Biomedical subjects

L T Williams

Publications and source records attributed to L T Williams.

At least 73 records · Page 4Linked to original sources

PubMed1994

An alternative to SH2 domains for binding tyrosine-phosphorylated proteins.

Src homology 2 (SH2) domains bind specifically to tyrosine-phosphorylated proteins that participate in signaling by growth factors and oncogenes. A protein domain was identified that bound specifically to the tyrosine-phosphorylated form of its target protein but differs from known SH2 sequences. Phosphotyrosine-binding (PTB) domains were found in two proteins: SHC, a protein implicated in signaling through Ras; and SCK, encoded by a previously uncharacterized gene. The PTB domain of SHC specifically bound to a tyrosine-phosphorylated 145-kilodalton protein. PTB domains are an alternative to SH2 domains for specifically recruiting tyrosine-phosphorylated proteins into signaling complexes and are likely to take part in signaling by many growth factors.

3T3 Cells↗

PubMed1994

Activation of Na+/H+ exchange by platelet-derived growth factor involves phosphatidylinositol 3'-kinase and phospholipase C gamma.

The effect of site-specific mutations in the mouse platelet-derived growth factor (PDGF) beta-receptor on activation of the Na+/H+ exchanger was examined in normal murine mammary gland epithelial (NMuMG) and Chinese hamster ovary (CHO) cells. These cells, which do not normally express PDGF receptors, were stably transfected with PDGF beta-receptor cDNA. Intracellular pH and Ca2+ were monitored using fluorescent probes. In both NMuMG and CHO cells expressing wild-type PDGF beta-receptors, PDGF B/B activated the amiloride-sensitive Na+/H+ exchanger. In both cell types, cell alkalinization was reduced by approximately 50% with a receptor mutant Y708F,Y719F which cannot bind phosphatidylinositol (PI) 3'-kinase. An inhibitor of PI 3'-kinase, LY294002, also inhibited alkalinization by 43% in cells with wild-type, but not Y708F,Y719F receptors. PDGF-induced intracellular Ca2+ release was not affected by this mutation. Both alkalinization and Ca2+ release were reduced by nearly 100% with the mutant Y977F,Y989F, which cannot bind phospholipase C gamma (PLC gamma). Y739F, a mutant that fails to bind the GTPase-activating protein did not affect PDGF-induced alkalinization. In protein kinase C (PKC) down-regulated NMuMG cells (wild-type receptor), PDGF no longer activated the Na+/H+ exchanger. In contrast, in PKC down-regulated CHO cells (wild-type receptor), PDGF-induced alkalinization was attenuated by only 37%. This residual activity was unaffected by the Y708F,Y719F mutation, but was completely eliminated by removal of medium Ca2+. These findings indicate that phospholipase C gamma is essential for activation of Na+/H+ exchange. PI 3'-kinase participates in PKC-dependent activation of Na+/H+ exchange by PDGF. In CHO cells, there is a second, Ca(2+)-dependent mechanism for activation of the exchanger.

Animals↗

PubMed1994

The function of KGF in morphogenesis of epithelium and reepithelialization of wounds.

The function of keratinocyte growth factor (KGF) in normal and wounded skin was assessed by expression of a dominant-negative KGF receptor transgene in basal keratinocytes. The skin of transgenic mice was characterized by epidermal atrophy, abnormalities in the hair follicles, and dermal hyperthickening. Upon skin injury, inhibition of KGF receptor signaling reduced the proliferation rate of epidermal keratinocytes at the wound edge, resulting in substantially delayed reepithelialization of the wound.

Aging↗

PubMed1994

Activation of Raf-1 by 14-3-3 proteins.

The protein Raf-1, a key mediator of mitogenesis and differentiation, associates with p21ras (refs 1-3). However, the regulation of the serine/threonine kinase activity of Raf-1 is still not understood. Using the yeast two-hybrid system, we identified two structurally related proteins that interact with the aminoterminal region of Raf-1. These proteins, 14-3-3 zeta (PLA2) and 14-3-3 beta (HS1), are members of the 14-3-3 family of proteins. Expression of 14-3-3 proteins in Xenopus oocytes enhanced Raf-1 activity and promoted Raf-1-dependent oocyte maturation. A dominant negative mutant of Raf-1 blocked the effects of 14-3-3 protein.

14-3-3 Proteins↗

PubMed1994

Tyrosine 508 of the 85-kilodalton subunit of phosphatidylinositol 3-kinase is phosphorylated by the platelet-derived growth factor receptor.

