Biomedical subjects

M Park

Publications and source records attributed to M Park.

At least 19 recordsLinked to original sources

PubMed2000

Identification of an atypical Grb2 carboxyl-terminal SH3 domain binding site in Gab docking proteins reveals Grb2-dependent and -independent recruitment of Gab1 to receptor tyrosine kinases.

The Gab family of docking proteins is phosphorylated in response to various growth factors and cytokines and serves to recruit multiple signaling proteins. Gab1 acts downstream from the Met-hepatocyte growth factor receptor, and Gab1 overexpression promotes Met-dependent morphogenesis of epithelial cells. Recruitment of Gab1 to Met or epidermal growth factor (EGF) receptors requires a receptor-binding site for the Grb2 adapter protein and a proline-rich domain in Gab1, defined as the Met-binding domain. To determine the requirement for Grb2 in Gab1 recruitment, we have mapped two Grb2 carboxyl-terminal SH3 domain binding sites conserved in Gab1 and related protein Gab2. One corresponds to a canonical Grb2-binding motif, whereas the second, located within the Gab1 Met-binding domain, requires the proline and arginine residues of an atypical PXXXR motif. The PXXXR motif is required but not sufficient for Grb2 binding, whereas an extended motif, PX3RX2KPX7PLD, conserved in Gab proteins as well as the Grb2/Gads-docking protein, Slp-76, efficiently competes binding of Grb2 or Gads adapter proteins. The association of Gab1 with Grb2 is required for Gab1 recruitment to the EGF receptor but not the Met receptor. Hence different mechanisms of Gab1 recruitment may reflect the distinct biological functions for Gab1 downstream from the EGF and Met receptors.

Adaptor Proteins, Signal Transducing↗

PubMed2000

The MADS-Box factor CeMEF2 is not essential for Caenorhabditis elegans myogenesis and development.

MEF2 is an evolutionarily conserved MADS (MCM1, Agamous, Deficiens, and serum response factor) box-type transcription factor that plays a critical role in vertebrate and Drosophila melanogaster myogenesis. We have addressed the developmental role of the single MEF2-like factor, CeMEF2, in Caenorhabditis elegans. Using expression assays and two mef-2 deletion alleles, we show that CeMEF2 is not required for proper myogenesis or development. Moreover, a putative null mef-2 allele fails to enhance or suppress the phenotypes of mutants in CeMyoD or CeTwist. Our results suggest that despite its evolutionary conservation of sequence and DNA binding properties, CeMEF2 has adopted a divergent role in development in the nematode compared with Drosophila and vertebrates.

Amino Acid Sequence↗

PubMed2000

Design and synthesis of anticonvulsive agents as gamma-vinyl GABA-based potential dual acting prodrugs and their biological activities.

For the development of new anticonvulsive agents, gamma-vinyl GABA (vigabatrin) and GABA mimetics derivatives were covalently coupled as potential dual acting prodrugs and evaluated for their anticonvulsive activities. Among the prepared compounds, 11 showed the most potent anticonvulsive activity, a shorter onset time and a broader spectrum compared to vigabatrin.

Animals↗

PubMed2000

Constitutive activation of cyclin B1-associated cdc2 kinase overrides p53-mediated G2-M arrest.

Recent studies have suggested that p53 regulates the G2 checkpoint in the cell cycle and that this function is required for the maintenance of genomic integrity. In this study, we investigated a regulatory role of p53 specifically in G2-M transition. Human bladder carcinoma cells lacking functional p53 were synchronized at G1-S, which is preceded by p53-mediated G1 arrest. p53 expression in the synchronized cells was induced by infection with a recombinant adenovirus that encodes p53. After release from the G1-S arrest, the cells progressed to S-phase and G2 but failed to enter mitosis. Biochemical analysis showed that p53 inhibits cell cycle-dependent expression of cdc2 and cyclin B1 and consequently inhibits cdc2 kinase. The role of cyclin B1-associated cdc2 kinase in p53-mediated G2-M arrest was further investigated by expression of both cyclin B1 and cdc2AF, in which inhibitory phosphorylation sites were substituted. The cells expressing both cdc2AF and cyclin B1 showed a constitutive activation of cdc2 kinase during cell cycle progression and passed through G2-M regardless of p53 expression. Therefore, inactivation of cdc2 kinase through cdc2 and cyclin B1 repression is an essential step in p53-mediated G2-M arrest.

