VEGF receptor signaling in vertebrate development

The secreted glycoprotein vascular endothelial growth factor A (VEGF or VEGFA) affects many different cell types and modifies a wide spectrum of cellular behaviors in tissue culture models, including proliferation, migration, differentiation and survival. The versatility of VEGF signaling is reflected in the complex composition of its cell surface receptors and their ability to activate a variety of different downstream signaling molecules. A major challenge for VEGF research is to determine which of the specific signaling pathways identified in vitro control development and homeostasis of tissues containing VEGF-responsive cell types in vivo.     

P-Rex1, a PtdIns(3,4,5)P3- and Gbetagamma-regulated guanine-nucleotide exchange factor for Rac

Rac, a member of the Rho family of monomeric GTPases, is an integrator of intracellular signaling in a wide range of cellular processes. We have purified a PtdIns(3,4,5)P3-sensitive activator of Rac from neutrophil cytosol. It is an abundant, 185 kDa guanine-nucleotide exchange factor (GEF), which we cloned and named P-Rex1. The recombinant enzyme has Rac-GEF activity that is directly, substantially, and synergistically activated by PtdIns(3,4,5)P3 and Gbetagammas both in vitro and in vivo. P-Rex1 antisense oligonucleotides reduced endogenous P-Rex1 expression and C5a-stimulated reactive oxygen species formation in a neutrophil-like cell line. P-Rex1 appears to be a coincidence detector in PtdIns(3,4,5)P3 and Gbetagamma signaling pathways that is particularly adapted to function downstream of heterotrimeric G proteins in neutrophils.     

A signal transduction pathway model prototype I: From agonist to cellular endpoint

The postgenomic era is providing a wealth of information about the genes involved in many cellular processes. However, the ability to apply this information to understanding cellular signal transduction is limited by the lack of tools that quantitatively describe cellular signaling processes. The objective of the current studies is to provide a framework for modeling cellular signaling processes beginning at a plasma membrane receptor and ending with a measurable endpoint in the signaling process. Agonist-induced Ca(2+) mobilization coupled to down stream phosphorylation events was modeled using knowledge of in vitro and in vivo process parameters. The simulation process includes several modules that describe cellular processes involving receptor activation phosphoinositide metabolism, Ca(2+)-release, and activation of a calmodulin-dependent protein kinase. A Virtual Cell-based simulation was formulated using available literature data and compared to new and existing experimental results. The model provides a new approach to facilitate hypothesis-driven investigation and experimental design based upon simulation results. These investigations may be directed at the timing of multiple phosphorylation/dephosphorylation events affecting key enzymatic activities in the signaling pathway.     

Activated mutants of SHP-2 preferentially induce elongation of Xenopus animal caps

In Xenopus ectodermal explants (animal caps), fibroblast growth factor (FGF) evokes two major events: induction of ventrolateral mesodermal tissues and elongation. The Xenopus FGF receptor (XFGFR) and certain downstream components of the XFGFR signal transduction pathway (e.g., members of the Ras/Raf/MEK/mitogen-activated protein kinase [MAPK] cascade) are required for both of these processes. Likewise, activated versions of these signaling components induce mesoderm and promote animal cap elongation. Previously, using a dominant negative mutant approach, we showed that the protein-tyrosine phosphatase SHP-2 is necessary for FGF-induced MAPK activation, mesoderm induction, and elongation of animal caps. Taking advantage of recent structural information, we now have generated novel, activated mutants of SHP-2. Here, we show that expression of these mutants induces animal cap elongation to an extent comparable to that evoked by FGF. Surprisingly, however, activated mutant-induced elongation can occur without mesodermal cytodifferentiation and is accompanied by minimal activation of the MAPK pathway and mesodermal marker expression. Our results implicate SHP-2 in a pathway(s) directing cell movements in vivo and identify potential downstream components of this pathway. Our activated mutants also may be useful for determining the specific functions of SHP-2 in other signaling systems.     

Nanostructures of designed geometry and functionality enable regulation of cellular signaling processes

Extracellular matrices (ECM) triggered cellular signaling processes often begin with the clustering of the cellular receptors such as integrin and FcεRI. The sizes of these initial protein complexes or clusters are tens to 100 nm in dimension; therefore, engineered nanostructures could provide effective mimics of ECM for investigation and control of the initial and downstream specific signaling processes. This current topic discusses recent advances in nanotechnology in the context of design and production of matching chemical functionality and geometry for control of specific cellular signaling processes. Two investigations are reported to demonstrate this concept: (a) how the presentation of antigen at the nanometer scale would influence the aggregation of FcεRI, which would impact the formation of activation complexes, leading to the rearrangement of actin in cytoskeleton and degranulation or activation of mast cells; (b) how the engineered nanostructure could guide the initial integrin clustering, which would impact the formation of focal adhesion and downstream cell signaling cascades, leading to polarization, migration, and morphological changes. Complementary to engineered ECMs using synthetic ligands or peptides, or topographic control at the micrometer scale, nanostructures of designed geometry and chemical functionality provide new and effective biochemical cues for regulation of cellular signaling processes and downstream behaviors.     

