Dennis Warner - Academia.edu (original) (raw)

Papers by Dennis Warner

Molecular Endocrinology, 1997

A novel G(S alpha) mutation encoding the substitution of arginine for serine 250 (G[S alpha] S250... more A novel G(S alpha) mutation encoding the substitution of arginine for serine 250 (G[S alpha] S250R) was identified in a patient with pseudohypoparathyroidism type Ia. Both G(S) activity and G(S alpha) expression were decreased by about 50% in erythrocyte membranes from the affected patient. The cDNA of this G(S alpha) mutant, as well as one in which the S250 residue is deleted (G[S alpha]-deltaS250), was generated, and the biochemical properties of the products of in vitro transcription/translation were examined. Both mutants had a sedimentation coefficient similar to that of wild type G(S alpha) (approximately 3.7S) when kept at 0 C after synthesis. However when maintained for 1-2 h at 30-37 C, both mutants aggregated to a material sedimenting at approximately 6.3S or greater (G[S alpha]-S250R to a greater extent than G(S alpha]-deltaS250), while wild type G(S alpha) sedimented at approximately 3.7S, suggesting that the mutants were thermolabile. Incubation in the presence of high doses of guanine nucleotide partially prevented heat denaturation of G(S alpha) deltaS250 but had no protective effect on G(S alpha-S250R. Sucrose density gradient centrifugation at 0 C in the presence and absence of beta gamma-dimers demonstrated that, in contrast to wild type G(S alpha) neither mutant could interact with beta gamma. Trypsin protection assays revealed no protection of G(S alpha)-S250R by GTPgammaS or AIF4- at any temperature. GTPgammaS conferred modest protection of G(S alpha)-deltaS250 (approximately 50% of wild-type G[S alpha]) at 30 C but none at 37 C, while AIF4- conferred slight protection at 20 C but none at 30 C or above. Consistent with this result, G(S alpha)-deltaS250 was able to stimulate adenylyl cyclase at 30 C when reconstituted with cyc- membranes in the presence of GTPgammaS but not in the presence of AIF4-. G(S alpha)-S250R showed no ability to stimulate adenylyl cyclase in the presence of either agent. Stable transfection of mutant and wild-type G(S alpha) into cyc- S49 lymphoma cells revealed that the majority of wild type G(S alpha) localized to membranes, while little or no membrane localization occurred for either mutant. Modeling of G(S alpha) based upon the crystal structure of G(t alpha) or G(i alpha) suggests that Ser250 interacts with several residues within and around the conserved NKXD motif, which directly interacts with the guanine ring of bound GDP or GTP. It is therefore possible that substitution or deletion of this residue may alter guanine nucleotide binding, which could lead to thermolability and impaired function.

Febs Letters, 2003

A yeast two-hybrid screen was utilized to identify novel Smad 3 binding proteins expressed in dev... more A yeast two-hybrid screen was utilized to identify novel Smad 3 binding proteins expressed in developing mouse orofacial tissue. Three proteins (Erbin, Par-3, and Dishevelled) were identi¢ed that share several similar structural and functional characteristics. Each contains at least one PDZ domain and all have been demonstrated to play a role in the establishment and maintenance of cell polarity. In GST (glutathione S-transferase) pull-down assays, Erbin, Par-3, and Dishevelled bound strongly to the isolated MH2 domain of Smad 3, with weaker binding to a full-length Smad 3 protein. Failure of Erbin, Par-3, and Dishevelled to bind to a Smad 3 mutant protein that was missing the MH2 domain con¢rms that the binding site resides within the MH2 domain. Erbin, Par-3, and Dishevelled also interacted with the MH2 domains of other Smads, suggesting broad Smad binding speci¢city. Dishevelled and Erbin mutant proteins, in which the PDZ domain was removed, still retained their ability to bind Smad 3, albeit with lower a⁄nity. While transforming growth factor L L (TGFL L) has been suggested to alter cell polarity through a Smad-independent mechanism involving activation of members of the RhoA family of GTP binding proteins, the observation that Smads can directly interact with proteins involved in cell polarity, as shown in the present report, suggests an additional means by which TGFL L could alter cell polarity via a Smad-dependent signaling mechanism.

Biochemical and Biophysical Research Communications, 2003

Ontogenesis of the mammalian orofacial region is controlled by numerous developmental signals, in... more Ontogenesis of the mammalian orofacial region is controlled by numerous developmental signals, including those initiated by the transforming growth factors b (TGFbs). Targeted deletion of the genes encoding several of the TGFbs in mice has been shown to result in clefts of the secondary palate. Members of the TGFb family of growth factors utilize intracellular Smads as signal transducers. Smads 2 and 3 are transcriptional regulators that bind DNA through their conserved MH1 domains and activate/ inhibit transcription of TGFb-responsive genes through their MH2 domains. Using a yeast two-hybrid screen of a cDNA expression library constructed from fetal murine orofacial tissue, we have identified three types of collagens (types I, III, and V) that are capable of binding to the MH2 domain of Smad 3. These interactions were confirmed by glutathione S-transferase (GST) pull-down assays in which the MH2 domain of Smad 3 fused to GST interacted strongly with in vitro translated, 35 S-labeled collagen types I, III, and V. Each collagen also bound to the MH2 domains of Smads 4 and 7 and, to a lesser extent, full-length Smads 1, 2, 3, and 4. Binding of Smads to collagen is a novel observation. Moreover, TGFb is a potent regulator of collagen synthesis and turnover during mammalian orofacial development. These data thus suggest an important means of feedback regulation of the TGFb signaling cascade.