CDC2 Protein Kinase↗

PubMed2000

Structural and functional evidence for microglial expression of C1qR(P), the C1q receptor that enhances phagocytosis.

Microglial activation has been associated with several degenerative diseases of the central nervous system (CNS). One consequence of activation is the induction of a more efficient phagocytic response, and it is therefore important to determine what factors regulate microglial phagocytosis and whether this capacity influences the progression of neurodegenerative changes. Previous studies have demonstrated that complement component C1q enhances Fc receptor- and CR1-mediated phagocytosis in cells of the myeloid lineage via a cell surface receptor, C1qRp. Because C1q has been found in the area of lesions in several degenerative CNS diseases, the current investigations were carried out to characterize the effects of C1q on microglial phagocytosis. Neonatal rat microglia were shown to express C1qRp, as assessed by flow cytometry and immunocytochemistry. Interaction of these cells with substrate-bound C1q was shown to enhance both FcR-and CR1-mediated phagocytosis two- to fourfold. In addition, introduction of an antibody raised against the carboxy-terminal, cytoplasmic domain of C1qRp into microglia by electroporation markedly diminished the ability of C1q to enhance uptake of IgG-coated targets, whereas nonspecific IgG had no such effect. These results suggest that C1q in areas of active degeneration may promote the phagocytic capacity of microglia via interaction with microglial C1qRp.

Animals↗

PubMed2000

The Wnt/Ca2+ pathway: a new vertebrate Wnt signaling pathway takes shape.

Members of the vertebrate Wnt family have been subdivided into two functional classes according to their biological activities. Some Wnts signal through the canonical Wnt-1/wingless pathway by stabilizing cytoplasmic beta-catenin. By contrast other Wnts stimulate intracellular Ca2+ release and activate two kinases, CamKII and PKC, in a G-protein-dependent manner. Moreover, putative Wnt receptors belonging to the Frizzled gene family have been identified that preferentially couple to the two prospective pathways in the absence of ectopic Wnt ligand and that might account for the signaling specificity of the Wnt pathways. As Ca2+ release was the first described feature of the noncanonical pathway, and as Ca2+ probably plays a key role in the activation of CamKII and PKC, we have named this Wnt pathway the Wnt/Ca2+ pathway.

Animals↗

PubMed2000

Ontogeny of (D-Ala(2))-deltorphin I-like immunoreactive neurones in foetal rat brain.

Foetal rat brain from embryonic day (ED) 12-22 was immunohistochemically studied to describe the time of first appearance and further distribution patterns of (D-Ala(2))-deltorphin-I-immunoreactive (DADTI-IR) nerve elements. The primary antiserum used in this study was a polyclonal antibody against DADTI previously used in adult and postnatal rat brain mapping. DADTI-IR nerve elements first appeared in the neuroepithelium of ventral mesencephalon on ED 13. From there, positive cell bodies migrated towards the mantle layer until they invaded the whole ventral mesencephalic tegmentum. They then reached their definitive position, corresponding to a subpopulation of the A8, A9 and A10 dopaminergic neurones that had been constantly observed also in the adult age. From ED 15-17, DADTI-positive nerve fibres appeared in the medial forebrain bundle, the neostriatum anlage, the accumbens nucleus, the olfactory tubercle, the fasciculus retroflexus, and the prefrontal cortex. All these locations have also been found in adult rats. From ED 14 onwards, transient DADTI-IR somata and nerve fibres were observed in retinal neuroepithelium, optic pathways as far as the superior colliculus, CA3 hippocampal field, reticular formation in the medulla oblongata. All these locations gradually disappeared either before birth (medulla oblongata) or within the first 3 weeks after birth. These results suggest that the DADT-like molecule recognised by our antibody has during the embryonic development a regulatory function in neuronal growth and differentiation.