The mechanisms by which growth factors and oncogenic agents activate phosphatidylinositol 3-kinase (PI3 kinase) are unknown. Previously, we reported that the 85-kDa regulatory subunit of PI3 kinase is tyrosine-phosphorylated both in vitro by the platelet-derived growth factor beta-receptor (PDGFR) tyrosine kinase and in fibroblasts in response to PDGF. As a first step in determining the role of tyrosine phosphorylation in PDGF signaling through PI3 kinase, we investigated which tyrosines on p85 are phosphorylated by the PDGFR. Recombinant p85 was phosphorylated with recombinant PDGF receptors, and tryptic phosphopeptides were purified by HPLC and analyzed by Edman degradation. By this approach and by mutational analysis, Y508 was identified as the major in vitro phosphorylation site. Tryptic phosphopeptide mapping demonstrated Y508 to also be phosphorylated in vivo in COS cells. Comparison of these data with a previous report [Hayashi, H., Nishioka, Y., Kamohara, S., Kanai, F., Ishii, K., Fukui, Y., Shibasaki, F., Takenawa, T., Kido, H., Katsunuma, N., & Ebina, Y. (1993) J. Biol. Chem. 268, 7107-7117] suggests that p85 is phosphorylated differently by the PDGF and insulin receptor tyrosine kinases. Therefore, p85 may be regulated differently by PDGF and insulin. Mapping of phosphorylation sites on p85 may lead to new insights into the regulation of signal transduction through PI3 kinase.

Amino Acid Sequence↗

PubMed1994

The post-translational modification of ras p21 is important for Raf-1 activation.

Raf-1, a serine/threonine kinase, is required for the mitogenic action of ras p21. It has been recently demonstrated that ras p21 directly associates with Raf-1. The C-terminal region of ras p21 is modified by farnesylation and carboxyl methylation. This modification is necessary for ras p21 function. To elucidate the role of post-translational modification of ras p21 in Raf-1 activation, we examined ras p21-dependent Raf-1 activity in baculovirus/Sf9 cells overexpressing Raf-1 and ras p21. Coexpression of Raf-1 with v-ras p21 in Sf9 cells stimulated the autophosphorylating activity of Raf-1. The activity of Raf-1, as assessed by its ability to activate extracellular signal-regulated kinase kinase (MEK) in vitro, was also increased when Raf-1 was coexpressed with v-ras p21. However, neither the autophosphorylating activity of Raf-1 nor its ability to activate MEK was stimulated by v-ras p21 mutants which are not post-translationally modified. Raf-1 formed a complex with v-ras p21 and the v-ras p21 mutants in Sf9 cells. These results indicate that the post-translational modification of ras p21 is necessary for Raf-1 activation but that the association of Raf-1 with ras p21 is not sufficient to activate Raf-1.

Animals↗

PubMed1994

Regulation of chemotaxis by the platelet-derived growth factor receptor-beta.

Chemotaxis is an important component of wound healing, development, immunity and metastasis, yet the signalling pathways that mediate chemotaxis are poorly understood. Platelet-derived growth factor (PDGF) acts both as a mitogen and a chemoattractant. Upon stimulation, the tyrosine kinase PDGF receptor-beta (PDGFR-beta) autophosphorylates and forms a complex that includes SII2(Src homology 2)-domain-containing proteins such as the phosphatidylinositol-specific phospholipase C-gamma, Ras-GTPase-activating protein (GAP), and phosphatidylinositol-3-OH kinase. Specific tyrosine-to-phenylalanine substitutions in the PDGFR-beta can prevent binding of one SH2-domain-containing protein without affecting binding of other receptor-associated proteins. Here we use phospholipase C-gamma and PDGFR-beta mutants to map specific tyrosines involved in both positive and negative regulation of chemotaxis towards the PDGF-BB homodimer. Our results indicate that a delicate balance of migration-promoting (phospholipase C-gamma and phosphatidylinositol-3-OH kinase) and migration-suppressing (GAP) activities are recruited by the PDGFR-beta to drive chemotaxis towards PDGF-BB.

3T3 Cells↗

PubMed1994

Expression of the platelet-derived growth factor beta receptor during organogenesis and tissue differentiation in the mouse embryo.

