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Biomedical subjects

L F Lau

Publications and source records attributed to L F Lau.

At least 73 records · Page 4Linked to original sources
PubMed1993

Functional domains and phosphorylation of the orphan receptor Nur77.

Nur77 represents a unique class within the steroid receptor superfamily since its synthesis is tightly regulated by extracellular signals and it is capable of potent transactivation activity in the absence of an exogenously added ligand. In this study, we sought to dissect the functional domains regulating the activities of Nur77 by deletion mapping. We demonstrate that whereas the transactivation activity of Nur77 resides in the amino-terminal domain, the carboxy-terminal domain regulates this activity. A short deletion from the carboxy terminus eliminates transactivation activity while a further deletion restores the activity. Deletion of the domain immediately carboxyl to the zinc fingers motif eliminates both DNA binding activity and nuclear localization, thus abolishing transactivation. Nur77 is posttranslationally modified predominantly by phosphorylation, which occurs primarily at the N-terminal domain. The growth-related kinase pp90rsk, but neither the pp44mapk nor the pp70s6k, can phosphorylate recombinant Nur77 in vitro. Furthermore, we have identified a site within the region required for sequence-specific DNA binding, Ser-354, that is phosphorylated by pp90rsk in vitro; this site is also phosphorylated in vivo. The possibility that phosphorylation might affect DNA binding is discussed.

3T3 Cells↗
PubMed1993

Genomic structure, cDNA sequence, and expression of gly96, a growth factor-inducible immediate-early gene encoding a short-lived glycosylated protein.

We report the cDNA sequence and genomic structure of gly96, an immediate early gene inducible by serum growth factors in mouse fibroblasts. It encodes a 153-amino acid protein that does not share significant sequence similarity with any known protein. In the adult mouse, gly96 is expressed predominantly in the lung, testes and the uterus. We have identified the Gly96 protein in Balb/c 3T3 cells using affinity-purified antibodies recognizing the Gly96 polypeptide. We show that Gly96 is glycosylated and has a short half-life in serum stimulated fibroblasts.

3T3 Cells↗
PubMed1992

Activation of signal transduction in platelets by the tyrosine phosphatase inhibitor pervanadate (vanadyl hydroperoxide).

The protein tyrosine phosphatase (PTPase) inhibitor pervanadate (vanadyl hydroperoxide) stimulated protein tyrosine phosphorylation 29-fold more than did thrombin in intact and saponin-permeabilized platelets. Increased tyrosine phosphorylation preceded, or was coincident with, a fall in PtdIns(4,5)P2 levels, production of PtdIns(3,4)P2 and phosphatidic acid, mobilization of intracellular Ca2+, stimulation of protein kinase C-dependent protein phosphorylation, secretion of dense and alpha-granules, increased actin polymerization, shape change and aggregation which required fibrinogen and was mediated by increased surface expression of GPIIb-IIIa. The tyrosine kinase inhibitor RG 50864 totally prevented induction of tyrosine phosphorylation by pervanadate, as well as all other responses measured; in contrast, the inactive structural analogue, tyrphostin #1, had no effect. Dense-granule secretion induced by pervanadate required protein kinase C activity; however, aggregation and alpha-granule secretion were independent of protein kinase C. In saponin-permeabilized platelets pervanadate and thrombin stimulated phospholipase C activity by GTP-independent and GTP-dependent mechanisms respectively. We conclude that PTPases are important regulators of signal transduction in platelets.

Actins↗
PubMed1992

Expression of the growth factor-inducible immediate early gene cyr61 correlates with chondrogenesis during mouse embryonic development.

cyr61 is a growth factor-inducible immediate early gene initially identified in serum-stimulated mouse fibroblasts. It encodes a member of an emerging family of cysteine-rich secreted proteins that includes a connective tissue growth factor. We show here that cyr61 is expressed in the developing mouse embryo and extraembryonic tissues. In the placenta, cyr61 is expressed in regions of trophoblastic origin, including the ectoplacental cone and the trophoblastic giant cells. In the midgestation embryo, cyr61 is expressed in the smooth muscle vessel walls of the arterial circulatory system. Most notably, expression is found in developing cartilaginous elements, including the limbs, ribs, and prevertebrae. In addition, regions of the chondrocranium and craniofacial elements, such as Meckel's cartilage, also express cyr61. Thus, cyr61 transcript is found in mesenchymal cells of both mesodermal and ectodermal origin during their differentiation into chondrocytes. The temporal and spatial regulation of cyr61 expression and the biochemical features of its encoded protein suggest that cyr61 may be important for the normal growth, differentiation, or morphogenesis of the cartilaginous skeleton of the embryo.

