Biomedical subjects

R J Lechleider

Publications and source records attributed to R J Lechleider.

13 recordsLinked to original sources

PubMed1998

SMAD3/4-dependent transcriptional activation of the human type VII collagen gene (COL7A1) promoter by transforming growth factor beta.

The human type VII collagen gene (COL7A1) recently has been identified as an immediate-early response gene for transforming growth factor beta (TGF-beta)/SMAD signaling pathway. In this study, by using MDA-MB-468 SMAD4-/- breast carcinoma cells, we demonstrate that expression of SMAD4 is an absolute requirement for SMAD-mediated promoter activity. We also demonstrate that the SMAD binding sequence (SBS) representing the TGF-beta response element in the region -496/-444 of the COL7A1 promoter functions as an enhancer in the context of a heterologous promoter. Electrophoretic mobility-shift assays with nuclear extracts from COS-1 cells transfected with expression vectors for SMADs 1-5 indicate that SMAD3 forms a complex with a migration similar to that of the endogenous TGF-beta-specific complex observed in fibroblast extracts. Electrophoretic mobility-shift assays using recombinant glutathione S-transferase-SMAD fusion proteins indicate that both SMAD4 and C-terminally truncated SMAD3, but not SMAD2, can bind the COL7A1 SBS. Coexpression of SMAD3 and SMAD4 in COS-1 cells leads to the formation of two complexes: a DNA/protein complex containing SMAD3 alone and another slower-migrating complex containing both SMAD3 and SMAD4, the latter complex not being detected in fibroblasts. Maximal transactivation of COL7A1 SBS-driven promoters in either MDA-MB-468 carcinoma cells or fibroblasts requires concomitant overexpression of SMAD3 and SMAD4. These data may represent the first identification of a functional homomeric SMAD3 complex regulating a human gene.

Animals↗

PubMed1998

Transcriptional activating activity of Smad4: roles of SMAD hetero-oligomerization and enhancement by an associating transactivator.

Smad4 plays a pivotal role in signal transduction of the transforming growth factor beta superfamily cytokines by mediating transcriptional activation of target genes. Hetero-oligomerization of Smad4 with the pathway-restricted SMAD proteins is essential for Smad4-mediated transcription. We provide evidence that SMAD hetero-oligomerization is directly required for the Smad4 C-terminal domain [Smad4(C)] to show its transcriptional transactivating activity; this requirement obtains even when Smad4(C) is recruited to promoters by heterologous DNA-binding domains and in the absence of the inhibitory Smad4 N-terminal domain. Defined mutations of GAL4 DNA-binding domain fusion of Smad4(C) that disrupt SMAD hetero-oligomerization suppressed transcriptional activation. Importantly, we found that an orphan transcriptional activator MSG1, a nuclear protein that has strong transactivating activity but apparently lacks DNA-binding activity, functionally interacted with Smad4 and enhanced transcription mediated by GAL4 DNA-binding domain-Smad4(C) and full-length Smad4. Transcriptional enhancement by MSG1 depended on transforming growth factor beta signaling and was suppressed by Smad4(C) mutations disrupting SMAD hetero-oligomerization or by the presence of Smad4 N-terminal domain. Furthermore, Smad4(C) did not show any detectable transactivating activity in yeast when fused to heterologous DNA-binding domains. These results demonstrate additional roles of SMAD hetero-oligomerization in Smad4-mediated transcriptional activation. They also suggest that the transcriptional-activating activity observed in the presence of Smad4 in mammalian cells may be derived, at least in part, from endogenously expressed separate transcriptional activators, such as MSG1.

3T3 Cells↗

PubMed1998

Smad2 transduces common signals from receptor serine-threonine and tyrosine kinases.

SMAD proteins mediate signals from receptor serine-threonine kinases (RSKs) of the TGF-beta superfamily. We demonstrate here that HGF and EGF, which signal through RTKs, can also mediate SMAD-dependent reporter gene activation and induce rapid phosphorylation of endogenous SMAD proteins by kinase(s) downstream of MEK1. HGF induces phosphorylation and nuclear translocation of epitope-tagged Smad2 and a mutation that blocks TGF-beta signaling also blocks HGF signal transduction. Smad2 may thus act as a common positive effector of TGF-beta- and HGF-induced signals and serve to modulate cross talk between RTK and RSK signaling pathways.

