulin-dependent calcium pump involves a structure in the stalk that connects the transmembrane domain to the ATPase catalytic domain.

The regulation of Ca(2+)-pumps is important for controlling [Ca(2+)] in the cytosol and organelles of all eukaryotes. Here, we report a genetic strategy to identify residues that function in autoinhibition of a novel calmodulin-activated Ca(2+)-pump with an N-terminal regulatory domain (isoform ACA2 from Arabidopsis). Mutant pumps with constitutive activity were identified by complementation of a yeast (K616) deficient in two Ca(2+)-pumps. Fifteen mutations were found that disrupted a segment of the N-terminal autoinhibitor located between Lys(23) and Arg(54). Three mutations (E167K, D219N, and E341K) were found associated with the stalk that connects the ATPase catalytic domain (head) and with the transmembrane domain. Enzyme assays indicated that the stalk mutations resulted in calmodulin-independent activity, with V(max), K(mATP), and K(mCa(2+)) similar to that of a pump in which the N-terminal autoinhibitor had been deleted. A highly conservative substitution at Asp(219) (D219E) still produced a deregulated pump, indicating that the autoinhibitory structure in the stalk is highly sensitive to perturbation. In plasma membrane H(+)-ATPases from yeast and plants, similarly positioned mutations resulted in hyperactive pumps. Together, these results suggest that a structural feature of the stalk is of general importance in regulating diverse P-type ATPases.

ATP-Binding Cassette Transporters↗
PubMed2000

Suppressor of cytokine signaling-3 preferentially binds to the SHP-2-binding site on the shared cytokine receptor subunit gp130.

Suppressor of cytokine signaling-3 (SOCS-3) is one member of a family of intracellular inhibitors of signaling pathways initiated by cytokines that use, among others, the common receptor subunit gp130. The SH2 domain of SOCS-3 has been shown to be essential for this inhibitory activity, and we have used a quantitative binding analysis of SOCS-3 to synthetic phosphopeptides to map the potential sites of interaction of SOCS-3 with different components of the gp130 signaling pathway. The only high-affinity ligand found corresponded to the region of gp130 centered around phosphotyrosine-757 (pY757), previously shown to be a docking site for the tyrosine phosphatase SHP-2. By contrast, phosphopeptides corresponding to other regions within gp130, Janus kinase, or signal transducer and activator of transcription proteins bound to SOCS-3 with weak or undetectable affinity. The significance of pY757 in gp130 as a biologically relevant SOCS-3 docking site was investigated by using transfected 293T fibroblasts. Although SOCS-3 inhibited signaling in cells transfected with a chimeric receptor containing the wild-type gp130 intracellular domain, inhibition was considerably impaired for a receptor carrying a Y-->F point mutation at residue 757. Taken together, these data suggest that the mechanism by which SOCS-3 inhibits the gp130 signaling pathway depends on recruitment to the phosphorylated gp130 receptor, and that some of the negative regulatory roles previously attributed to the phosphatase SHP-2 might in fact be caused by the action of SOCS-3.

Amino Acid Sequence↗
PubMed2000

Halogenations of anthracenes and Dibenz

Halogenation of dibenz[a,c]anthracene (1) by NBS in CCl(4) affords the products of 9- and 10-monobromination in the ratio of 9:1. The reaction is accelerated by iodine, and HBr effects rearrangement of 9-bromo product to the sterically less crowded 10-bromo isomer. The mechanism is proposed to involve reversible addition of Br(2), followed by elimination of HBr. Reaction of NCS with 1 in CCl(4) requires addition of HCl and affords exclusively 9-chlorination. The different reactivities of NBS and NCS are ascribed to the relative amounts of free halogen produced (due to differences in N-X bond strengths involving Br and Cl), and the different sizes of the halogens. Under similar conditions, NCS chlorinates 9-bromoanthracene (2a) to afford 9,10-dichloroanthracene and 9-bromo-10-chloroanthracene in the ratio of 65:35. This reaction ostensibly occurs by addition of Cl(2) to 2a, followed by preferential loss of HBr rather than HCl. 9-Methylanthracene (3) affords exclusively 9-(bromomethyl)anthracene with NBS in the absence of iodine, but mainly (67%) 9-bromo-10-methylanthracene in the presence of iodine. Chlorination of 3 with NCS in the presence of HCl also affords mostly (65%) nuclear halogenation. Nuclear bromination of anthracene, 9-methylanthracene, and dibenz[a, c]anthracene by NBS in the absence of added HBr is accelerated by iodine. This effect is probably due to an increase in the amount of bromine produced from NBS in the presence of iodine.