Amino Acid Sequence↗
PubMed1992

cDNA sequence of a growth factor-inducible immediate early gene and characterization of its encoded protein.

We report the cDNA sequence of 3CH134, an immediate early gene whose transcription is rapidly and transiently stimulated by serum growth factors. 3CH134 encodes a 367 amino acid protein that does not share significant sequence similarity with any known protein. 3CH134 is inducible through multiple signal transduction pathways, and in the adult mouse is expressed predominantly in the lung. Using affinity-purified antibodies, we have identified the 3CH134 protein in serum-stimulated Balb/c 3T3 cells and determined that it has a short half-life.

3T3 Cells↗
PubMed1991

Promoter function and structure of the growth factor-inducible immediate early gene cyr61.

cyr61 is an immediate early gene that is transcriptionally activated in 3T3 fibroblasts by serum, platelet-derived growth factor, fibroblast growth factor, and the tumor promoter TPA with kinetics similar to the induction of c-fos. cyr61 encodes a secreted protein that is associated with the cell surface and the extracellular matrix, and may play a role in cell-cell communication. We report here the complete nucleotide sequence of the mouse cyr61 gene, which contains four short introns. The transcription start site was mapped by S1 nuclease and primer extension analyses. A 2 kb 5' flanking DNA fragment functions as a serum-inducible promoter. This DNA fragment contains a poly(CA) sequence that can adopt the Z DNA form. In addition, it contains a sequence that resembles the serum response element (SRE) originally identified in the c-fos promoter. We show that deletion of the cry61 SRE-like sequence abrogates serum inducibility. Furthermore, this SRE-like sequence is sufficient to confer serum and growth factor inducibility when linked to a basal promoter, and binds the 67 kD serum response factor in vitro. We conclude that the cyr61 SRE functions as a serum response element and may account for the coordinate activation of cyr61 and c-fos.

Amino Acid Sequence↗
PubMed1991

An Id-related helix-loop-helix protein encoded by a growth factor-inducible gene.

An mRNA encoding a helix-loop-helix protein that we have named HLH462 is induced in mouse 3T3 cells as part of the immediate early transcriptional response to growth factors and other signaling agents. The RNA is present in a number of mouse tissues and in the developing mouse fetus. The HLH462 gene has been mapped by interspecific backcross analysis to the distal region of mouse chromosome 4. In its helix-loop-helix region HLH462 is closely related to the Id protein and the Drosophila emc protein. Like Id, HLH462 lacks a basic region required for DNA binding, and it inhibits the DNA-binding activities of other helix-loop-helix proteins. On the basis of its structural and functional similarity to Id, we suggest that HLH462 may inhibit the activities of helix-loop-helix transcription factors during the cellular growth response and during development.

Amino Acid Sequence↗
PubMed1991

Rapid and preferential activation of the c-jun gene during the mammalian UV response.

Exposure of mammalian cells to DNA-damaging agents leads to activation of a genetic response known as the UV response. Because several previously identified UV-inducible genes contain AP-1 binding sites within their promoters, we investigated the induction of AP-1 activity by DNA-damaging agents. We found that expression of both c-jun and c-fos, which encode proteins that participate in formation of the AP-1 complex, is rapidly induced by two different DNA-damaging agents: UV and H2O2. Interestingly, the c-jun gene is far more responsive to UV than any other immediate-early gene that was examined, including c-fos. Other jun and fos genes were only marginally affected by UV or H2O2. Furthermore, UV is a much more efficient inducer of c-jun than phorbol esters, the standard inducers of c-jun expression. This preferential response of the c-jun gene is mediated by its 5' control region and requires the TPA response element, suggesting that this element also serves as an early target for the signal transduction pathway elicited by DNA damage. Both UV and H2O2 lead to a long-lasting increase in AP-1 binding activity, suggesting that AP-1 may mediate the induction of other damage-inducible genes such as human collagenase.

DNA-Binding Proteins↗
PubMed1991

Nur77 is differentially modified in PC12 cells upon membrane depolarization and growth factor treatment.

The rat pheochromocytoma cell line PC12 can be induced by growth factors to undergo proliferation and neuronal differentiation. These cells also have excitable membranes that can be depolarized by neurotransmitters or elevated levels of extracellular KCl. Treatment of PC12 cells with growth factors or membrane-depolarizing agents rapidly activates the expression of specific genes whose products are thought to mediate the subsequent biological responses. One such gene, nur77, is a member of the steroid and thyroid hormone receptor gene superfamily. We have identified the Nur77 protein and shown that it is synthesized rapidly and transiently in PC12 cells following stimulation, has a short half-life of 30 to 40 min, and is located in both the nucleus and the cytoplasm. Nur77 is posttranslationally modified, primarily by phosphorylation on serine residues. Phosphopeptide analysis reveals that Nur77 is modified differently upon membrane depolarization than after treatment with growth factors. We hypothesize that the activity of Nur77 is regulated by both differential gene expression and posttranslational modification and that these modes of regulation contribute to distinct downstream responses specific to membrane depolarization and growth factor treatment.