Animals↗

PubMed1998

Smad-dependent transcriptional activation of human type VII collagen gene (COL7A1) promoter by transforming growth factor-beta.

We have previously shown that transforming growth factor-beta (TGF-beta) increases type VII collagen gene (COL7A1) expression in human dermal fibroblasts in culture (Mauviel, A., Lapière, J.-C., Halcin, C., Evans, C. H., and Uitto, J. (1994) J. Biol. Chem. 269, 25-28). To gain insight into the molecular mechanisms underlying the up-regulation of COL7A1 by this growth factor, we performed transient cell transfections with a series of 5'-deletion promoter/chloramphenicol acetyltransferase reporter gene constructs. We identified a 68-base pair region between nucleotides -524 and -456, relative to the transcription start site, as critical for TGF-beta response. Using electrophoresis mobility shift assays (EMSAs) with an oligonucleotide spanning the region from -524 to -444, we discovered that a TGF-beta-specific protein-DNA complex was formed as early as 11 min after TGF-beta stimulation and persisted for 1 h after addition of the growth factor. Deletion analysis of the TGF-betaresponsive region of the COL7A1 promoter by EMSA identified segment -496/-444 as the minimal fragment capable of binding the TGF-beta-induced complex. Furthermore, two distinct segments, -496/-490 and -453/-444, appeared to be necessary for TGF-beta-induced DNA binding activity, suggesting a bipartite element. Supershift experiments with a pan-Smad antibody unambiguously identified the TGF-beta-induced complex as containing a Smad member. This is the first direct identification of binding of endogenous Smad proteins to regulatory sequences of a human gene.

Base Sequence↗

PubMed1997

Characterization of functional domains within Smad4/DPC4.

Smad proteins are a family of highly conserved, intracellular proteins that signal cellular responses downstream of transforming growth factor-beta (TGF-beta) family serine/threonine kinase receptors. One of these molecules, Smad4, originally identified as the candidate tumor suppressor gene dpc-4, reconstitutes TGF-beta- and activin-dependent transcriptional responses in Smad4 null cell lines and interacts in a ligand-dependent manner with other Smad family members in both TGF-beta, activin, and bone morphogenetic protein-2/-4 pathways. Here, we used an assay based on the restoration of ligand-dependent transcriptional responses in a Smad4 null cell line to characterize functional domain structures within Smad4. We showed that restoration of TGF-beta-induced transcriptional responses by Smad4 was inhibited by co-transfection with a kinase dead TGF-beta type II receptor and that constitutive activation was blocked with TGF-beta neutralizing antibodies, confirming the essential role of Smad4 in TGF-beta signaling. Using a series of Smad4 mutation, deletion, and Smad1/Smad4 chimera constructs we identified a 47-amino acid deletion within the middle-linker region of Smad4 that is essential for the mediation of signaling responses. In addition, we showed that the NH2-terminal domain of Smad4 augments ligand-dependent activation associated with the middle-linker region, indicating that there is a distinct ligand-response domain within the N terminus of this molecule.

Binding Sites↗

PubMed1996

The immunophilin FKBP12 functions as a common inhibitor of the TGF beta family type I receptors.

The immunophilin FKBP12 is an evolutionarily conserved abundant protein; however, its physiological roles remain poorly defined. Here we report that FKBP12 is a common cytoplasmic interactor of TGF beta family type I receptors. FKBP12 binds to ligand-free TGF beta type I receptor, from which it is released upon a ligand-induced, type II receptor mediated phosphorylation of the type I receptor. Blocking FKBP12/type I receptor interaction with FK506 nonfunctional derivatives enhances the ligand activity, indicating that FKBP12 binding is inhibitory to the signaling pathways of the TGF beta family ligands. Overexpression of a myristylated FKBP12 in Mv1Lu cell specifically inhibits two separate pathways activated by TGF beta, and two point mutations on FKBP12 (G89P, I90K) abolish the inhibitory activity of FKBP12, suggesting that FKBP12 may dock a cytoplasmic protein to the type I receptors to inhibit TGF beta family mediated signaling.

Activin Receptors, Type I↗

PubMed1996

Serine phosphorylation, chromosomal localization, and transforming growth factor-beta signal transduction by human bsp-1.