In this study we used in situ hybridization to localize expression of the platelet-derived growth factor beta (PDGF beta) receptor mRNA during organogenesis in the mouse embryo (E 9.5-16.5). Expression was first seen in periaortic mesenchyme (E 9.5-10.5). Later (E 12.5-E 16.5), the receptor was expressed in the mesenchymal component of many developing tissues and organs, particularly derivatives of the primitive gut. The expression was exceptionally high in mesenchyme directly supporting an epithelium, typical of many developing organs such as the trachea and intestine. However, as the mesenchyme differentiated into smooth muscle, PDGF beta receptor mRNA was no longer detected. The expression of the PDGF beta receptor mRNA in mesenchymal components of developing organs, along with its absence in epithelial tissues, indicates that it may play a role in mesenchymal-epithelial interactions during organ development. Somewhat unexpectedly, the PDGF beta receptor was highly expressed in the endothelium of small blood vessels and vascular structures such as the hyaloid plexus and choroid plexus. In large blood vessels, PDGF beta receptor mRNA was found in the mesenchyme surrounding the endothelium. This suggests that the PDGF beta receptor is involved in growth and development of blood vessels.

Animals↗

PubMed1994

Evidence for both tyrosine kinase and G-protein-coupled pathways leading to starfish egg activation.

To investigate possible pathways leading to egg activation at fertilization, the ability of exogenously introduced tyrosine kinase and G-protein-coupled receptors to mimic events of fertilization was examined. Oocytes of the starfish Asterina miniata were injected with RNA for a chimeric receptor consisting of the extracellular domain of the beta form of the mouse platelet-derived growth factor (PDGF) receptor and the transmembrane/intracellular domain of the human fibroblast growth factor (FGF) receptor, or with RNA for the rat serotonin 1c receptor. These oocytes were cultured for 1 to 3 days and then matured with 1-methyladenine. In response to PDGF or serotonin, the injected eggs underwent responses like those at fertilization: cortical granule exocytosis, a rise in intracellular free calcium, and DNA synthesis. Some of these artificially activated eggs cleaved, and some of the PDGF-activated eggs were observed to form larvae. A PDGF/FGF receptor with a point mutation which eliminated its ability to interact with phospholipase C-gamma did not cause fertilization-like responses. Thus components of a signaling pathway involving phospholipase C-gamma, characteristic of tyrosine kinase receptors, as well as components of a pathway involving a G-protein and phospholipase C-beta, characteristic of G-protein-coupled receptors, appear to be present in starfish eggs. Either or both could function in egg activation at fertilization.

Animals↗

PubMed1994

ralGDS family members interact with the effector loop of ras p21.

Using a yeast two-hybrid system, we identified a novel protein which interacts with ras p21. This protein shares 69% amino acid homology with ral guanine nucleotide dissociation stimulator (ralGDS), a GDP/GTP exchange protein for ral p24. We designated this protein RGL, for ralGDS-like. Using the yeast two-hybrid system, we found that an effector loop mutant of ras p21 was defective in interacting with the ras p21-interacting domain of RGL, suggesting that this domain binds to ras p21 through the effector loop of ras p21. Since ralGDS contained a region highly homologous with the ras p21-interacting domain of RGL, we examined whether ralGDS could interact with ras p21. In the yeast two-hybrid system, ralGDS failed to interact with an effector loop mutant of ras p21. In insect cells, ralGDS made a complex with v-ras p21 but not with a dominant negative mutant of ras p21. ralGDS interacted with the GTP-bound form of ras p21 but not with the GDP-bound form in vitro. ralGDS inhibited both the GTPase-activating activity of the neurofibromatosis gene product (NF1) for ras p21 and the interaction of Raf with ras p21 in vitro. These results demonstrate that ralGDS specifically interacts with the active form of ras p21 and that ralGDS can compete with NF1 and Raf for binding to the effector loop of ras p21. Therefore, ralGDS family members may be effector proteins of ras p21 or may inhibit interactions between ras p21 and its effectors.

Amino Acid Sequence↗

PubMed1994

The interaction of small domains between the subunits of phosphatidylinositol 3-kinase determines enzyme activity.

Previous studies have suggested that the two subunits of phosphatidylinositol (PI) 3-kinase, p85 and p110, function as localizing and catalytic subunits, respectively. Using recombinant p85 and p110 molecules, we have reconstituted the specific interaction between the two subunits of mouse PI 3-kinase in cells and in vitro. We have previously shown that the region between the two Src homology 2 (SH2) domains of p85 is able to form a functional complex with the 110-kDa subunit in vivo. In this report, we identify the corresponding domain in p110 which directs the binding to p85. We demonstrate that the interactive domains in p85 and p110 are less than 103 and 124 amino acids, respectively, in size. We also show that the association of p85 and p110 mediated by these domains is critical for PI 3-kinase activity. Surprisingly, a complex between a 102-amino-acid segment of p85 and the full-length p110 molecule is catalytically active, whereas p110 alone has no activity. In addition to the catalytic domain in the carboxy-terminal region, 123 amino acids at the amino terminus of p110 were required for catalytic activity and were sufficient for the interaction with p85. These results indicate that the 85-kDa subunit, previously thought to have only a linking role in localizing the p110 catalytic subunit, is an important component of the catalytic complex.