Biochemical and Biophysical Research Communications, 2004

The transforming growth factors b control a diversity of biological processes including cellular ... more The transforming growth factors b control a diversity of biological processes including cellular proliferation, differentiation, apoptosis, and extracellular matrix production, and are critical effectors of embryonic patterning and development, including that of the orofacial region. TGFb superfamily members signal through specific cell surface receptors that phosphorylate the cytoplasmic Smad proteins, resulting in their translocation to the nucleus and interaction with promoters of TGFb-responsive genes. Subsequent alterations in transcription are cell type-specific and dependent on recruitment to the Smad/transcription factor complex of coactivators, such as CBP and p300, or corepressors, such as c-ski and SnoN. Since the affinity of Smads for DNA is generally low, additional accessory proteins that facilitate Smad/DNA binding are required, and are often cell-and tissue-specific. In order to identify novel Smad 3 binding proteins in developing orofacial tissue, a yeast two hybrid assay was employed in which the MH2 domain of Smad 3 was used to screen an expression library derived from mouse embryonic orofacial tissue. The RNA helicase, p68, was identified as a unique Smad binding protein, and the specificity of the interaction was confirmed through various in vitro and in vivo assays. Co-expression of Smad 3 and a CBP-Gal4 DNA binding domain fusion protein in a Gal4-luciferase reporter assay resulted in increased TGFb-stimulated reporter gene transcription. Moreover, co-expression of p68 RNA helicase along with Smad 3 and CBP-Gal4 resulted in synergistic activation of Gal4-luciferase reporter expression. Collectively, these data indicate that the RNA helicase, p68, can directly interact with Smad 3 resulting in formation of a transcriptionally active ternary complex containing Smad 3, p68, and CBP. This offers a means of enhancing TGFb-mediated cellular responses in developing orofacial tissue.

Journal of Interferon and Cytokine Research, 2005

cAMP response element binding protein (CREB)-binding protein (CBP) is a multifunctional transcrip... more cAMP response element binding protein (CREB)-binding protein (CBP) is a multifunctional transcriptional coactivator that plays important roles in gene regulation. CBP is expressed in murine embryonic orofacial tissue, where it is developmentally regulated. To identify nuclear factors associated with CBP in developing orofacial tissue, a yeast two-hybrid screen of a cDNA library derived from embryonic orofacial tissue from gestational days 11-13 mouse embryos was conducted. Using the carboxy terminal region of CBP as bait, the protein inhibitor of activated Stat1 (PIAS1) was identified as a novel CBP binding protein. The association of PIAS1 with CBP was confirmed in vitro by glutathione S-transferase (GST) pull-down assays and in vivo by coimmunoprecipitation. Reporter assays demonstrated that PIAS1 inhibited CBP-mediated transcriptional activation in the presence or absence of transforming growth factor-beta (TGF-beta). These results identify PIAS1 as a novel binding partner for CBP and inhibitor of CBP-mediated transcription, suggesting that PIAS1 might play a role in regulating cell proliferation, migration, and differentiation during embryonic development.

Biochimica Et Biophysica Acta-molecular Cell Research, 2007

TGFβ signaling regulates central cellular processes such as proliferation and extracellular matri... more TGFβ signaling regulates central cellular processes such as proliferation and extracellular matrix production during development of the orofacial region. Extracellular TGFβ binds to cell surface receptors to activate the nucleocytoplasmic Smad proteins that, along with other transcription factors and cofactors, bind specific DNA sequences in the promoters of target genes to regulate their expression. To determine the identity of Smad binding proteins that regulate TGFβ signaling in developing murine orofacial tissue, a yeast two-hybrid screening approach was employed. The PR-domain containing protein, PRDM16/MEL1 was identified as a novel Smad binding protein. The interaction between PRDM16/MEL1 and Smad 3 was confirmed by GST pull-down assays. The expression of PRDM16/MEL1 was detected in developing orofacial tissue by both Northern blot and in situ hybridization. PRDM16/MEL1 was constitutively expressed in orofacial tissue on E12.5-E14.5 as well as other embryonic tissues such as heart, brain, liver, and limb buds. Taken together, these results demonstrate that PRDM16/MEL1 is a Smad binding protein that may be important for development of orofacial structures through modulation of the TGFβ signaling pathway.

Biochemical and Biophysical Research Communications, 2003

The TGFbs, a family of secreted polypeptide growth factors, are critical regulators of mammalian ... more The TGFbs, a family of secreted polypeptide growth factors, are critical regulators of mammalian orofacial development. The importance of the TGFbs in development of the orofacial region in mice is underscored by the resulting orofacial clefts in mice with targeted deletion of either TGFb2 or TGFb3 and most recently, a conditional knockout of the type II TGFb receptor (TbRII) gene. The TGFbs signal via binding to specific cell surface receptors which, in turn, activates translocation of the nucleocytoplasmic Smad transcriptional regulators. Smads 2 and 3 are TGFb-specific transcriptional regulators that bind DNA through their conserved MH1 domains and activate or inhibit transcription of TGFb-responsive genes through their MH2 domains. To search for novel Smad binding proteins expressed in developing murine orofacial tissue, a yeast two-hybrid assay was utilized to screen a cDNA expression library constructed from fetal murine orofacial tissue. Several novel Smad binding proteins were identified. These include a putative zinc finger protein (ZNF198), peroxisomal biogenesis factor 6 (Pex6), eucaryotic translation initiation factor 4E nuclear import factor 1 (4-ET), and splicing factor 3b subunit 2 (SF3b2). Results of the yeast two-hybrid screen were verified by GST pulldown assays which confirmed the interaction of these proteins with the MH2 domain of Smad 3, and also indicated interaction of these proteins with additional Smad family members. The identification of these proteins as Smad binding partners allows exploration of new mechanisms whereby TGFb signaling may be regulated, and reveals additional potential interactions with other signaling pathways.

Febs Letters, 2005

The transforming growth factor beta (TGFb) and Wnt signaling pathways play central roles regulati... more The transforming growth factor beta (TGFb) and Wnt signaling pathways play central roles regulating embryogenesis and maintaining adult tissue homeostasis. TGFb mediates its cellular effects through types I and II cell surface receptors coupled to the nucleocytoplasmic Smad proteins. Wnt signals via binding to a cell surface receptor, Frizzled, which in turn activates intracellular Dishevelled, ultimately leading to stabilization and nuclear translocation of b-catenin. Previous studies have demonstrated several points of cross-talk between the TGFb and Wnt signaling pathways. In yeast two-hybrid and GST-pull down assays, Dishevelled-1 and Smad 3 have been shown to physically interact through the C-terminal one-half of Dishevelled-1 and the MH2 domain of Smad 3. The current study demonstrates that co-treatment of murine embryonic maxillary mesenchyme (MEMM) cells with Wnt-3a and TGFb leads to enhanced reporter activity from TOPflash, a Wnt-responsive reporter plasmid. Transcriptional cooperation between TGFb and Wnt did not require the presence of a Smad binding element, nor did it occur when a TGFb-responsive reporter plasmid (p3TP-lux) was transfected. Overexpression of Smad 3 further enhanced the cooperation between Wnt and TGFb while overexpression of dominant-negative Smads 2 and 3 inhibited this effect. Co-stimulation with TGFb led to greater nuclear translocation of b-catenin, providing explanation for the effect of TGFb on Wnt-3a reporter activity. Wnt-3a exerted antiproliferative activity in MEMM cells, similar to that exerted by TGFb. In addition, Wnt-3a and TGFb in combination led to synergistic decreases in MEMM cell proliferation. These data demonstrate a functional interaction between the TGFb and Wnt signaling pathways and suggest that Wnt activation of the canonical pathway is an important mediator of MEMM cell growth.