Adrenal Gland Neoplasms↗
PubMed1991

Transcriptional activation by Nur77, a growth factor-inducible member of the steroid hormone receptor superfamily.

nur77 is a growth factor-inducible immediate early gene that encodes a protein with extensive sequence homology to members of the steroid/thyroid hormone receptor superfamily. By analogy to steroid receptors, the Nur77 protein is thought to act as a ligand-dependent transcription factor that regulates the genomic response to growth factors. Using chimeric gene constructs, we show that Nur77 can indeed function as a transcription activator. Furthermore, Nur77 chimeras can activate transcription in the absence of an exogenously added ligand.

Animals↗
PubMed1991

Cyr61, product of a growth factor-inducible immediate early gene, is associated with the extracellular matrix and the cell surface.

cyr61 is a specific target for activation by platelet-derived growth factor and fibroblast growth factor and is inducible by the oncogene v-src. It is a member of the class of immediate early genes that includes those encoding protooncogene products, transcription factors, and cytokines. We have previously characterized the synthesis and degradation of the cyr61-encoded mRNA and protein. Although the deduced Cyr61 protein sequence contains an NH2-terminal secretory signal, it is not detectable in the conditioned medium of serum-stimulated cells. We show here that in rapidly growing cell cultures, newly synthesized Cyr61 is secreted and is associated with both the extracellular matrix and the cell surface. In contrast, Cyr61 secreted in serum-stimulated quiescent cells is directed to the cell surface and is hot incorporated into the extracellular matrix. Once associated with the extracellular matrix, Cyr61 has a half-life of greater than 24 h, whereas intracellular and cell surface-associated Cyr61 has an apparent half-life of approximately 30 min. Furthermore, Cyr61 appears to bind heparin with high affinity. These observations suggest similarities among Cyr61, the fibroblast growth factors (heparin-binding growth factors), and the protooncogene product Int-1 and are consistent with the hypothesis that Cyr61 plays a role in cell-cell communication involving the interaction of neighboring cells.

Animals↗
PubMed1990

A growth factor-inducible nuclear protein with a novel cysteine/histidine repetitive sequence.

Growth factors rapidly induce transcription of a set of genes that encode regulatory proteins, many of which have been identified by cDNA cloning. Here we report the analysis of a cDNA corresponding to a gene induced in mouse 3T3 cells by growth factors and a variety of other extracellular signaling agents. The cDNA encodes a proline-, serine-, and glycine-rich nuclear protein designated Nup475 of 319 amino acids that contains two tandemly repeated cysteine- and histidine-containing sequences (CX8CX5CX3H) suggestive of a novel heavy metal-binding domain. Nup475 produced in Escherichia coli binds zinc. Its mRNA is present in a number of mouse tissues and cell lines, being especially abundant in intestine, thymus, and regenerating liver and in a macrophage cell line stimulated by gamma-interferon. We hypothesize that Nup475 is a regulatory protein with a novel zinc finger structure.

Amino Acid Sequence↗
PubMed1990

Synergistic effect of phorbol myristate acetate on bacterial lipopolysaccharide induced rat splenocyte proliferation.

1. Bacterial lipopolysaccharide (LPS) stimulated [3H]TdR incorporation of rat splenocytes in a concentration dependent manner. 2. Phorbol 12-myristate 13-acetate (PMA) alone has little effect on rat splenocyte proliferation but it exerted a marked synergistic effect on LPS-induced [3H]TdR incorporation when added at the first few hours of incubation with LPS. Minimal synergistic effect of PMA was observed if it was added later than 4 hr after LPS application. 3. Both LPS-stimulated and PMA synergized incorporation of [3H]TdR in rat splenocytes were inhibited by H-7, a protein kinase C inhibitor. 4. The results support the notion that the activation of protein kinase C is a necessary but insufficient cellular signal in the initiation of proliferative response of rat splenocytes by LPS.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

Pip92: a short-lived, growth factor-inducible protein in BALB/c 3T3 and PC12 cells.

pip92 is a cellular immediate-early gene inducible by serum growth factors in fibroblasts. It is also induced in the rat pheochromocytoma cell line PC12 by agents that cause proliferation, neuronal differentiation, and membrane depolarization. We show that the pip92-encoded polypeptide is a proline-rich protein of 221 amino acids, has an extremely short half-life, and is localized in the cytoplasm. We hypothesize that Pip92 plays a role in mediating the cellular responses to a variety of extracellular signals.