3T3 Cells↗

PubMed1994

Direct activation of phospholipase C-gamma by fibroblast growth factor receptor is not required for mesoderm induction in Xenopus animal caps.

Members of the fibroblast growth factor (FGF) family induce mesoderm formation in explants of Xenopus embryonic ectoderm (animal caps). Recent studies have been directed at determining signaling pathways downstream of the FGF receptor that are important in mesoderm induction. We have recently shown that a point mutation in the FGF receptor changing tyrosine 766 to phenylalanine (Y/F mutation) abolishes phospholipase C-gamma (PLC-gamma) activation in mammalian cells. To explore the role of PLC-gamma activation in FGF-stimulated mesoderm induction, we constructed two chimeric receptors, each consisting of the extracellular portion of the platelet-derived growth factor beta receptor, with one having the transmembrane and intracellular portions of the wild-type FGF receptor 1 (PR-FR wt) and the other having the corresponding region of the Y/F766 mutant FGF receptor 1 (PR-FR Y/F766). When expressed in Xenopus oocytes, only PR-FR wt was able to mediate PLC gamma phosphorylation, inositol-1,4,5-trisphosphate accumulation, and calcium efflux in response to platelet-derived growth factor stimulation. However, both receptors mediated mesoderm induction in Xenopus animal caps as measured by cap elongation, muscle-specific actin mRNA induction, and skeletal muscle formation. These results demonstrate that PLC gamma activation by the FGF receptor is not required for FGF-stimulated mesoderm induction.

Animals↗

PubMed1994

Phospholipase C gamma activation, phosphotidylinositol hydrolysis, and calcium mobilization are not required for FGF receptor-mediated chemotaxis.

Basic fibroblast growth factor (FGF) is a potent angiogenic factor that stimulates several cell types to migrate along a chemotactic gradient. Most chemoattractant receptors appear to share a common mechanism that involves activation of phospholipase C (PLC), hydrolysis of phosphotidylinositol, and mobilization of intracellular calcium. We transfected two different cell lines with either human FGF receptor-1 cDNA or chimeric FGF receptor cDNA. Ligand stimulation induced chemotaxis, activation of PLC gamma, phosphotidylinositol hydrolysis, and calcium mobilization in both wild-type receptor cell lines. No such response was elicited in control cells. Mutation of the two fibroblast growth factor receptors at residue 766, replacing tyrosine with phenylalanine, made the receptors incapable of associating with and activating PLC gamma following ligand stimulation. These mutant receptors also failed to mediate phosphotidylinositol hydrolysis and calcium mobilization. However, cells transfected with the mutant fibroblast growth factor receptors were as chemotactically responsive to the appropriate ligand as were cells transfected with the wild-type receptors. These findings demonstrate that the ability of the fibroblast growth factor receptor to promote chemotaxis is not dependent on increased activation of PLC gamma, increased hydrolysis of phosphotidylinositol, or increased global mobilization of calcium.

Animals↗

PubMed1994

Signaling molecules that mediate the actions of FGF.

Signaling by fibroblast growth factor (FGF) is critical for cellular growth and differentiation. We have studied the role of FGF and its receptor in development and also the signaling pathways utilized by these molecules. Transgenic mice expressing a dominant negative FGF receptor specifically in lung tissue fail to develop lungs. These findings suggest that FGF signaling is important for airway branching and alveolar formation. In Xenopus embryos, FGF is involved in the development of posterior structures. Induction of mesoderm by FGF is mediated by the serine-threonine kinase RAF. The expression of a dominant negative RAF selectively blocks the mesoderm-inducing action of FGF. Expression of activated RAF accentuated the development of posterior structures Other intracellular molecules that are involved with FGF signaling include RAS, a low-molecular weight GTP-binding protein. We have identified a novel gene (RIP, for RAS-interacting protein) that contains a RAS-binding domain. This domain is found in at least one other protein, ralGDS. Both RIP and ralGDS bind to activated RAS suggesting that these proteins are likely candidates for mediators of RAS function. These findings help elucidate the role of FGF signaling in development.

Animals↗

PubMed1993

Dominant-negative mutations of platelet-derived growth factor (PDGF) receptors. Inhibition of receptor function by ligand-dependent formation of heterodimers between PDGF alpha- and beta-receptors.