Journal of Cellular Physiology, 2008

The bone morphogenetic protein (BMP) family represents a class of signaling molecules, that plays... more The bone morphogenetic protein (BMP) family represents a class of signaling molecules, that plays key roles in morphogenesis, cell proliferation, survival and differentiation during normal development. Members of this family are essential for the development of the mammalian orofacial region where they regulate cell proliferation, extracellular matrix synthesis, and cellular differentiation. Perturbation of any of these processes results in orofacial clefting. Embryonic orofacial tissue expresses BMP mRNAs, their cognate proteins, and BMP-specific receptors in unique temporo-spatial patterns, suggesting functional roles in orofacial development. However, specific genes that function as downstream mediators of BMP action during orofacial ontogenesis have not been well defined. In the current study, elements of the Smad component of the BMP intracellular signaling system were identified and characterized in embryonic orofacial tissue and functional activation of the Smad pathway by BMP2 and BMP4 was demonstrated. BMP2 and BMP4-initiated Smad signaling in cells derived from embryonic orofacial tissue was found to result in: (1) phosphorylation of Smads 1 and 5; (2) nuclear translocation of Smads 1, 4, and 5; (3) binding of Smads 1, 4, and 5 to a consensus Smad binding element (SBE)-containing oligonucleotide; (4) transactivation of transfected reporter constructs, containing BMP-inducible Smad response elements; and (5) increased expression at transcriptional as well as translational levels of Id3 (endogenous gene containing BMP receptor-specific Smad response elements). Collectively, these data document the existence of a functional Smad-mediated BMP signaling system in cells of the developing murine orofacial region. J. Cell. Physiol. 216: 771–779, 2008, © 2008 Wiley-Liss, Inc.

Biochemical and Biophysical Research Communications, 2005

cAMP response element-binding protein (CREB)-binding protein (CBP) plays an important role as a g... more cAMP response element-binding protein (CREB)-binding protein (CBP) plays an important role as a general co-integrator of multiple signaling pathways and interacts with a large number of transcription factors and co-factors, through its numerous protein-binding domains. To identify nuclear factors associated with CBP in developing orofacial tissue, a yeast two-hybrid screen of a cDNA library derived from orofacial tissue from gestational day 11 to 13 mouse embryos was conducted. Using the carboxy terminus (amino acid residues 1676-2441) of CBP as bait, several novel proteins that bind CBP were identified, including an Msxinteracting-zinc finger protein, CDC42 interaction protein 4/thyroid hormone receptor interactor 10, SH3-domain GRB2-like 1, CCR4-NOT transcription complex subunit 3, adaptor protein complex AP-1 b1 subunit, eukaryotic translation initiation factor 2B subunit 1 (a), and cyclin G-associated kinase. Results of the yeast two-hybrid screen were confirmed by glutathione S-transferase pull-down assays. The identification of these proteins as novel CBP-binding partners allows exploration of new mechanisms by which CBP regulates and integrates diverse cell signaling pathways.

Journal of Cellular Physiology, 2003

The transforming growth factor-β (TGFβ) family represents a class of signaling molecules that pla... more The transforming growth factor-β (TGFβ) family represents a class of signaling molecules that plays a central role in morphogenesis, growth, and cell differentiation during normal embryonic development. Members of this growth factor family are particularly vital to development of the mammalian secondary palate where they regulate palate mesenchymal cell proliferation and extracellular matrix synthesis. Such regulation is particularly critical since perturbation of either cellular process results in a cleft of the palate. While the cellular and phenotypic effects of TGFβ on embryonic craniofacial tissue have been extensively catalogued, the specific genes that function as downstream mediators of TGFβ action in the embryo during palatal ontogenesis are poorly defined. Embryonic palatal tissue in vivo and murine embryonic palate mesenchymal (MEPM) cells in vitro secrete and respond to TGFβ. In the current study, elements of the Smad component of the TGFβ intracellular signaling system were identified and characterized in cells of the embryonic palate and functional activation of the Smad pathway by TGFβ1, TGFβ2, and TGFβ3 was demonstrated. TGFβ-initiated Smad signaling in cells of the embryonic palate was found to result in: (1) phosphorylation of Smad 2; (2) nuclear translocation of the Smads 2, 3, and 4 protein complex; (3) binding of Smads 3 and 4 to a consensus Smad binding element (SBE) oligonucleotide; (4) transactivation of transfected reporter constructs, containing TGFβ-inducible Smad response elements; and (4) increased expression of gelatinases A and B (endogenous genes containing Smad response elements) whose expression is critical to matrix remodeling during palatal ontogenesis. Collectively, these data point to the presence of a functional Smad-mediated TGFβ signaling system in cells of the developing murine palate. J. Cell. Physiol. 197: 261–271, 2003. © 2003 Wiley-Liss, Inc.

International Journal of Developmental Biology, 2009

Morphogenesis of the mammalian secondary palate requires coordination of cell migration, prolifer... more Morphogenesis of the mammalian secondary palate requires coordination of cell migration, proliferation, differentiation, apoptosis and synthesis of extracellular matrix molecules by numerous signal transduction pathways. Recent evidence suggests a role for members of the Wnt family of secreted cytokines in orofacial development. However, no study has systematically or comprehensively examined the expression of Wnts in embryonic orofacial tissue. We thus conducted a survey of the expression of all known Wnt genes in the developing murine secondary palate. Using an RT-PCR strategy to assay gene expression, 12 of the 19 known members of the Wnt family were found to be expressed in embryonic palatal tissue during key phases of its development. The expression of 5 Wnt family members was found to be temporally regulated. Moreover, these Wnts had unique spatio-temporal patterns of expression which suggested possible roles in palatal ontogeny.

