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L T Williams

Publications and source records attributed to L T Williams.

At least 91 records · Page 5Linked to original sources
PubMed1993

Targeted expression of a dominant-negative FGF receptor mutant in the epidermis of transgenic mice reveals a role of FGF in keratinocyte organization and differentiation.

In this study we used a dominant-negative FGF receptor mutant to block FGF function in a specific tissue of transgenic mice. The mutant receptor, which is known to block signal transduction in cells when co-expressed with wild-type receptors, was targeted to suprabasal keratinocytes using a keratin 10 promoter. The transgene was expressed specifically in the skin and highest expression levels were found in the tail. Expression of the mutant receptor disrupted the organization of epidermal keratinocytes, induced epidermal hyperthickening and resulted in an aberrant expression of keratin 6. This suggests that FGF is essential for the morphogenesis of suprabasal keratinocytes and for the establishment of the normal program of keratinocyte differentiation. Our study demonstrates that dominant-negative growth factor receptors can be used to block selectively the action of a growth factor in specific tissues of transgenic mice.

Animals↗
PubMed1993

Phosphatidylinositol 3-kinase activity is important for progesterone-induced Xenopus oocyte maturation.

In somatic cells, phosphatidylinositol 3-kinase (PI3 kinase) is a critical intermediary in growth factor-induced mitogenesis. We have examined the role of this enzyme in meiotic maturation of Xenopus laevis oocytes. PI3 kinase activity was present in immunoprecipitates of the p85 subunit of PI3 kinase from immature oocytes and markedly increased following progesterone stimulation. Injection of bacterially expressed protein corresponding to the C-terminal SH2 domain of p85 (SH2-C) inhibited progesterone-induced PI3 kinase activation and meiotic maturation. Injection of protein corresponding to the N-terminal SH2 domain or the SH3 domain of p85 did not inhibit PI3 kinase activation or maturation. SH2-C did not inhibit oocyte maturation induced by c-mos RNA injection. In addition, radiolabelled SH2-C was used to probe oocyte lysates, revealing that a novel 200-kDa protein bound to SH2-C. This protein may be an important mediator of progesterone-induced lipid metabolism in oocytes.

Animals↗
PubMed1993

Platelet-derived growth factor receptor mediates activation of ras through different signaling pathways in different cell types.

A series of pieces of evidence have shown that Ras protein acts as a transducer of the platelet-derived growth factor (PDGF) receptor-mediated signaling pathway: (i) formation of Ras.GTP is detected immediately on PDGF stimulation, and (ii) a dominant inhibitory mutant Ras, as well as a neutralizing anti-Ras antibody, can interfere with PDGF-induced responses. On the other hand, several signal transducing molecules including phosphatidylinositol 3-kinase (PI3-K), GTPase-activating protein (GAP), and phospholipase C gamma (PLC gamma) bind directly to the PDGF receptor and become tyrosine phosphorylated. Recently, it was shown that specific phosphorylated tyrosines of the PDGF receptor are responsible for interaction between the receptor and each signaling molecule. However, the roles of these signaling molecules have not been elucidated, and it remains unclear which molecules are implicated in the Ras pathway. In this study, we measured Ras activation in cell lines expressing mutant PDGF receptors that are deficient in coupling with specific molecules. In fibroblast CHO cells, a mutant receptor (Y708F/Y719F [PI3-K-binding sites]) was unable to stimulate Ras, whereas another mutant (Y739F [the GAP-binding site]) could do so, suggesting an indispensable role of PI3-K or a protein that binds to the same sites as PI3-K for PDGF-stimulated Ras activation. By contrast, both of the above mutants were capable of stimulating Ras protein in a pro-B-cell line, BaF3. Furthermore, a mutant receptor (Y977F/Y989F [PLC gamma-binding sites]) could fully activate Ras, and the direct activation of protein kinase C and calcium mobilization had almost no effect on the GDP/GTP state of Ras in this cell line. These results suggest that, in the pro-B-cell transfectants, each of the above pathways (PI3-K, GAP, and PLC gamma) can be eliminated without a loss of Ras activation. It remains unclear whether another unknown essential pathway which regulates Ras protein exists within BaF3 cells. Therefore, it is likely that several different PDGF receptor-mediated signaling pathways function upstream of Ras, and the extent of the contribution of each pathway for the regulation of Ras may differ among different cell types.

Animals↗
PubMed1993

Raf-1 protein kinase is important for progesterone-induced Xenopus oocyte maturation and acts downstream of mos.