Analgesics, Opioid↗

PubMed2000

In vitro DNA deamination by alpha-nitrosaminoaldehydes determined by GC/MS-SIM quantitation.

The deamination of DNA bases by three alpha-nitrosaminoaldehydes, butylethanalnitrosamine, methylethanalnitrosamine, and N-nitroso-2-hydroxymorpholine (NHMOR), the direct metabolite of potent animal carcinogen N-nitrosodiethanolamine, was demonstrated by a set of in vitro experiments. The deamination of guanine, adenine, and cytosine bases in nucleotides, oligonucleotides, and calf thymus DNA gave xanthine, hypoxanthine, and uracil, respectively. The order of relative reactivities of the bases was as listed above. Deamination of cytosine to uracil was detected by the reaction of (32)P-labeled oligonucleotide ([5'-(32)P]CGAT) followed by enzymatic hydrolysis. Quantitative analysis of deamination of guanine and adenine in calf thymus DNA was performed by a gas chromatography/mass spectrometry-selected ion monitoring method. Both the extent and the rate of the deamination reactions which occur by transnitrosation from the alpha-nitrosaminoaldehyde to the base were determined for formation of xanthine and hypoxanthine. The deamination of guanine by NHMOR remained significant at low substrate levels.

Adenine↗

PubMed2000

Effects of phenethylisothiocyanate on the expression of glutathione S-transferases and hepatotoxicity induced by acetaminophen.

1. The effect of PEITC on the expression of hepatic glutathione S-transferases (GST) and the glutathione (GSH) conjugation has been investigated in the Sprague-Dawley rat, and it has been determined whether hepatotoxicity of acetaminophen (AA) could be inhibited through the induction of GST expression in mouse. 2. The hepatic GST activity and protein levels of alpha class (Ya, Yc) and mu class (Yb1, Yb2) of GST were elevated in a dose-dependent manner after treatment with PEITC (0, 3.16, 10, 31.6, 100 and 200 mg/kg, p.o., 3 days). The mRNA levels of GST Ya and GST Yb1 were also markedly increased 1 day after treatment with PEITC at dosages ranging from 31.6 to 200 mg/kg. The hepatic GSH content was significantly increased to 200% of control at dose of 200 mg/kg PEITC. 3. Pretreatment with 100 mg/kg PEITC significantly enhanced the biliary excretion of glutathione conjugate of AA 2-fold, whereas treatment with 200 mg/kg did not affect it. 4. In mouse, PEITC (100 and 200 mg/kg, 3 days) decreased the lethality and hepatotoxicity caused by AA. 5. These results indicate that (1) the induction of GST by PEITC is presumably under transcriptional regulation, and (2) PEITC may have a protective function against AA-induced hepatotoxicity by induction effect on GST, in combination of enhancement of hepatic GSH.

Acetaminophen↗

PubMed2000

Activation of cdc42, rac, PAK, and rho-kinase in response to hepatocyte growth factor differentially regulates epithelial cell colony spreading and dissociation.

Hepatocyte growth factor (HGF), the ligand for the Met receptor tyrosine kinase, is a potent modulator of epithelial-mesenchymal transition and dispersal of epithelial cells, processes that play crucial roles in tumor development, invasion, and metastasis. Little is known about the Met-dependent proximal signals that regulate these events. We show that HGF stimulation of epithelial cells leads to activation of the Rho GTPases, Cdc42 and Rac, concomitant with the formation of filopodia and lamellipodia. Notably, HGF-dependent activation of Rac but not Cdc42 is dependent on phosphatidylinositol 3-kinase. Moreover, HGF-induced lamellipodia formation and cell spreading require phosphatidylinositol 3-kinase and are inhibited by dominant negative Cdc42 or Rac. HGF induces activation of the Cdc42/Rac-regulated p21-activated kinase (PAK) and c-Jun N-terminal kinase, and translocation of Rac, PAK, and Rho-dependent Rho-kinase to membrane ruffles. Use of dominant negative and activated mutants reveals an essential role for PAK but not Rho-kinase in HGF-induced epithelial cell spreading, whereas Rho-kinase activity is required for the formation of focal adhesions and stress fibers in response to HGF. We conclude that PAK and Rho-kinase play opposing roles in epithelial-mesenchymal transition induced by HGF, and provide new insight regarding the role of Cdc42 in these events.