Biochemistry, 1996

Thioether S-methyltransferase is an important enzyme in the metabolism of sulfur and selenium-con... more Thioether S-methyltransferase is an important enzyme in the metabolism of sulfur and selenium-containing compounds in animals. Ethyl vinyl sulfide was previously shown to be a substrate for this enzyme yielding methyl ethyl vinyl sulfonium ion (MEVS+) upon reaction with S-adenosylmethionine. Since vinyl sulfonium ions are reactive toward nucleophiles, the inactivation of thioether S-methyltransferase as a result of its methylation of ethyl vinyl sulfide was investigated. Ethyl vinyl sulfide was found to inactivate thioether S-methyltransferase in a time-dependent, pseudo-first-order process with k(inact) and KI values of 0.05 min(-1) and 0.275 mM, respectively. Calculation of the partition ratio revealed one inactivation event for every 100 turnovers. Dimethyl sulfide, an alternate substrate for thioether S-methyltransferase which yields the nonreactive product trimethyl sulfonium ion, protected the enzyme from inactivation by ethyl vinyl sulfide. The inactivation is a result of covalent reaction of methyl ethyl vinyl sulfonium ion with the enzyme as shown by comigration of radioactivity with the enzyme during denaturing gel filtration of reaction mixtures containing thioether S-methyltransferase, ethyl vinyl sulfide, and S-adenosyl[methyl-3H]methionine. Using this method the stoichiometry of inactivation was determined to be 1 mol of [3H]-methyl group/mol of thioether S-methyltransferase inactivated. Both the alternate substrate, dimethyl sulfide, and the competitive product inhibitor, S-adenosylhomocysteine, inhibited such covalent labeling of the enzyme by ethyl vinyl sulfide and S-adenosyl[methyl-3H]methionine. Chemically synthesized MEVS+ inactivated thioether S-methyltransferase, and [methyl-14C]MEVS+ covalently labeled the enzyme with 14C. These results reveal a previously unrecognized mechanism for biochemical activation of vinyl thioethers by methylation to form reactive vinyl sulfonium ions.

Biochimica Et Biophysica Acta-protein Structure and Molecular Enzymology, 1995

Thioether S-methyltransferase catalyzes transfer of the methyl group from S-adenosylmethionine to... more Thioether S-methyltransferase catalyzes transfer of the methyl group from S-adenosylmethionine to X in compounds of the structure R-X-R', where X may be sulfur, selenium, or tellurium, and R and R' may be various organic groups. To obtain a cDNA clone of thioether S-methyltransferase, a mouse lung cDNA library in lambda gt11 was screened with a 99 base-pair probe obtained by performing the polymerase chain reaction on oligo(dT) primed, reverse transcribed, mouse lung RNA using two degenerate primers designed from partial amino-acid sequences of the enzyme. The entire coding and 3'-untranslated regions were obtained and sequenced. The predicted protein contains 264 amino-acid residues and has a calculated M(r) of 29,460. The amino-acid sequence of thioether S-methyltransferase contains three motifs characteristic of many methyltransferases and has a high level of identity with the amino-acid sequences of nicotinamide N-methyltransferase and phenylethanolamine N-methyltransferase. However, in spite of the fact that they are both mammalian cytosolic sulfur methyltransferases, the sequences of thioether S-methyltransferase and thiopurine S-methyltransferase share little identity.

Biochemistry, 1996

Thioether S-methyltransferase is an important enzyme in the metabolism of sulfur and selenium-con... more Thioether S-methyltransferase is an important enzyme in the metabolism of sulfur and selenium-containing compounds in animals. Ethyl vinyl sulfide was previously shown to be a substrate for this enzyme yielding methyl ethyl vinyl sulfonium ion (MEVS+) upon reaction with S-adenosylmethionine. Since vinyl sulfonium ions are reactive toward nucleophiles, the inactivation of thioether S-methyltransferase as a result of its methylation of ethyl vinyl sulfide was investigated. Ethyl vinyl sulfide was found to inactivate thioether S-methyltransferase in a time-dependent, pseudo-first-order process with k(inact) and KI values of 0.05 min(-1) and 0.275 mM, respectively. Calculation of the partition ratio revealed one inactivation event for every 100 turnovers. Dimethyl sulfide, an alternate substrate for thioether S-methyltransferase which yields the nonreactive product trimethyl sulfonium ion, protected the enzyme from inactivation by ethyl vinyl sulfide. The inactivation is a result of covalent reaction of methyl ethyl vinyl sulfonium ion with the enzyme as shown by comigration of radioactivity with the enzyme during denaturing gel filtration of reaction mixtures containing thioether S-methyltransferase, ethyl vinyl sulfide, and S-adenosyl[methyl-3H]methionine. Using this method the stoichiometry of inactivation was determined to be 1 mol of [3H]-methyl group/mol of thioether S-methyltransferase inactivated. Both the alternate substrate, dimethyl sulfide, and the competitive product inhibitor, S-adenosylhomocysteine, inhibited such covalent labeling of the enzyme by ethyl vinyl sulfide and S-adenosyl[methyl-3H]methionine. Chemically synthesized MEVS+ inactivated thioether S-methyltransferase, and [methyl-14C]MEVS+ covalently labeled the enzyme with 14C. These results reveal a previously unrecognized mechanism for biochemical activation of vinyl thioethers by methylation to form reactive vinyl sulfonium ions.

Biochimica Et Biophysica Acta-protein Structure and Molecular Enzymology, 1995

Thioether S-methyltransferase catalyzes transfer of the methyl group from S-adenosylmethionine to... more Thioether S-methyltransferase catalyzes transfer of the methyl group from S-adenosylmethionine to X in compounds of the structure R-X-R', where X may be sulfur, selenium, or tellurium, and R and R' may be various organic groups. To obtain a cDNA clone of thioether S-methyltransferase, a mouse lung cDNA library in lambda gt11 was screened with a 99 base-pair probe obtained by performing the polymerase chain reaction on oligo(dT) primed, reverse transcribed, mouse lung RNA using two degenerate primers designed from partial amino-acid sequences of the enzyme. The entire coding and 3'-untranslated regions were obtained and sequenced. The predicted protein contains 264 amino-acid residues and has a calculated M(r) of 29,460. The amino-acid sequence of thioether S-methyltransferase contains three motifs characteristic of many methyltransferases and has a high level of identity with the amino-acid sequences of nicotinamide N-methyltransferase and phenylethanolamine N-methyltransferase. However, in spite of the fact that they are both mammalian cytosolic sulfur methyltransferases, the sequences of thioether S-methyltransferase and thiopurine S-methyltransferase share little identity.