We showed previously that a truncated form of the platelet-derived growth factor (PDGF) beta-receptor lacking its kinase region can form a nonfunctional heterodimer with the wild-type beta-receptor and thereby inhibit its signal transduction. In this paper we investigated whether the truncated form of either alpha- or beta-receptor could block the function of the other type of wild-type PDGF receptor. When the truncated alpha-receptor was expressed in Xenopus oocytes in excess over either the wild-type alpha- or beta-receptor, the Ca2+ mobilization signal elicited by either the wild-type alpha- or beta-receptor was completely blocked. The truncated beta-receptor abolished signaling by the wild-type alpha-receptor in response to PDGF-AB or -BB. However signal transduction by the alpha-receptor in response to PDGF-AA was not affected by the truncated beta-receptor. In the presence of PDGF-AB or -BB, both the wild-type and truncated beta-receptors formed a heterologous complex with the alpha-receptor in intact cells. A kinase-inactive beta-receptor (an ATP-binding site mutation) became cross-phosphorylated on tyrosine residues by the co-expressed wild-type alpha-receptor in response to PDGF-AB or -BB but not in response to PDGF-AA. These findings indicate that the alpha-and beta-receptors interact in response to PDGF-AB or -BB and are consistent with the hypothesis that the truncated alpha-receptor inhibits function of the wild-type beta-receptor through formation of a ligand-dependent nonfunctional alpha beta-heterodimer. A similar heterodimer can form between the truncated beta-receptor and the wild-type alpha-receptor. These observations provide useful information for future studies using dominant-negative mutations of PDGF receptors to selectively inhibit the actions of specific PDGFs in animals.

Animals↗

PubMed1993

Vascular endothelial growth factor receptor expression during embryogenesis and tissue repair suggests a role in endothelial differentiation and blood vessel growth.

Vascular endothelial growth factor (VEGF) is a polypeptide mitogen that stimulates the growth of endothelial cells in vitro and promotes the growth of blood vessels in vivo. We have recently shown that the fms-like receptor tyrosine kinase (flt) is a receptor for VEGF. Here we used in situ hybridization to show that, in adult mouse tissues, the pattern of flt expression was consistent with localization in endothelium. We also show that flt was expressed in endothelium during neovascularization of healing skin wounds and during early vascular development in mouse embryos. Moreover, flt was expressed in populations of embryonic cells from which endothelium is derived such as early yolk sac mesenchyme. The expression of flt in the endothelium of both developing and mature blood vessels suggests that VEGF might regulate endothelial differentiation, blood vessel growth, and vascular repair.

Animals↗

PubMed1993

Fetal liver kinase 1 is a receptor for vascular endothelial growth factor and is selectively expressed in vascular endothelium.

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, induces endothelial proliferation in vitro and vascular permeability in vivo. The human transmembrane c-fms-like tyrosine kinase Flt-1 has recently been identified as a VEGF receptor. Flt-1 kinase has seven immunoglobulin-like extracellular domains and a kinase insert sequence, features shared by two other human gene-encoded proteins, kinase insert domain-containing receptor (KDR) and FLT-4. In this study we show that the mouse homologue of KDR, Flk-1, is a second functional VEGF receptor. Flk-1 binds VEGF with high affinity, undergoes autophosphorylation, and mediates VEGF-dependent Ca2+ efflux in Xenopus oocytes injected with Flk-1 mRNA. We also demonstrate by in situ hybridization that Flk-1 protein expression in the mouse embryo is restricted to the vascular endothelium and the umbilical cord stroma. VEGF and its receptors Flk-1/KDR and Flt-1 may play a role in vascular development and regulation of vascular permeability.

3T3 Cells↗

PubMed1993

Raf-1 kinase is essential for early Xenopus development and mediates the induction of mesoderm by FGF.

Animal cap explants from Xenopus embryos injected with a dominant negative Raf-1 mutant, termed NAF (not a functional Raf), demonstrated a complete block to basic fibroblast growth factor (FGF)-stimulated mesoderm induction. Activin induction of mesoderm was normal in embryos that expressed NAF. Injection of NAF RNA into 2-cell stage embryos blocked normal development during neurula stages and caused severe posterior truncations in tadpoles. The phenotype induced by NAF could be rescued by coinjection of wild-type raf-1 RNA. The NAF mutant functioned by specifically blocking the activation of endogenous Raf kinase activity. These findings suggest that Raf-1 mediates FGF, but not activin, receptor signaling during mesoderm induction and implicate Raf-1 as a key signaling molecule in the development of posterior structure.

Activins↗