Journal Article↗
PubMed1999

Murine Gcm1 gene is expressed in a subset of placental trophoblast cells.

The gcm gene of Drosophila melanogaster encodes a transcription factor that is an important component in cell fate specification within the nervous system. In the absence of a functional gcm gene, progenitor cells differentiate into neurons, whereas when the gene is ectopically expressed the cells produce excess glial cells at the expense of neuronal differentiation. Recent searches of databases have uncovered high sequence similarity between the Drosophila gem gene and an anonymous human placental cDNA clone (Altschuller et al., 1996; this communication). Here we report the molecular organization of the murine Gcm1, its spatio-temporal pattern of expression in developing placenta, and its map position at E1-E3 on murine chromosome 9. The murine gene is composed of at least 6 exons. The promoter region contains an "initiation sequence" and is GC rich, characteristics of the promoters of several transcription factors. The mRNA has a modest 5'UTR (ca. 200 bases) but an extensive 3' UTR (ca. 2 kb). Northern blot and mRNA in situ hybridization studies showed that Gcm1 expression was readily detectable only in the placenta. It began at embryonic day 7.5 within trophoblast cells of the chorion and continued to about embryonic day 17.5 within a subset of labyrinthine trophoblast cells. Comparison with other transcription factors revealed that Gcm1 expression defines a unique subset of trophoblast cells.

Animals↗
PubMed1999

Suppressors of cytokine signaling (SOCS): negative regulators of signal transduction.

SOCS-1 was originally identified as an inhibitor of interleukin-6 signal transduction and is a member of a family of proteins (SOCS-1 to SOCS-7 and CIS) that contain an SH2 domain and a conserved carboxyl-terminal SOCS box motif. Mutation studies have established that critical contributions from both the amino-terminal and SH2 domains are essential for SOCS-1 and SOCS-3 to inhibit cytokine signaling. Inhibition of cytokine-dependent activation of STAT3 occurred in cells expressing either SOCS-1 or SOCS-3, but unlike SOCS-1, SOCS-3 did not directly interact with or inhibit the activity of JAK kinases. Although the conserved SOCS box motif appeared to be dispensable for SOCS-1 and SOCS-3 action when overexpressed, this domain interacts with elongin proteins and may be important in regulating protein turnover. In gene knockout studies, SOCS-1(-/-) mice were born but failed to thrive and died within 3 weeks of age with fatty degeneration of the liver and hemopoietic infiltration of several organs. The thymus in SOCS-1(-/-) mice was small, the animals were lymphopenic, and deficiencies in B lymphocytes were evident within hemopoietic organs. We propose that the absence of SOCS-1 in these mice prevents lymphocytes and liver cells from appropriately controlling signals from cytokines with cytotoxic side effects.

Animals↗
PubMed1998

Identifying the cholesterol binding domain in the nicotinic acetylcholine receptor with [125I]azido-cholesterol.

A novel photoreactive analog of cholesterol, 3alpha-(4-azido-3-[125I]iodosalicylic)-cholest-5-ene ([125I]azido-cholesterol), was used to label both native acetylcholine receptor (AChR)-rich membranes from Torpedo californica and affinity-purified Torpedo AChRs reconstituted into lipid vesicles. In both cases all four AChR subunits incorporated [125I]azido-cholesterol on an equal molar basis and neither the pattern nor the extent of labeling was affected by the presence of the agonist carbamylcholine. Labeled regions in each of the AChR subunits were initially mapped by Staphylococcus aureus V8 protease digestion to large fragments which contain the AChR transmembrane segments. Sites of [125I]azido-cholesterol incorporation were further mapped by exhaustive tryptic digestion of the V8 protease subunit fragments alphaV8-20 (alphaSer-173-Glu-338), alphaV8-10 (alphaAsn-339-Gly-439), and gammaV8-14 (gammaLeu-373-Pro-489). The digests were separated by reverse-phase high-performance liquid chromatography and labeled peptides identified by amino-terminal sequence analysis. [125I]Azido-cholesterol labeling was localized to peptides that contain almost exclusively the alpha-M4, alpha-M1 and gamma-M4 membrane spanning segments. These results establish that the binding domain for cholesterol is at the lipid-protein interface of the AChR.