Molecular Endocrinology, 1997

A novel G(S alpha) mutation encoding the substitution of arginine for serine 250 (G[S alpha] S250... more A novel G(S alpha) mutation encoding the substitution of arginine for serine 250 (G[S alpha] S250R) was identified in a patient with pseudohypoparathyroidism type Ia. Both G(S) activity and G(S alpha) expression were decreased by about 50% in erythrocyte membranes from the affected patient. The cDNA of this G(S alpha) mutant, as well as one in which the S250 residue is deleted (G[S alpha]-deltaS250), was generated, and the biochemical properties of the products of in vitro transcription/translation were examined. Both mutants had a sedimentation coefficient similar to that of wild type G(S alpha) (approximately 3.7S) when kept at 0 C after synthesis. However when maintained for 1-2 h at 30-37 C, both mutants aggregated to a material sedimenting at approximately 6.3S or greater (G[S alpha]-S250R to a greater extent than G(S alpha]-deltaS250), while wild type G(S alpha) sedimented at approximately 3.7S, suggesting that the mutants were thermolabile. Incubation in the presence of high doses of guanine nucleotide partially prevented heat denaturation of G(S alpha) deltaS250 but had no protective effect on G(S alpha-S250R. Sucrose density gradient centrifugation at 0 C in the presence and absence of beta gamma-dimers demonstrated that, in contrast to wild type G(S alpha) neither mutant could interact with beta gamma. Trypsin protection assays revealed no protection of G(S alpha)-S250R by GTPgammaS or AIF4- at any temperature. GTPgammaS conferred modest protection of G(S alpha)-deltaS250 (approximately 50% of wild-type G[S alpha]) at 30 C but none at 37 C, while AIF4- conferred slight protection at 20 C but none at 30 C or above. Consistent with this result, G(S alpha)-deltaS250 was able to stimulate adenylyl cyclase at 30 C when reconstituted with cyc- membranes in the presence of GTPgammaS but not in the presence of AIF4-. G(S alpha)-S250R showed no ability to stimulate adenylyl cyclase in the presence of either agent. Stable transfection of mutant and wild-type G(S alpha) into cyc- S49 lymphoma cells revealed that the majority of wild type G(S alpha) localized to membranes, while little or no membrane localization occurred for either mutant. Modeling of G(S alpha) based upon the crystal structure of G(t alpha) or G(i alpha) suggests that Ser250 interacts with several residues within and around the conserved NKXD motif, which directly interacts with the guanine ring of bound GDP or GTP. It is therefore possible that substitution or deletion of this residue may alter guanine nucleotide binding, which could lead to thermolability and impaired function.

Febs Letters, 2003

A yeast two-hybrid screen was utilized to identify novel Smad 3 binding proteins expressed in dev... more A yeast two-hybrid screen was utilized to identify novel Smad 3 binding proteins expressed in developing mouse orofacial tissue. Three proteins (Erbin, Par-3, and Dishevelled) were identi¢ed that share several similar structural and functional characteristics. Each contains at least one PDZ domain and all have been demonstrated to play a role in the establishment and maintenance of cell polarity. In GST (glutathione S-transferase) pull-down assays, Erbin, Par-3, and Dishevelled bound strongly to the isolated MH2 domain of Smad 3, with weaker binding to a full-length Smad 3 protein. Failure of Erbin, Par-3, and Dishevelled to bind to a Smad 3 mutant protein that was missing the MH2 domain con¢rms that the binding site resides within the MH2 domain. Erbin, Par-3, and Dishevelled also interacted with the MH2 domains of other Smads, suggesting broad Smad binding speci¢city. Dishevelled and Erbin mutant proteins, in which the PDZ domain was removed, still retained their ability to bind Smad 3, albeit with lower a⁄nity. While transforming growth factor L L (TGFL L) has been suggested to alter cell polarity through a Smad-independent mechanism involving activation of members of the RhoA family of GTP binding proteins, the observation that Smads can directly interact with proteins involved in cell polarity, as shown in the present report, suggests an additional means by which TGFL L could alter cell polarity via a Smad-dependent signaling mechanism.

Biochemical and Biophysical Research Communications, 2003

Ontogenesis of the mammalian orofacial region is controlled by numerous developmental signals, in... more Ontogenesis of the mammalian orofacial region is controlled by numerous developmental signals, including those initiated by the transforming growth factors b (TGFbs). Targeted deletion of the genes encoding several of the TGFbs in mice has been shown to result in clefts of the secondary palate. Members of the TGFb family of growth factors utilize intracellular Smads as signal transducers. Smads 2 and 3 are transcriptional regulators that bind DNA through their conserved MH1 domains and activate/ inhibit transcription of TGFb-responsive genes through their MH2 domains. Using a yeast two-hybrid screen of a cDNA expression library constructed from fetal murine orofacial tissue, we have identified three types of collagens (types I, III, and V) that are capable of binding to the MH2 domain of Smad 3. These interactions were confirmed by glutathione S-transferase (GST) pull-down assays in which the MH2 domain of Smad 3 fused to GST interacted strongly with in vitro translated, 35 S-labeled collagen types I, III, and V. Each collagen also bound to the MH2 domains of Smads 4 and 7 and, to a lesser extent, full-length Smads 1, 2, 3, and 4. Binding of Smads to collagen is a novel observation. Moreover, TGFb is a potent regulator of collagen synthesis and turnover during mammalian orofacial development. These data thus suggest an important means of feedback regulation of the TGFb signaling cascade.

