Biomedical subjects

A J Muslin

Publications and source records attributed to A J Muslin.

28 records · Page 2Linked to original sources

PubMed1995

Regulation of Raf-1-dependent signaling during early Xenopus development.

The Raf-1 gene product is activated in response to cellular stimulation by a variety of growth factors and hormones. Raf-1 activity has been implicated in both cellular differentiation and proliferation. We have examined the regulation of the Raf-1/MEK/MAP kinase (MAPK) pathway during embryonic development in the frog Xenopus laevis. We report that Raf-1, MEK, and MAPK activities are turned off following fertilization and remain undetectable up until blastula stages (stage 8), some 4 h later. Tight regulation of the Raf-1/MEK/MAPK pathway following fertilization is crucial for embryonic cell cycle progression. Inappropriate reactivation of MAPK activity by microinjection of oncogenic Raf-1 RNA results in metaphase cell cycle arrest and, consequently, embryonic lethality. Our findings demonstrate an absolute requirement, in vivo, for inactivation of the MAPK signaling pathway to allow normal cell cycle progression during the period of synchronous cell divisions which occur following fertilization. Further, we show that cytostatic factor effects are mediated through MEK and MAPK.

Animals↗

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

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

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

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

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↗

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

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↗

PubMed1991

Well-defined growth factors promote cardiac development in axolotl mesodermal explants.

The effect of growth factors on the formation of cardiac mesoderm in the urodele, Ambystoma mexicanum (axolotl), has been examined using an in vitro explant system. It has previously been shown that cardiac mesoderm is induced by pharyngeal endoderm during neurula stages in urodeles. In this study, explants of prospective cardiac mesoderm from early neurula stage embryos rarely formed beating cardiac tissue in culture. When transforming growth factor beta-1 (TGF-beta 1) or platelet-derived growth factor BB (PDGF) was added to such explants, the frequency of heart tissue formation increased markedly. The addition of other growth factors to these explants did not enhance cardiac mesoderm formation. The addition of basic fibroblast growth factor (bFGF) to prospective heart mesoderm derived from later stage embryos resulted in a decreased tendency to form cardiac tissue. These results suggest that growth factors analogous to TGF-beta 1, PDGF, and bFGF may regulate the initial stages of vertebrate cardiac development in vivo.

Ambystoma↗

PubMed1989

Spontaneously hypertensive rat vascular smooth muscle cells in culture exhibit increased growth and Na+/H+ exchange.

The cellular mechanisms responsible for abnormalities in spontaneously hypertensive rat (SHR) vascular smooth muscle cell (VSMC) growth and vasoreactivity are not defined. Because Na+/H+ exchange, which we have previously demonstrated in cultured VSMC, plays an essential role in mediating growth factor responses, we hypothesized that abnormalities in SHR growth regulation might be reflected in the activity of this transporter. To test this hypothesis, we studied DNA synthesis and Na+/H+ exchange (measured as the rate of amiloride-sensitive intracellular alkalinization or Na+ influx) in early subcultures (less than 6) of aortic VSMC from 12-wk-old SHR and Wistar Kyoto (WKY) animals. Serum-deprived SHR VSMC grew more rapidly in response to 10% serum with an increase in [3H]thymidine incorporation of 439% compared with 191% in WKY controls. Basal intracellular pH (pHi) values determined by fluorescent pH measurements were 7.37 +/- 0.04 and 7.27 +/- 0.03 (P less than 0.05) in early passage SHR and WKY, respectively. Acid recovery (initial pHi = 6.8) by SHR VSMC was faster than by WKY VSMC as measured by alkalinization (1.8 +/- 0.6 vs. 0.8 +/- 0.2 mmol H+/liter.min, P less than 0.05) or by amiloride-sensitive 22Na+ influx (14.5 +/- 1.2 vs. 4.0 +/- 0.5 nmol Na+/mg protein.min, P less than 0.05). In comparison to WKY cells early passage SHR VSMC exhibited 2.5-fold greater alkalinization and amiloride-sensitive 22Na+ influx in response to 100 nM angiotensin II. During serial passage, WKY cells acquired enhanced Na+/H+ exchange and growth rates so that by passage 6, these differences were no longer present. These findings in early cultures of SHR VSMC, removed from the in vivo neurohumoral milieu, suggest that increased Na+/H+ exchange in SHR may reflect alterations in Na+ homeostasis that might contribute to altered SHR VSMC function such as enhanced growth and vasoreactivity.

Amiloride↗