Affinity Labels↗
PubMed1998

Secondary structure analysis of individual transmembrane segments of the nicotinic acetylcholine receptor by circular dichroism and Fourier transform infrared spectroscopy.

Circular dichroism (CD) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy are used to establish the secondary structure of peptides containing one or more transmembrane segments (M1-M4) of the Torpedo californica nicotinic acetylcholine receptor (AChR). Peptides containing the M2-M3 and M1-M2-M3 transmembrane segments of the AChR beta-subunit and the M4 segment of the alpha- and gamma-subunits were isolated from proteolytic digests of receptor subunits, purified, and reconstituted into lipid vesicles. For each peptide, an amide I vibrational frequency centered between 1650 and 1656 cm-1 and negative CD absorption bands at 208 and 222 nm indicate that the peptide is largely alpha-helical. In addition, the CD spectrum of a tryptic peptide of the alpha-subunit containing the M1 segment is also consistent with a largely alpha-helical structure. However, secondary structure analysis of the alpha-M1 CD spectrum indicates the presence of other structures, suggesting that the M1 segment may represent either a distorted alpha-helix, likely the consequence of several proline residues, or may not be entirely alpha-helical. Overall, these findings are consistent with studies that indicate that the transmembrane region of the AChR comprises predominantly, if not exclusively, membrane-spanning alpha-helices.

Animals↗
PubMed1997

Progressive cyclic nucleotide-induced conformational changes in the cGMP-dependent protein kinase studied by small angle X-ray scattering in solution.

Small angle scattering data from bovine lung type Ialpha cGMP-dependent protein kinase (PKG) in the absence of cGMP show the protein to have a highly asymmetric structure with a radius of gyration (Rg) of 45 A and a maximum linear dimension (dmax) of 165 A. The addition of cGMP induces a marked conformational change in PKG. The Rg and dmax increase 25-30%, and the protein's mass moves further away from the center of mass; this results in an even more asymmetric structure. Fourier transform infrared spectroscopy data suggest that the conformational change induced by cGMP binding is primarily due to a topographical movement of the structural domains of PKG rather than to secondary structural changes within one or more of the individual domains. Each monomer of the dimeric PKG contains one high and one low affinity cGMP-binding site. A prominent increase in the asymmetry of PKG occurs with binding to high affinity cGMP-binding sites alone, but the full domain movements require the binding to both sets of sites. These conformational changes occurring in PKG with the progressive binding of cGMP to both sets of cGMP-binding sites correlate with past data, which have indicated that cGMP binding to both sets of sites is required for the full activation of the enzyme. These results provide the first quantitative measurement of the overall PKG structure, as well as an assessment of the structural events that accompany the activation of a protein kinase upon binding a small molecular weight ligand.

Animals↗
PubMed1997

Activation of cyclic AMP-dependent kinase is required but may not be sufficient to mimic cyclic AMP-dependent DNA synthesis and thyroglobulin expression in dog thyroid cells.

Thyrotropin (TSH), via a cyclic AMP (cAMP)-dependent pathway, induces cytoplasmic retractions, proliferation, and differentiation expression in dog thyroid cells. The role of cAMP-dependent protein kinase (PKA) in the induction of these events was assessed by microinjection into living cells. Microinjection of the heat-stable inhibitor of PKA (PKI) inhibited the effects of TSH, demonstrating that activation of PKA was required in this process. Overexpression of the catalytic (C) subunit of PKA brought about by microinjection of the expression plasmid pC alpha ev or of purified C subunit itself was sufficient to mimic the cAMP-dependent cytoplasmic changes and thyroperoxidase mRNA expression but not to induce DNA synthesis and thyroglobulin (Tg) expression. The cAMP-dependent morphological effect was not observed when C subunit was coinjected with the regulatory subunit (RI or RII subunit) of PKA. To mimic the cAMP-induced PKA dissociation into free C and R subunits, the C subunit was coinjected with the regulation-deficient truncated RI subunit (RIdelta1-95) or with wild-type RI or native RII subunits, followed by incubation with TSH at a concentration too low to stimulate the cAMP-dependent events by itself. Although the cAMP-dependent morphology changes were still observed, neither DNA synthesis nor Tg expression was stimulated in these cells. Taken together, these data suggest that in addition to PKA activation, another cAMP-dependent mechanism could exist and play an important role in the transduction of the cAMP signal in thyroid cells.