Biochemical and Biophysical Research Communications, 2004

The transforming growth factors b control a diversity of biological processes including cellular ... more The transforming growth factors b control a diversity of biological processes including cellular proliferation, differentiation, apoptosis, and extracellular matrix production, and are critical effectors of embryonic patterning and development, including that of the orofacial region. TGFb superfamily members signal through specific cell surface receptors that phosphorylate the cytoplasmic Smad proteins, resulting in their translocation to the nucleus and interaction with promoters of TGFb-responsive genes. Subsequent alterations in transcription are cell type-specific and dependent on recruitment to the Smad/transcription factor complex of coactivators, such as CBP and p300, or corepressors, such as c-ski and SnoN. Since the affinity of Smads for DNA is generally low, additional accessory proteins that facilitate Smad/DNA binding are required, and are often cell-and tissue-specific. In order to identify novel Smad 3 binding proteins in developing orofacial tissue, a yeast two hybrid assay was employed in which the MH2 domain of Smad 3 was used to screen an expression library derived from mouse embryonic orofacial tissue. The RNA helicase, p68, was identified as a unique Smad binding protein, and the specificity of the interaction was confirmed through various in vitro and in vivo assays. Co-expression of Smad 3 and a CBP-Gal4 DNA binding domain fusion protein in a Gal4-luciferase reporter assay resulted in increased TGFb-stimulated reporter gene transcription. Moreover, co-expression of p68 RNA helicase along with Smad 3 and CBP-Gal4 resulted in synergistic activation of Gal4-luciferase reporter expression. Collectively, these data indicate that the RNA helicase, p68, can directly interact with Smad 3 resulting in formation of a transcriptionally active ternary complex containing Smad 3, p68, and CBP. This offers a means of enhancing TGFb-mediated cellular responses in developing orofacial tissue.

Journal of Interferon and Cytokine Research, 2005

cAMP response element binding protein (CREB)-binding protein (CBP) is a multifunctional transcrip... more cAMP response element binding protein (CREB)-binding protein (CBP) is a multifunctional transcriptional coactivator that plays important roles in gene regulation. CBP is expressed in murine embryonic orofacial tissue, where it is developmentally regulated. To identify nuclear factors associated with CBP in developing orofacial tissue, a yeast two-hybrid screen of a cDNA library derived from embryonic orofacial tissue from gestational days 11-13 mouse embryos was conducted. Using the carboxy terminal region of CBP as bait, the protein inhibitor of activated Stat1 (PIAS1) was identified as a novel CBP binding protein. The association of PIAS1 with CBP was confirmed in vitro by glutathione S-transferase (GST) pull-down assays and in vivo by coimmunoprecipitation. Reporter assays demonstrated that PIAS1 inhibited CBP-mediated transcriptional activation in the presence or absence of transforming growth factor-beta (TGF-beta). These results identify PIAS1 as a novel binding partner for CBP and inhibitor of CBP-mediated transcription, suggesting that PIAS1 might play a role in regulating cell proliferation, migration, and differentiation during embryonic development.

Biochimica Et Biophysica Acta-molecular Cell Research, 2007

TGFβ signaling regulates central cellular processes such as proliferation and extracellular matri... more TGFβ signaling regulates central cellular processes such as proliferation and extracellular matrix production during development of the orofacial region. Extracellular TGFβ binds to cell surface receptors to activate the nucleocytoplasmic Smad proteins that, along with other transcription factors and cofactors, bind specific DNA sequences in the promoters of target genes to regulate their expression. To determine the identity of Smad binding proteins that regulate TGFβ signaling in developing murine orofacial tissue, a yeast two-hybrid screening approach was employed. The PR-domain containing protein, PRDM16/MEL1 was identified as a novel Smad binding protein. The interaction between PRDM16/MEL1 and Smad 3 was confirmed by GST pull-down assays. The expression of PRDM16/MEL1 was detected in developing orofacial tissue by both Northern blot and in situ hybridization. PRDM16/MEL1 was constitutively expressed in orofacial tissue on E12.5-E14.5 as well as other embryonic tissues such as heart, brain, liver, and limb buds. Taken together, these results demonstrate that PRDM16/MEL1 is a Smad binding protein that may be important for development of orofacial structures through modulation of the TGFβ signaling pathway.

Biochemical and Biophysical Research Communications, 2003

The TGFbs, a family of secreted polypeptide growth factors, are critical regulators of mammalian ... more The TGFbs, a family of secreted polypeptide growth factors, are critical regulators of mammalian orofacial development. The importance of the TGFbs in development of the orofacial region in mice is underscored by the resulting orofacial clefts in mice with targeted deletion of either TGFb2 or TGFb3 and most recently, a conditional knockout of the type II TGFb receptor (TbRII) gene. The TGFbs signal via binding to specific cell surface receptors which, in turn, activates translocation of the nucleocytoplasmic Smad transcriptional regulators. Smads 2 and 3 are TGFb-specific transcriptional regulators that bind DNA through their conserved MH1 domains and activate or inhibit transcription of TGFb-responsive genes through their MH2 domains. To search for novel Smad binding proteins expressed in developing murine orofacial tissue, a yeast two-hybrid assay was utilized to screen a cDNA expression library constructed from fetal murine orofacial tissue. Several novel Smad binding proteins were identified. These include a putative zinc finger protein (ZNF198), peroxisomal biogenesis factor 6 (Pex6), eucaryotic translation initiation factor 4E nuclear import factor 1 (4-ET), and splicing factor 3b subunit 2 (SF3b2). Results of the yeast two-hybrid screen were verified by GST pulldown assays which confirmed the interaction of these proteins with the MH2 domain of Smad 3, and also indicated interaction of these proteins with additional Smad family members. The identification of these proteins as Smad binding partners allows exploration of new mechanisms whereby TGFb signaling may be regulated, and reveals additional potential interactions with other signaling pathways.

Febs Letters, 2005

The transforming growth factor beta (TGFb) and Wnt signaling pathways play central roles regulati... more The transforming growth factor beta (TGFb) and Wnt signaling pathways play central roles regulating embryogenesis and maintaining adult tissue homeostasis. TGFb mediates its cellular effects through types I and II cell surface receptors coupled to the nucleocytoplasmic Smad proteins. Wnt signals via binding to a cell surface receptor, Frizzled, which in turn activates intracellular Dishevelled, ultimately leading to stabilization and nuclear translocation of b-catenin. Previous studies have demonstrated several points of cross-talk between the TGFb and Wnt signaling pathways. In yeast two-hybrid and GST-pull down assays, Dishevelled-1 and Smad 3 have been shown to physically interact through the C-terminal one-half of Dishevelled-1 and the MH2 domain of Smad 3. The current study demonstrates that co-treatment of murine embryonic maxillary mesenchyme (MEMM) cells with Wnt-3a and TGFb leads to enhanced reporter activity from TOPflash, a Wnt-responsive reporter plasmid. Transcriptional cooperation between TGFb and Wnt did not require the presence of a Smad binding element, nor did it occur when a TGFb-responsive reporter plasmid (p3TP-lux) was transfected. Overexpression of Smad 3 further enhanced the cooperation between Wnt and TGFb while overexpression of dominant-negative Smads 2 and 3 inhibited this effect. Co-stimulation with TGFb led to greater nuclear translocation of b-catenin, providing explanation for the effect of TGFb on Wnt-3a reporter activity. Wnt-3a exerted antiproliferative activity in MEMM cells, similar to that exerted by TGFb. In addition, Wnt-3a and TGFb in combination led to synergistic decreases in MEMM cell proliferation. These data demonstrate a functional interaction between the TGFb and Wnt signaling pathways and suggest that Wnt activation of the canonical pathway is an important mediator of MEMM cell growth.