Actins↗

PubMed2000

Enhanced transformation by a plasma membrane-associated met oncoprotein: activation of a phosphoinositide 3'-kinase-dependent autocrine loop involving hyaluronic acid and CD44.

A Met-hepatocyte growth factor receptor oncoprotein, Tpr-Met, generated by chromosomal rearrangement, fuses a protein dimerization motif with the cytoplasmic domain of the Met receptor, producing a cytosolic, constitutively activated tyrosine kinase. Although both the Met receptor and the Tpr-Met oncoprotein associate with the same substrates, activating mutations of the Met receptor in hereditary papillary renal carcinomas have different signaling requirements for transformation than Tpr-Met. This suggests differential activation of membrane-localized pathways by oncogenic forms of the membrane-bound Met receptor but not by the cytoplasmic Tpr-Met oncoprotein. To establish which pathways might be differentially regulated, we have localized the constitutively activated Tpr-Met oncoprotein to the membrane using the c-src myristoylation signal. Membrane localization enhances cellular transformation, focus formation, and anchorage-independent growth and induces tumors with a distinct myxoid phenotype. This correlates with the induction of hyaluronic acid (HA) and the presence of a distinct form of its receptor, CD44. A pharmacological inhibitor of phosphoinositide 3' kinase (PI3'K), inhibits the production of HA, and conversely, an activated, plasma membrane-targeted form of PI3'K is sufficient to enhance HA production. Furthermore, the multisubstrate adapter protein Gab-1, which couples the Met receptor with PI3'K, enhances Met receptor-dependent HA synthesis in a PI3'K-dependent manner. These results provide a positive link to a role for HA and CD44 in Met receptor-mediated oncogenesis and implicate PI3'K in these events.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Molecular mechanism for the Shp-2 tyrosine phosphatase function in promoting growth factor stimulation of Erk activity.

We have previously shown that activation of extracellular signal-regulated kinase (Erk) by epidermal growth factor (EGF) treatment was significantly decreased in mouse fibroblast cells expressing a mutant Shp-2 molecule lacking 65 amino acids in the SH2-N domain, Shp-2(Delta46-110). To address the molecular mechanism for the positive role of Shp-2 in mediating Erk induction, we evaluated the activation of signaling components upstream of Erk in Shp-2 mutant cells. EGF-stimulated Ras, Raf, and Mek activation was significantly attenuated in Shp-2 mutant cells, suggesting that Shp-2 acts to promote Ras activation or to suppress the down-regulation of activated Ras. Biochemical analyses indicate that upon EGF stimulation, Shp-2 is recruited into a multiprotein complex assembled on the Gab1 docking molecule and that Shp-2 seems to exert its biological function by specifically dephosphorylating an unidentified molecule of 90 kDa in the complex. The mutant Shp-2(Delta46-110) molecule failed to participate in the Gab1-organized complex for dephosphorylation of p90, correlating with a defective activation of the Ras-Raf-Mek-Erk cascade in EGF-treated Shp-2 mutant cells. Evidence is also presented that Shp-2 does not appear to modulate the signal relay from EGF receptor to Ras through the Shc, Grb2, and Sos proteins. These results begin to elucidate the mechanism of Shp-2 function downstream of a receptor tyrosine kinase to promote the activation of the Ras-Erk pathway, with potential therapeutic applications in cancer treatment.

Animals↗

PubMed2000

Primary structure and function of three gonadotropin-releasing hormones, including a novel form, from an ancient teleost, herring.