Adrenal Gland Neoplasms↗
PubMed1990

Expression of cyr61, a growth factor-inducible immediate-early gene.

A set of immediate-early genes that are rapidly activated by serum or purified platelet-derived growth factor in mouse 3T3 fibroblasts has been previously identified. Among these genes, several are related to known or putative transcription factors and growth factors, supporting the notion that some of these genes encode regulatory molecules important to cell growth. We show here that a member of this set of genes, cyr61 (originally identified by its cDNA 3CH61), encodes a 379-amino-acid polypeptide rich in cysteine residues. cyr61 can be induced through protein kinase C-dependent and -independent pathways. Unlike many immediate-early genes that are transiently expressed, the cyr61 mRNA is accumulated from the G0/G1 transition through mid-G1. This expression pattern is due to persistent transcription, while the mRNA is rapidly turned over during the G0/G1 transition and in mid-G1 at the same rate. In logarithmically growing cells, the cyr61 mRNA level is constant throughout the cell cycle. Cyr61 contains an N-terminal secretory signal sequence; however, it is not detected in the culture medium by immunoprecipitation. Cyr61 is synthesized maximally at 1 to 2 h after serum stimulation and has a short half-life within the cell.

Amino Acid Sequence↗
PubMed1989

Synergistic release of arachidonic acid from platelets by activators of protein kinase C and Ca2+ ionophores. Evidence for the role of protein phosphorylation in the activation of phospholipase A2 and independence from the Na+/H+ exchanger.

The protein kinase C activators phorbol myristate acetate (PMA), mezerein, oleoylacetylglycerol, and (-)-indolactam V, although without direct effect on arachidonic acid release, greatly enhance the release of platelet arachidonic acid caused by the Ca2+ ionophores A23187 and ionomycin. In contrast, 4 alpha-phorbol 12,13-didecanoate and (+)-indolactam V, which lack the ability to activate kinase C, do not potentiate arachidonate release. Release of arachidonic acid occurs without activation of phospholipase C and is therefore mediated by phospholipase A2. Synergism between PMA and A23187 is not affected by inactivation of the Na+/H+ exchanger with dimethylamiloride. The time course and dose-response for the effect of PMA at 23 degrees C closely correlate with the phosphorylation of a set of relatively "slowly" phosphorylated proteins (P20, P35, P41, P60), but not the rapidly phosphorylated P47 protein. P20 is myosin light chain, and P41 is probably Gi alpha, but the other proteins have not been positively identified. Depletion of metabolic ATP stores by antimycin A plus 2-deoxyglucose abolishes both protein phorphorylation and the potentiation of arachidonate release by PMA, but does not prevent fatty acid release by the ionophores. Similarly, the kinase C inhibitors H-7 and staurosporine produce, respectively, partial and complete inhibition of PMA-potentiated arachidonic acid release and protein phosphorylation, without affecting the direct response to ionophores. These results indicate that protein phosphorylation, mediated by kinase C, promotes the phospholipase A2 dependent release of arachidonic acid in platelets when intracellular Ca2+ is elevated by Ca2+ ionophores.

Adenosine Triphosphate↗
PubMed1989

Growth factors and membrane depolarization activate distinct programs of early response gene expression: dissociation of fos and jun induction.

A set of early response genes has been identified whose transcription in fibroblasts is rapidly induced in response to growth factors. Prototype members of this group, c-fos and c-jun, encode products that form a heterodimer and have been implicated in the regulation of gene expression and cell growth. It is thought that other early response genes also encode critical mediators of the cell's response to external stimuli. We have used PC12 pheochromocytoma cells as a model system to test the hypothesis that different extracellular signals induce distinct patterns of expression of early response genes. Our results indicate that membrane depolarization, induced either by potassium chloride or by the neurotransmitter analog nicotine, activates a program of gene expression distinct from that activated by nerve growth factor or epidermal growth factor. Notably, c-fos and c-jun activation can be dissociated; whereas c-jun is coinduced with c-fos and jun-B after growth factor stimulation, membrane depolarization activates c-fos and jun-B without stimulating c-jun. Fos may therefore form transcription complexes with alternative cofactors under different stimulation conditions. nur/77 and zif/268, which encode putative transcription factors, also show markedly different responses to growth factors and depolarization. We conclude that multiple nonconvergent signal transduction pathways control early response gene expression. Our findings also indicate that the diversity and specificity of cellular response to environmental change can be accounted for by the differential combinatorial induction of a relatively small number of early response genes.

Animals↗