Animals↗
PubMed1997

Porcine aromatases: studies on tissue-specific, functionally distinct isozymes from a single gene?

Aromatase cytochrome P450 (P450arom) is expressed in a variety of tissues. Pigs express P450arom as bilaminar blastocysts in utero, and thereafter in the gonads, adrenal glands and placenta. Our studies also demonstrate the existence of porcine isozymes of P450arom which differ substantially in their amino acid composition and function. The placental isoform, most similar to P450arom in other mammals, consists of 503 amino acids. The ovarian isoform, expressed in both theca and granulosa cells, is a 501 amino acid protein exhibiting less than 20% of the activity of the placental isozyme. Furthermore, it is inhibited not only by CGS16949A but also by etomidate which does not inhibit the placental P450arom. Partial sequences generated by the rapid amplification of the cDNA ends (RACE) procedure indicate that the expression of a third isoform in the blastocyst is switched to the placental isozyme during differentiation of the fetal membranes. In addition, these transcripts, and others from the theca, granulosa, testes, adrenal glands and placenta demonstrate differences in the 5'-untranslated region (putative exon I) suggestive of tissue-specific alternative splicing. An identical 5'-untranslated sequence was obtained from transcripts expressed in the theca and granulosa. Testes and adrenal transcripts also have identical 5' ends, which differ substantially from the ovarian sequence. Blastocyst and placenta 5'-untranslated sequences differ from each other and from those expressed in the gonads and adrenals. Several tissue-specific transcripts thus encode porcine P450arom. Interestingly, distinct 5' sequences exist for ovarian and testes P450arom mRNAs, suggesting different promoters and therefore regulation in the male and female gonads. The molecular origins of the functional isoforms and the tissue-specific transcripts are uncertain, however partial genomic sequence and other genetic analyses suggest the existence of multiple genes. However, sequence alignment of the placental and ovarian isoforms indicates complete conservation of putative exon III, so that complex splicing remains a possibility. Clearly, the regulation of P450arom expression is more complex in the pig than in other vertebrates investigated to date.

Amino Acid Sequence↗
PubMed1996

Ser-27, Tyr-10 and Tyr-7 in the alpha-chain of pig stomach H+,K(+)-ATPase as Ca(2+)-dependent phosphorylatable sites by intrinsic and extrinsic protein kinases.

When pig stomach membrane H+,K(+)-ATPase preparations were incubated with [gamma-32P]ATP, Mg2+ and Ca2+, reversible phosphorylation of specific Tyr and Ser residues in the N-terminal alpha-chain of H+,K(+)-ATPase occurred without any detectable phosphorylation in other regions of the alpha-chain. Mild tosylphenylalanyl chloromethyl ketone trypsin treatment followed by reverse-phase column chromatography yielded three radioactive peptide peaks. The first peak contained both Tyr10(32P) and Tyr7(32P) and the second peak contained Tyr10(32P). The third peak contained Ser27(32P) which was also obtained after trypsin treatment of partially purified H+,K(+)-ATPase preparations phosphorylated with protein kinase-C + Ca2+ or protein kinase-A. This is the first demonstration of Ca2(+)-dependent phosphorylation of the alpha-chain of H+,K(+)-ATPase by protein kinases.

Amino Acid Sequence↗
PubMed1996

Proximal regions of the olfactory marker protein gene promoter direct olfactory neuron-specific expression in transgenic mice.