The transforming growth factor-beta (TGF-beta) superfamily regulates a multitude of cellular and developmental events. TGF-beta family ligands signal through transmembrane serine/threonine kinase receptors whose downstream effectors are largely unknown. Using genetic data from the fruit fly, we have identified a downstream effector of TGF-beta-induced signaling. TGF-beta signaling protein-1 (BSP-1) is rapidly phosphorylated in response to TGF-beta. Localization of bsp-1 to chromosome 4q28 suggests a role in carcinogenesis. These data suggest that BSP-1 is the prototype of a new class of signaling molecules.

Amino Acid Sequence↗

PubMed1994

SH-PTP2/Syp SH2 domain binding specificity is defined by direct interactions with platelet-derived growth factor beta-receptor, epidermal growth factor receptor, and insulin receptor substrate-1-derived phosphopeptides.

Signaling by tyrosine kinases involves direct associations between proteins with Src homology 2 (SH2) domains and sites of tyrosine phosphorylation. Specificity in signaling pathways results in part from inherent selectivity in interactions between particular SH2 domains and phosphopeptide sequences. The cytoplasmic phosphotyrosine phosphatase SH-PTP2 (Syp, PTP 1D, PTP-2C) contains two SH2 domains (N and C) which mediate its association with and activation by the platelet-derived growth factor (PDGF) and epidermal growth factor receptors and IRS-1. We have developed a competitive phosphopeptide binding assay to analyze specificity of the SH-PTP2 N-SH2 domain for phosphorylation sites of these phosphoproteins. The sequence surrounding Tyr1009 bound with greatest affinity (ID50 = 14 microM) of eight PDGF receptor-derived phosphopeptides tested. No peptides corresponding to known epidermal growth factor receptor phosphorylation sites bound with high affinity. However, an alternative sequence surrounding Tyr954 bound tightly (ID50 = 21 microM). Of the 13 IRS-1-related peptides analyzed, sequences surrounding Tyr546, Tyr895, and Tyr1172 bound with highest affinity (ID50 = 11, 4, and 1 microM, respectively). Alternative phosphopeptides generally bound with much weaker affinity (ID50 > 150 microM). These findings are consistent with recent mutational analyses of the PDGF receptor and predict site-specific interactions between SH-PTP2 and each of these phosphoproteins. Comparisons between peptide sequences suggest that the N-terminal SH2 domain of SH-PTP2 binds with highest affinity to phosphotyrosine (pY) followed by a beta-branched residue (Val, Ile, Thr) at pY+1 and a hydrophobic residue (Val, Leu, Ile) at pY+3 positions. Peptide truncation studies also indicate that residues outside of the pY-1 to pY+4 motif are required for high affinity interactions.

Amino Acid Sequence↗

PubMed1993

Expression, purification, and characterization of SH2-containing protein tyrosine phosphatase, SH-PTP2.

A human protein tyrosine phosphatase containing two src homology 2 (SH2) domains (SH-PTP2) was expressed in Escherichia coli under T7 promoter control and purified to near homogeneity. The purified protein, with molecular mass of 68 kDa on SDS-polyacrylamide gel electrophoresis, was identified as SH-PTP2 by its protein tyrosine phosphatase activity and N-terminal amino acid sequence analysis. Its protein tyrosine phosphatase activity was sensitive to pH and salt concentration. Whereas its optimum pH for the low molecular weight substrate para-nitrophenyl phosphate is 5.6, the pH optima for peptide substrates were shifted toward neutral. With the artificial protein substrate reduced, carboxyamidomethylated, and maleylated lysozyme, it displays 2000-fold lower Km (1.7 microM) and 2.4-fold higher kcat (0.11 s-1) than with para-nitrophenyl phosphate. Among the phosphopeptides from autophosphorylation sites of receptors for epidermal growth factor and platelet-derived growth factor, SH-PTP2 displayed high activity toward phosphopeptides corresponding to pY992 of the epidermal growth factor receptor and pY1009 and pY1021 of the platelet-derived growth factor receptor. In further enzymatic studies with phosphopeptides corresponding to pY1009, SH-PTP2 showed nonlinear Line-weaver-Burk double-reciprocal plots, suggesting that the phosphopeptide corresponding to pY1009 may have a substrate and allosteric effect.

Amino Acid Sequence↗

PubMed1993

Activation of the SH2-containing phosphotyrosine phosphatase SH-PTP2 by its binding site, phosphotyrosine 1009, on the human platelet-derived growth factor receptor.