The evolution of GnRH and the role of multiple forms within the brain are examined. Three forms of GnRH were purified from the brain of Pacific herring (Clupea harengus pallasi) and characterized using Edman degradation and mass spectrometry. Two forms correspond with the known structures of chicken GnRH-II and salmon GnRH that are found in many vertebrate species. The third form, designated herring GnRH (hrGnRH), has a primary structure of pGlu-His-Trp-Ser-His-Gly-Leu-Ser-Pro-Gly-NH2. This novel peptide is a potent stimulator of gonadotropin II and GH release from dispersed fish pituitary cells. The content of hrGnRH in the pituitary was 8-fold that of salmon GnRH and 43-fold that of chicken GnRH-II, which provides supporting evidence that hrGnRH is involved in the release of gonadotropin. Herring is the most phylogenetically ancient animal in which three forms of GnRH have been isolated and sequenced. Our evidence suggests that the existence of three GnRHs in the brain of one species 1) is an ancestral condition for teleosts, 2) has the potential for separate regulation of the distinct GnRHs, and 3) may be an evolutionary advantage for refined control of reproduction in different environments.

Amino Acid Sequence↗

PubMed2000

Cardiac enhancer activity of the homeobox gene tinman depends on CREB consensus binding sites in Drosophila.

The Drosophila homeobox gene tinman plays a critical role in subdividing the early mesoderm. In particular, tinman is absolutely required for formation of the heart and visceral mesoderm. tinman expression is initiated throughout the mesoderm of the trunk region under the control of the bHLH transcription factor encoded by the twist gene, a determinant of all mesoderm. Later, tinman expression is restricted to the dorsal portion of the mesoderm, a process that is directed by decapentaplegic (dpp) whose product (a TGF-beta-related protein) is secreted by the overlaying ectoderm. Further restriction of tinman expression to the cardiac progenitors, in which it will persist throughout development, involves the secreted segmentation gene product encoded by wingless (wg, a Drosophila Wnt gene). Here, we show that strong early expression depends on the synergistic action of an enhancer element at the 5' end of the gene in conjunction with an element in the first intron. Moreover, two distinct enhancer regions are responsible for tinman expression in the heart: one region confers expression in the heart-tube-associated pericardial cells, the other element drives expression in the contractile, myocardial cells. The latter element contains two CREB consensus binding sites that are essential for cardiac-specific expression. genesis 26:55-66, 2000.

Animals↗

PubMed1999

The fourth immunoglobulin-like loop in the extracellular domain of FLT-1, a VEGF receptor, includes a major heparin-binding site.

We found that heparin at low concentrations increases the vascular endothelial growth factor (VEGF)-induced proliferation of human umbilical vein endothelial cells (HUVECs) but at high concentrations decreases it. To examine whether FLT-1, a VEGF receptor, interacts with heparin and which domain of FLT-1 binds to heparin, various extracellular domains of FLT-1 were expressed in a baculovirus/insect cell system: sFLT-1(1-7), sFLT-1(1-4), sFLT-1(1-3), and sFLT-1(1-2) containing immunoglobulin (Ig)-like loop one to seven, one to four, one to three, and one to two, respectively. The sFLT-1(1-7) and sFLT-1(1-4) readily bound heparin at the physiological salt concentration and half-dissociated from heparin at 0.65 M and 0.57 M NaCl, respectively. In contrast, the sFLT-1(1-3) and sFLT-1(1-2) poorly bound heparin at the physiological salt concentration. In addition, the interaction of sFLT-1(1-7) with heparin was not affected by EDTA up to 80 mM. We thus concluded that the fourth Ig-like loop of FLT-1 is a major heparin-binding site and divalent cations are not involved in the interaction of FLT-1 and heparin.

Binding Sites↗

PubMed1999

A conserved inositol phospholipid binding site within the pleckstrin homology domain of the Gab1 docking protein is required for epithelial morphogenesis.