Journal of Cellular Physiology, 2008

The bone morphogenetic protein (BMP) family represents a class of signaling molecules, that plays... more The bone morphogenetic protein (BMP) family represents a class of signaling molecules, that plays key roles in morphogenesis, cell proliferation, survival and differentiation during normal development. Members of this family are essential for the development of the mammalian orofacial region where they regulate cell proliferation, extracellular matrix synthesis, and cellular differentiation. Perturbation of any of these processes results in orofacial clefting. Embryonic orofacial tissue expresses BMP mRNAs, their cognate proteins, and BMP-specific receptors in unique temporo-spatial patterns, suggesting functional roles in orofacial development. However, specific genes that function as downstream mediators of BMP action during orofacial ontogenesis have not been well defined. In the current study, elements of the Smad component of the BMP intracellular signaling system were identified and characterized in embryonic orofacial tissue and functional activation of the Smad pathway by BMP2 and BMP4 was demonstrated. BMP2 and BMP4-initiated Smad signaling in cells derived from embryonic orofacial tissue was found to result in: (1) phosphorylation of Smads 1 and 5; (2) nuclear translocation of Smads 1, 4, and 5; (3) binding of Smads 1, 4, and 5 to a consensus Smad binding element (SBE)-containing oligonucleotide; (4) transactivation of transfected reporter constructs, containing BMP-inducible Smad response elements; and (5) increased expression at transcriptional as well as translational levels of Id3 (endogenous gene containing BMP receptor-specific Smad response elements). Collectively, these data document the existence of a functional Smad-mediated BMP signaling system in cells of the developing murine orofacial region. J. Cell. Physiol. 216: 771–779, 2008, © 2008 Wiley-Liss, Inc.

Biochemical and Biophysical Research Communications, 2005

cAMP response element-binding protein (CREB)-binding protein (CBP) plays an important role as a g... more cAMP response element-binding protein (CREB)-binding protein (CBP) plays an important role as a general co-integrator of multiple signaling pathways and interacts with a large number of transcription factors and co-factors, through its numerous protein-binding domains. To identify nuclear factors associated with CBP in developing orofacial tissue, a yeast two-hybrid screen of a cDNA library derived from orofacial tissue from gestational day 11 to 13 mouse embryos was conducted. Using the carboxy terminus (amino acid residues 1676-2441) of CBP as bait, several novel proteins that bind CBP were identified, including an Msxinteracting-zinc finger protein, CDC42 interaction protein 4/thyroid hormone receptor interactor 10, SH3-domain GRB2-like 1, CCR4-NOT transcription complex subunit 3, adaptor protein complex AP-1 b1 subunit, eukaryotic translation initiation factor 2B subunit 1 (a), and cyclin G-associated kinase. Results of the yeast two-hybrid screen were confirmed by glutathione S-transferase pull-down assays. The identification of these proteins as novel CBP-binding partners allows exploration of new mechanisms by which CBP regulates and integrates diverse cell signaling pathways.

Journal of Cellular Physiology, 2003

The transforming growth factor-β (TGFβ) family represents a class of signaling molecules that pla... more The transforming growth factor-β (TGFβ) family represents a class of signaling molecules that plays a central role in morphogenesis, growth, and cell differentiation during normal embryonic development. Members of this growth factor family are particularly vital to development of the mammalian secondary palate where they regulate palate mesenchymal cell proliferation and extracellular matrix synthesis. Such regulation is particularly critical since perturbation of either cellular process results in a cleft of the palate. While the cellular and phenotypic effects of TGFβ on embryonic craniofacial tissue have been extensively catalogued, the specific genes that function as downstream mediators of TGFβ action in the embryo during palatal ontogenesis are poorly defined. Embryonic palatal tissue in vivo and murine embryonic palate mesenchymal (MEPM) cells in vitro secrete and respond to TGFβ. In the current study, elements of the Smad component of the TGFβ intracellular signaling system were identified and characterized in cells of the embryonic palate and functional activation of the Smad pathway by TGFβ1, TGFβ2, and TGFβ3 was demonstrated. TGFβ-initiated Smad signaling in cells of the embryonic palate was found to result in: (1) phosphorylation of Smad 2; (2) nuclear translocation of the Smads 2, 3, and 4 protein complex; (3) binding of Smads 3 and 4 to a consensus Smad binding element (SBE) oligonucleotide; (4) transactivation of transfected reporter constructs, containing TGFβ-inducible Smad response elements; and (4) increased expression of gelatinases A and B (endogenous genes containing Smad response elements) whose expression is critical to matrix remodeling during palatal ontogenesis. Collectively, these data point to the presence of a functional Smad-mediated TGFβ signaling system in cells of the developing murine palate. J. Cell. Physiol. 197: 261–271, 2003. © 2003 Wiley-Liss, Inc.

International Journal of Developmental Biology, 2009

Morphogenesis of the mammalian secondary palate requires coordination of cell migration, prolifer... more Morphogenesis of the mammalian secondary palate requires coordination of cell migration, proliferation, differentiation, apoptosis and synthesis of extracellular matrix molecules by numerous signal transduction pathways. Recent evidence suggests a role for members of the Wnt family of secreted cytokines in orofacial development. However, no study has systematically or comprehensively examined the expression of Wnts in embryonic orofacial tissue. We thus conducted a survey of the expression of all known Wnt genes in the developing murine secondary palate. Using an RT-PCR strategy to assay gene expression, 12 of the 19 known members of the Wnt family were found to be expressed in embryonic palatal tissue during key phases of its development. The expression of 5 Wnt family members was found to be temporally regulated. Moreover, these Wnts had unique spatio-temporal patterns of expression which suggested possible roles in palatal ontogeny.