Olfactory marker protein (OMP) expression is highly restricted to mature olfactory neurons (ON). Less than 0.3 kb of upstream 5' flanking sequence of the OMP gene directs lacZ expression preferentially to ON in several independently derived lines of transgenic mice. A larger transgene with 0.8 kb of upstream flanking sequence also gave lacZ expression in ON and in a few ectopic sites in the central nervous system (CNS). In addition to the main olfactory epithelium, endogenous OMP is also expressed in chemosensory neurons of the vomeronasal and septal organs, and lacZ expression was detected in neurons of these sites as well. This confirmed the presence of regulatory sequences in the proximal portion of the OMP gene. Endogenous OMP expression in ON was normal in all transgenic lines. Strikingly, in several transgenic lines lacZ expression was restricted to subsets of ON. In one such line, ON axons were intensely stained for lacZ and projected to a subset of olfactory bulb glomeruli. Although identifiable subsets of ON and their termination fields have been described previously, this is the first demonstration of this phenomenon in transgenic mice. These lines of transgenic mice thus provide in vivo models for characterization of genetic elements regulating developmental and functional organization of the olfactory neuroepithelium.

Animals↗
PubMed1989

Pairs of cyclic AMP analogs, that are specifically synergistic for type I and type II cAMP-dependent protein kinases, mimic thyrotropin effects on the function, differentiation expression and mitogenesis of dog thyroid cells.

The role of the two different isozymes of the cAMP-dependent protein kinase is still unclear. We have investigated the potential roles for each isozyme in dog thyroid cells, a model in which the function, expression of differentiation and proliferation are positively regulated by thyrotropin acting through cyclic AMP. The dog thyroid contains both type I and type II cAMP-dependent protein kinases. These isozymes were selectively activated in vitro by type-I-directed and type-II-directed analog pairs. In thyroid slices, both type-I directed and type II-directed analog pairs synergistically activated thyroid hormone synthesis, as measured by incorporation of 131I into proteins and thyroid hormone secretion as determined by the release of butanol-extractable 131I. In primary cultures of dog thyroid cells both isozyme-directed analog pairs synergistically enhanced iodide trapping, a marker of differentiation, and DNA synthesis, as measured by the percentage of cells incorporating [3H]thymidine into their nuclei. However, DNA synthesis was more sensitive to type-I-directed pairs. The results demonstrate that both cAMP-dependent protein kinase isozymes can mediate the action of cAMP on function, differentiation expression and cell proliferation in dog thyroid cells.

Animals↗
PubMed1989

Phosphorylation modulates the activity of glycine N-methyltransferase, a folate binding protein. In vitro phosphorylation is inhibited by the natural folate ligand.

Glycine N-methyltransferase (EC 2.1.1.20) was recently identified as a major folate binding protein of rat liver cytosol (Wagner, C., and Cook, R. J. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3631-3634). Activity of the enzyme is inhibited when the natural folate ligand, 5-methyltetrahydropteroylpentaglutamate (5-CH3-H4PteGlu5), is bound. It has been suggested that glycine N-methyltransferase plays a role in regulating the availability of methyl groups in the liver. Purified transferase was phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase. If 5-CH3-H4PteGlu5 was first bound to the transferase, phosphorylation was inhibited. Phosphorylation of glycine N-methyltransferase in vitro increased its activity approximately 2-fold. 5-CH3-H4PteGlu5 inhibited the activity of newly phosphorylated enzyme as well as native enzyme. Freshly isolated rat hepatocytes incorporated 32P-labeled inorganic phosphate into this folate binding protein. Chemical analysis of purified enzyme showed about 0.55 mol of phosphate present per mol of glycine N-methyltransferase subunit. These results indicate that phosphorylation of glycine N-methyltransferase may provide a mechanism for modulating the activity of this enzyme and support its role in regulating the availability of methyl groups.

Animals↗
PubMed1985

Inhibition of arachidonic acid metabolism by antipyrine and 4-aminoantipyrine.

Antipyrine and 4-aminoantipyrine are used to determine uterine and umbilical blood flows. When administered in vivo, these compounds produce a decrease in uterine contracture activity and maternal uterine vein 13,14-dihydro-15-keto-prostaglandin F2 alpha concentrations. In this paper we report that they also inhibit in vitro activity of prostaglandin synthase in bovine seminal microsomes.

Ampyrone↗