Much progress has been made in elucidating early events in signal transduction by growth factor receptors with intrinsic tyrosine kinase activity. Upon ligand addition, these receptors dimerize and activate, becoming phosphorylated at a number of tyrosyl residues. These phosphorylation sites serve as docking points for proteins containing src homology-2 (SH2) domains. However, little is known about how phosphotyrosine phosphatases (PTPs), participate in these events. Recently, we and others molecularly cloned a ubiquitously expressed SH2 domain-containing PTP, SH-PTP2 (Syp, PTP1D, PTP2C), and found that it interacts directly with several activated growth factor receptors via its SH2 domains. Using a peptide competition assay, we now demonstrate that the major binding site for SH-PTP2 on the platelet-derived growth factor receptor is phosphotyrosine 1009. Immunoprecipitation studies indicate that SH-PTP2 is the previously unidentified "64-kDa" protein known to bind at this site. Addition of a phosphotyrosyl peptide comprising the region around Tyr-1009 stimulates SH-PTP2 activity 5-10-fold, whereas other phosphotyrosyl peptides from the platelet-derived growth factor receptor have no stimulatory effect. Our data suggest that binding of SH-PTP2 to the activated receptor in vivo should result in stimulation of SH-PTP2 activity.

Animals↗

PubMed1993

Tyrosyl phosphorylation and growth factor receptor association of the human corkscrew homologue, SH-PTP2.

The pivotal role of tyrosine kinases in signal transduction is well established, but the role of tyrosine phosphatases remains obscure. The discovery of src homology 2 domain-containing protein tyrosine phosphatases suggested roles for these molecules in growth factor signaling pathways, since src homology 2 domains direct association of downstream signaling molecules with activated growth factor receptors and other phosphotyrosyl proteins. We have found that SH-PTP2, a putative homologue of Drosophila corkscrew, associates in vivo with the ligand-activated epidermal growth factor and platelet-derived growth factor receptors. The N-terminal src homology 2 domain of SH-PTP2 directly associates with activated receptors. SH-PTP2 itself is a phosphoprotein, and it becomes tyrosyl phosphorylated upon growth factor activation. These findings suggest several possible models for SH-PTP2 signaling.

3T3 Cells↗

PubMed1993

SH2 domains recognize specific phosphopeptide sequences.

A phosphopeptide library was used to determine the sequence specificity of the peptide-binding sites of SH2 domains. One group of SH2 domains (Src, Fyn, Lck, Fgr, Abl, Crk, and Nck) preferred sequences with the general motif pTyr-hydrophilic-hydrophilic-Ile/Pro while another group (SH2 domains of p85, phospholipase C-gamma, and SHPTP2) selected the general motif pTyr-hydrophobic-X-hydrophobic. Individual members of these groups selected unique sequences, except the Src subfamily (Src, Fyn, Lck, and Fgr), which all selected the sequence pTyr-Glu-Glu-Ile. The variability in SH2 domain sequences at likely sites of contact provides a structural basis for the phosphopeptide selectivity of these families. Possible in vivo binding sites of the SH2 domains are discussed.

Amino Acid Sequence↗

PubMed1987

Mechanism of proton-induced bone calcium release: calcium carbonate-dissolution.

Protons are buffered and calcium is released by bone during metabolic acidosis. Incubation of neonatal mouse calvariae in acid medium causes net calcium efflux from bone and net proton influx into bone, just as metabolic acidosis does in vivo. To determine whether the calcium carbonate phase of bone mineral is solubilized with increasing proton concentrations, we cultured calvariae for 3 h in medium in which the saturation was varied by changing pH or calcium and phosphate concentrations. We determined the driving force for crystallization by calculating the Gibbs free energy of formation (DG). With alteration of the medium pH, calcium carbonate entry or loss from bone varied linearly with the initial DG for medium calcium carbonate (r = -0.745, n = 41, P less than 0.001) as it did with alteration of the medium calcium and phosphate (r = -0.665, n = 118, P less than 0.001). There was dissolution of calcium carbonate into medium that was unsaturated with respect to calcium carbonate, net flux ceased at saturation, and calcium carbonate entered bone from supersaturated medium, indicating that the medium is in equilibrium with the calcium carbonate phase of bone mineral. Neither the mineral phase brushite nor apatite was in equilibrium with the medium. These observations indicate that in vitro, acute proton-induced calcium efflux is due to dissolution of bone calcium carbonate.

Acidosis↗