Biochemistry, 1996

Thioether S-methyltransferase is an important enzyme in the metabolism of sulfur and selenium-con... more Thioether S-methyltransferase is an important enzyme in the metabolism of sulfur and selenium-containing compounds in animals. Ethyl vinyl sulfide was previously shown to be a substrate for this enzyme yielding methyl ethyl vinyl sulfonium ion (MEVS+) upon reaction with S-adenosylmethionine. Since vinyl sulfonium ions are reactive toward nucleophiles, the inactivation of thioether S-methyltransferase as a result of its methylation of ethyl vinyl sulfide was investigated. Ethyl vinyl sulfide was found to inactivate thioether S-methyltransferase in a time-dependent, pseudo-first-order process with k(inact) and KI values of 0.05 min(-1) and 0.275 mM, respectively. Calculation of the partition ratio revealed one inactivation event for every 100 turnovers. Dimethyl sulfide, an alternate substrate for thioether S-methyltransferase which yields the nonreactive product trimethyl sulfonium ion, protected the enzyme from inactivation by ethyl vinyl sulfide. The inactivation is a result of covalent reaction of methyl ethyl vinyl sulfonium ion with the enzyme as shown by comigration of radioactivity with the enzyme during denaturing gel filtration of reaction mixtures containing thioether S-methyltransferase, ethyl vinyl sulfide, and S-adenosyl[methyl-3H]methionine. Using this method the stoichiometry of inactivation was determined to be 1 mol of [3H]-methyl group/mol of thioether S-methyltransferase inactivated. Both the alternate substrate, dimethyl sulfide, and the competitive product inhibitor, S-adenosylhomocysteine, inhibited such covalent labeling of the enzyme by ethyl vinyl sulfide and S-adenosyl[methyl-3H]methionine. Chemically synthesized MEVS+ inactivated thioether S-methyltransferase, and [methyl-14C]MEVS+ covalently labeled the enzyme with 14C. These results reveal a previously unrecognized mechanism for biochemical activation of vinyl thioethers by methylation to form reactive vinyl sulfonium ions.

Biochimica Et Biophysica Acta-protein Structure and Molecular Enzymology, 1995

Thioether S-methyltransferase catalyzes transfer of the methyl group from S-adenosylmethionine to... more Thioether S-methyltransferase catalyzes transfer of the methyl group from S-adenosylmethionine to X in compounds of the structure R-X-R', where X may be sulfur, selenium, or tellurium, and R and R' may be various organic groups. To obtain a cDNA clone of thioether S-methyltransferase, a mouse lung cDNA library in lambda gt11 was screened with a 99 base-pair probe obtained by performing the polymerase chain reaction on oligo(dT) primed, reverse transcribed, mouse lung RNA using two degenerate primers designed from partial amino-acid sequences of the enzyme. The entire coding and 3'-untranslated regions were obtained and sequenced. The predicted protein contains 264 amino-acid residues and has a calculated M(r) of 29,460. The amino-acid sequence of thioether S-methyltransferase contains three motifs characteristic of many methyltransferases and has a high level of identity with the amino-acid sequences of nicotinamide N-methyltransferase and phenylethanolamine N-methyltransferase. However, in spite of the fact that they are both mammalian cytosolic sulfur methyltransferases, the sequences of thioether S-methyltransferase and thiopurine S-methyltransferase share little identity.

Biochemistry, 1996

Thioether S-methyltransferase is an important enzyme in the metabolism of sulfur and selenium-con... more Thioether S-methyltransferase is an important enzyme in the metabolism of sulfur and selenium-containing compounds in animals. Ethyl vinyl sulfide was previously shown to be a substrate for this enzyme yielding methyl ethyl vinyl sulfonium ion (MEVS+) upon reaction with S-adenosylmethionine. Since vinyl sulfonium ions are reactive toward nucleophiles, the inactivation of thioether S-methyltransferase as a result of its methylation of ethyl vinyl sulfide was investigated. Ethyl vinyl sulfide was found to inactivate thioether S-methyltransferase in a time-dependent, pseudo-first-order process with k(inact) and KI values of 0.05 min(-1) and 0.275 mM, respectively. Calculation of the partition ratio revealed one inactivation event for every 100 turnovers. Dimethyl sulfide, an alternate substrate for thioether S-methyltransferase which yields the nonreactive product trimethyl sulfonium ion, protected the enzyme from inactivation by ethyl vinyl sulfide. The inactivation is a result of covalent reaction of methyl ethyl vinyl sulfonium ion with the enzyme as shown by comigration of radioactivity with the enzyme during denaturing gel filtration of reaction mixtures containing thioether S-methyltransferase, ethyl vinyl sulfide, and S-adenosyl[methyl-3H]methionine. Using this method the stoichiometry of inactivation was determined to be 1 mol of [3H]-methyl group/mol of thioether S-methyltransferase inactivated. Both the alternate substrate, dimethyl sulfide, and the competitive product inhibitor, S-adenosylhomocysteine, inhibited such covalent labeling of the enzyme by ethyl vinyl sulfide and S-adenosyl[methyl-3H]methionine. Chemically synthesized MEVS+ inactivated thioether S-methyltransferase, and [methyl-14C]MEVS+ covalently labeled the enzyme with 14C. These results reveal a previously unrecognized mechanism for biochemical activation of vinyl thioethers by methylation to form reactive vinyl sulfonium ions.

Biochimica Et Biophysica Acta-protein Structure and Molecular Enzymology, 1995

Thioether S-methyltransferase catalyzes transfer of the methyl group from S-adenosylmethionine to... more Thioether S-methyltransferase catalyzes transfer of the methyl group from S-adenosylmethionine to X in compounds of the structure R-X-R', where X may be sulfur, selenium, or tellurium, and R and R' may be various organic groups. To obtain a cDNA clone of thioether S-methyltransferase, a mouse lung cDNA library in lambda gt11 was screened with a 99 base-pair probe obtained by performing the polymerase chain reaction on oligo(dT) primed, reverse transcribed, mouse lung RNA using two degenerate primers designed from partial amino-acid sequences of the enzyme. The entire coding and 3'-untranslated regions were obtained and sequenced. The predicted protein contains 264 amino-acid residues and has a calculated M(r) of 29,460. The amino-acid sequence of thioether S-methyltransferase contains three motifs characteristic of many methyltransferases and has a high level of identity with the amino-acid sequences of nicotinamide N-methyltransferase and phenylethanolamine N-methyltransferase. However, in spite of the fact that they are both mammalian cytosolic sulfur methyltransferases, the sequences of thioether S-methyltransferase and thiopurine S-methyltransferase share little identity.