In somatic cells, the Raf-1 serine/threonine protein kinase is activated by several polypeptide growth factors. We investigated the role of Raf-1 in progesterone-induced meiotic maturation of Xenopus laevis oocytes. Raf-1 enzymatic activity and phosphorylation (reflected by a mobility shift on sodium dodecyl sulfate gels) were increased in oocytes following progesterone stimulation. The increase in Raf-1 activity was concurrent with an elevation in the activity of mitogen-activated protein (MAP) kinase. When RNA encoding an oncogenic form of Raf-1 (v-Raf) was injected into immature oocytes, MAP kinase mobility shift, germinal vesicle breakdown, and histone H1 phosphorylation increased markedly. When RNA encoding a dominant-negative version of Raf-1 was injected, progesterone-induced oocyte maturation was blocked. When RNA encoding Xenopus mos (mosxe) was injected into oocytes, Raf-1 and MAP kinase mobility shifts were observed after several hours. Also, when antisense mosxe oligonucleotides were injected into oocytes, progesterone-induced Raf-1 and MAP kinase mobility shifts were blocked. Finally, when antisense mosxe oligonucleotides were coinjected with v-Raf RNA into oocytes, histone H1 kinase activation, germinal vesicle breakdown, and MAP kinase mobility shift occurred. These findings suggest that Raf-1 activity is required for progesterone-induced oocyte maturation and that Raf-1 is downstream of mosxe activity.

Animals↗
PubMed1993

A region of the 85-kilodalton (kDa) subunit of phosphatidylinositol 3-kinase binds the 110-kDa catalytic subunit in vivo.

Phosphatidylinositol (PI) 3-kinase is a heterodimer consisting of an 85-kDa subunit (p85) and 110-kDa subunit (p110). The 85-kDa noncatalytic subunit, which contains two Src homology 2 (SH2) domains, one SH3 domain, and a domain homologous to the carboxy terminus of the breakpoint cluster region gene product, is known to mediate the association of the PI 3-kinase complex with activated growth factor receptors. We previously demonstrated that the C-terminal SH2 domain of p85 is responsible for the interaction of PI 3-kinase with phosphorylated platelet-derived growth factor receptor. To define the region in p85 that directs the complex formation with the PI 3-kinase catalytic subunit, a series of truncated p85 mutants was analyzed for association with p110 in vivo. We found that a fragment of p85 containing the region between the two SH2 domains was sufficient to promote the interaction with p110 in vivo. The complex between the fragment of p85 and p110 had PI 3-kinase activity that was comparable in magnitude to the activity of p110 associated with full-length p85. The binding with p110 was abolished when this domain in p85 was disrupted. These results identify a novel structural and functional element that is responsible for localizing the catalytic subunit of PI 3-kinase.

Animals↗
PubMed1992

A novel form of fibroblast growth factor receptor 2. Alternative splicing of the third immunoglobulin-like domain confers ligand binding specificity.

The fibroblast growth factor receptor 2 (FGFR2) gene is expressed as alternatively spliced mRNAs that encode bacterially expressed kinase, the keratinocyte growth factor receptor, or K-sam. We have now isolated a novel FGFR2 cDNA that is identical with the previously cloned human bacterially expressed kinase, except in the third immunoglobulin-like domain. The ligand binding properties of FGFR2 were studied by expressing the protein in rat L6 muscle myoblasts. Unlike human bacterially expressed kinase which binds acidic and basic FGF with similar affinities, FGFR2 bound acidic FGF with approximately 1000-fold higher affinity than basic FGF. These results indicate that alternative splicing of the FGFR2 gene in the region encoding the carboxyl-terminal half of the third immunoglobulin domain determines the ligand specificity of this group of receptors.

Alternative Splicing↗
PubMed1992

Point mutation of an FGF receptor abolishes phosphatidylinositol turnover and Ca2+ flux but not mitogenesis.

Stimulation of certain receptor tyrosine kinases results in the tyrosine phosphorylation and activation of phospholipase C gamma (PLC gamma), an enzyme that catalyses the hydrolysis of phosphatidylinositol (PtdIns). This hydrolysis generates diacylglycerol and free inositol phosphate, which in turn activate protein kinase C and increase intracellular Ca2+, respectively. PLC gamma physically associates with activated receptor tyrosine kinases, suggesting that it is a substrate for direct phosphorylation by these kinases. Here we report that a fibroblast growth factor (FGF) receptor with a single point mutation at residue 766 replacing tyrosine with phenylalanine fails to associate with PLC gamma in response to FGF. This mutant receptor also failed to mediate PtdIns hydrolysis and Ca2+ mobilization after FGF stimulation. However, the mutant receptor phosphorylated itself and several other cellular proteins, and it mediated mitogenesis in response to FGF. These findings show that a point mutation in the FGF receptor selectively eliminates activation of PLC gamma and that neither Ca2+ mobilization nor PtdIns hydrolysis are required for FGF-induced mitogenesis.

Amino Acid Sequence↗
PubMed1992

A naturally occurring secreted form of fibroblast growth factor (FGF) receptor 1 binds basic FGF in preference over acidic FGF.

Alternatively spliced variants of fibroblast growth factor receptor 1 mRNA are predicted to encode secreted forms and membrane-bound forms of receptors. The predicted amino acid sequences of these receptor variants differ in a portion of the extracellular region. In this study, we characterized the function of one of these splice variants which was predicted by its cDNA to be a secreted FGF receptor. We expressed this secreted form of the human FGFR1 (sFGFR1) in Chinese hamster ovary cells. The sFGFR1 protein oligomerized upon ligand binding. Surprisingly, the sFGFR1 preferentially bound basic FGF over acidic FGF. In cross-linking experiments, the sFGFR1 showed higher binding affinity for basic FGF (Kd approximately 30 nM) than for acidic FGF (Kd greater than 300 nM). These results suggest that this secreted form of FGF receptor has an unusual ligand binding specificity that may be important for its biological role in vivo.