Stimulation of the hepatocyte growth factor receptor tyrosine kinase, Met, induces the inherent morphogenic program of epithelial cells. The multisubstrate binding protein Gab1 (Grb2-associated binder-1) is the major phosphorylated protein in epithelial cells following activation of Met. Gab1 contains a pleckstrin homology domain and multiple tyrosine residues that act to couple Met with multiple signaling proteins. Met receptor mutants that are impaired in their association with Gab1 fail to induce a morphogenic program in epithelial cells, which is rescued by overexpression of Gab1. The Gab1 pleckstrin homology domain binds to phosphatidylinositol 3,4, 5-trisphosphate and contains conserved residues, shown from studies of other pleckstrin homology domains to be crucial for phospholipid binding. Mutation of conserved phospholipid binding residues tryptophan 26 and arginine 29, generates Gab1 proteins with decreased phosphatidylinositol 3,4,5-trisphosphate binding, decreased localization at sites of cell-cell contact, and reduced ability to rescue Met-dependent morphogenesis. We conclude that the ability of the Gab1 pleckstrin homology domain to bind phosphatidylinositol 3,4,5-trisphosphate is critical for subcellular localization of Gab1 and for efficient morphogenesis downstream from the Met receptor.

Amino Acid Sequence↗

PubMed1999

DNA contacts by protein domains of the molluscum contagiosum virus type-1B topoisomerase.

All poxviruses studied encode a type 1B topoisomerase that introduces transient nicks into DNA and thereby relaxes DNA supercoils. Here we present a study of the protein domains of the topoisomerase of the poxvirus molluscum contagiosum (MCV), which allows us to specify DNA contacts made by different domains. Partial proteolysis of the enzyme revealed two stable domains separated by a protease-sensitive linker. A fragment encoding the linker and carboxyl-terminal domain (residues 82-323) was overexpressed in Escherichia coli and purified. MCV topoisomerase (MCV-TOP)(82-323) could relax supercoiled plasmids in vitro, albeit with a slower rate than the wild-type enzyme. MCV-TOP(82-323) was sensitive to sequences in the favored 5'-(T/C)CCTT-3' recognition site and also flanking DNA, indicating that some of the sequence-specific contacts are made by residues 82-323. Assays of initial binding and covalent catalysis by MCV-TOP(82-323) identified the contacts flanking the 5'-CCCTT-3' sequence at +10, +9, -2, and -3 to be important. Tests with substrates containing a 5-bridging phosphorothiolate that trap the cleaved complex revealed that correct contacts to the flanking sequences were important in the initial cleavage step. MCV-TOP(82-323) differed from the full-length protein in showing reduced sensitivity to mutations at a position within the 5'-(T/C)CCTT-3' recognition site, consistent with a model in which the amino-terminal domain contacts this region. These findings provide insight into the division of labor within the MCV-TOP enzyme.

Base Sequence↗

PubMed1999

Protein kinase C is differentially stimulated by Wnt and Frizzled homologs in a G-protein-dependent manner.

In studies of developmental signaling pathways stimulated by the Wnt proteins and their receptors, Xenopus Wnt-5A (Xwnt-5A) and a prospective Wnt receptor, rat Frizzled 2 (Rfz2), have been shown to stimulate inositol signaling and Ca2+ fluxes in zebrafish [1] [2] [3]. As protein kinase C (PKC) isoforms can respond to Ca2+ signals [4], we asked whether expression of different Wnt and Frizzled homologs modulates PKC. Expression of Rfz2 and Xwnt-5A resulted in translocation of PKC to the plasma membrane, whereas expression of rat Frizzled 1 (Rfz1), which activates a Wnt pathway using beta-catenin but not Ca2+ fluxes [5], did not. Rfz2 and Xwnt-5A were also able to stimulate PKC activity in an in vitro kinase assay. Agents that inhibit Rfz2-induced signaling through G-protein subunits blocked Rfz2-induced translocation of PKC. To determine if other Frizzled homologs differentially stimulate PKC, we tested mouse Frizzled (Mfz) homologs for their ability to induce PKC translocation relative to their ability to induce the expression of two target genes of beta-catenin, siamois and Xnr3. Mfz7 and Mfz8 stimulated siamois and Xnr3 expression but not PKC activation, whereas Mfz3, Mfz4 and Mfz6 reciprocally stimulated PKC activation but not expression of siamois or Xnr3. These results demonstrate that some but not all Wnt and Frizzled signals modulate PKC localization and stimulate PKC activity via a G-protein-dependent mechanism. In agreement with other studies [1] [2] [3]. [6] [7] these data support the existence of multiple Wnt and Frizzled signaling pathways in vertebrates.

Animals↗