Animals↗
PubMed1992

Large induction of keratinocyte growth factor expression in the dermis during wound healing.

Recent studies have shown that application of basic fibroblast growth factor (basic FGF) to a wound has a beneficial effect. However, it has not been assessed whether endogenous FGF also plays a role in tissue repair. In this study we found a 160-fold induction of mRNA encoding keratinocyte growth factor (KGF) 1 day after skin injury. This large induction was unique within the family of FGFs, since mRNA levels of acidic FGF, basic FGF, and FGF-5 were only slightly induced (2- to 10-fold) during wound healing, and there was no expression of FGF-3, FGF-4, and FGF-6 detected in normal and wounded skin. High levels of FGF receptor 1 and FGF receptor 2 mRNA and low levels of FGF receptor 3 mRNA were found in both normal and wounded skin. No change in the levels of these transcripts was detected during wound healing. In situ hybridization studies revealed highest levels of KGF mRNA expression in the dermis at the wound edge and in the hypodermis below the wound. In contrast, mRNA encoding the receptor of this growth factor (a splice variant of FGF receptor 2) was predominantly expressed in the epidermis. These results suggest that basal keratinocytes are stimulated by dermally derived KGF during wound healing and implicate a unique role of this member of the FGF family in wound repair.

Animals↗
PubMed1992

Distinct phosphotyrosines on a growth factor receptor bind to specific molecules that mediate different signaling pathways.

The receptor for platelet-derived growth factor (PDGF) binds two proteins containing SH2 domains, GTPase activating protein (GAP) and phosphatidylinositol 3-kinase (PI3-kinase). The sites on the receptor that mediate this interaction were identified by using phosphotyrosine-containing peptides representing receptor sequences to block specifically binding of either PI3-kinase or GAP. These results suggested that PI3-kinase binds two phosphotyrosine residues, each located in a 5 aa motif with an essential methionine at the fourth position C-terminal to the tyrosine. Point mutations at these sites caused a selective elimination of PI3-kinase binding and loss of PDGF-stimulated DNA synthesis. Mutation of the binding site for GAP prevented the receptor from associating with or phosphorylating GAP, but had no effect on PI3-kinase binding and little effect on DNA synthesis. Therefore, GAP and PI3-kinase interact with the receptor by binding to different phosphotyrosine-containing sequence motifs.

3T3 Cells↗
PubMed1992

The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor.

The fms-like tyrosine kinase (Flt) is a transmembrane receptor in the tyrosine kinase family. Expression of flt complementary DNA in COS cells conferred specific, high-affinity binding of vascular endothelial growth factor, also known as vascular permeability factor (VEGF-VPF), a factor that induces vascular permeability when injected in the guinea pig skin and stimulates endothelial cell proliferation. Expression of Flt in Xenopus laevis oocytes caused the oocytes to release calcium in response to VEGF-VPF. These findings show that flt encodes a receptor for VEGF-VPF.

Animals↗
PubMed1992

Triggering signaling cascades by receptor tyrosine kinases.

Growth factor receptors that are tyrosine kinases (RTKs) regulate growth and differentiation of cells in many organisms, including flies, worms, frogs, mice and humans. There has been recent progress in understanding the mechanism by which these receptors transduce signals. Worm and insect studies on RTKs have relied primarily on genetics, while the mammalian studies have employed a combination of molecular genetics and biochemistry. While many RTKs seem to have unique features, there are also many general signal transduction principles that emerge from these studies. In this review, we will focus on common signaling molecules, using RTKs from both vertebrates and invertebrates as examples.

Amino Acid Sequence↗
PubMed1992

The fibroblast growth factor receptor is not required for herpes simplex virus type 1 infection.

The early events mediating herpes simplex virus type 1 (HSV-1) infection include virion attachment to cell surface heparan sulfates and subsequent penetration. Recent evidence has suggested that the high-affinity fibroblast growth factor (FGF) receptor mediates HSV-1 entry. This report presents three lines of experimental evidence showing that the high-affinity FGF receptor is not required for HSV-1 infection. First, rat L6 myoblasts lacking FGF receptors were as susceptible to HSV-1 infection as L6 cells genetically engineered to express the FGF receptor. Second, a soluble FGF receptor fragment that inhibited FGF binding and receptor activation did not inhibit HSV-1 infection. Finally, basic FGF (but not acidic FGF) inhibited HSV-1 infection in L6 cells lacking FGF receptors, presumably by blocking cell surface heparan sulfates also required for HSV-1 infection. These results show that the high-affinity FGF receptor is not required for HSV-1 infection but instead that specific low-affinity basic FGF binding sites are used for HSV-1 infection.

Animals↗