Cell biology of human salivary secretion

We treated surgical specimens of human parotid and submandibular glands in vitro to manipulate the receptor-signaling cascade pharmacologically and analyzed cellular responses by light microscopy on epoxy embedded sections. Treatment of specimens with the b-agonist, isoproterenol, and with the second messenger analog, dibutyryl cyclic AMP, stimulated serous acinar cells to engage in exocytosis and degranulation. The muscarinic agonist, carbachol, and the calcium ionophore, A23187, on the other hand, elicited formation of "vacuoles" in the cytoplasm of serous acinar cells. Taking previous in vivo human and animal studies into account, these changes are suggested as the morphological expression of enzyme release and fluid secretion, respectively. Specimens obtained from patients over 70 years old exhibited poor response even though their morphological appearance remained intact. Aged salivary glands are thus suggested to experience a decline in their secretory activity at the cellular level, probably by impairment of the signaling processes downstream to the receptor activation and second messenger production.     

Graded mitogen-activated protein kinase activity precedes switch-like c-Fos induction in mammalian cells

The mitogen-activated protein kinase (MAPK) pathway is an evolutionarily conserved signaling module that controls important cell fate decisions in a variety of physiological contexts. During Xenopus oocyte maturation, the MAPK cascade converts an increasing progesterone stimulus into a switch-like, all-or-nothing response. While the importance of such switch-like behavior is widely discussed in the literature, it is not known whether the MAPK pathway in mammalian cells exhibits a switch-like or graded response. For this study, we used flow cytometry and immunofluorescence to generate single-cell measurements of MAPK signaling in Swiss 3T3 fibroblasts. In contrast to the case in Xenopus oocytes, we found that ERK activation in individual mammalian cells is not ultrasensitive and shows a graded response to changes in agonist concentration. Thus, the conserved MAPK signaling module exhibits different systems-level properties in different cellular contexts. Furthermore, the graded ERK response was converted into a more switch-like behavior at the level of immediate-early gene induction and cell cycle progression. Thus, while MAPK signaling is involved in all-or-nothing cell fate decisions for both Xenopus oocyte maturation and mammalian fibroblast proliferation, the underlying mechanisms responsible for the switch-like nature of the cellular responses are different in these two systems, with the mechanism appearing to lie downstream of the kinase cascade in mammalian fibroblasts.     

Tob proteins enhance inhibitory Smad-receptor interactions to repress BMP signaling

Tob inhibits bone morphogenetic protein (BMP) signaling by interacting with receptor-regulated Smads in osteoblasts. Here we provide evidence that Tob also interacts with the inhibitory Smads 6 and 7. A yeast two-hybrid screen identified Smad6 as a protein interacting with Tob. Tob co-localizes with Smad6 at the plasma membrane and enhances the interaction between Smad6 and activated BMP type I receptors. Furthermore, we have isolated Xenopus Tob2, and show that it cooperates with Smad6 in inducing secondary axes when expressed in early Xenopus embryos. Finally, Tob and Tob2 cooperate with Smad6 to inhibit endogenous BMP signaling in Xenopus embryonic explants and in cultured mammalian cells. Our results provide both in vitro and in vivo evidence that Tob inhibits endogenous BMP signaling by facilitating inhibitory Smad functions.     

Activated Src tyrosine kinase phosphorylates Tyr-457 of bovine GTPase-activating protein (GAP) in vitro and the corresponding residue of rat GAP in vivo.

GTPase-activating protein (GAP) is a key regulator of the cellular Ras protein, which is implicated in oncogenic signal transduction pathways downstream of the viral Src (v-Src) kinase. Previous studies demonstrated that v-Src induces tyrosine phosphorylation of GAP, suggesting that GAP may provide a biochemical link between v-Src and Ras signaling pathways. To determine the precise residues in GAP phosphorylated by Src kinases, we used a baculovirus/insect cell expression system for investigating in vitro phosphorylation of GAP. Phosphopeptide mapping analysis revealed that v-Src and normal cellular Src (c-Src) phosphorylate tyrosine residues in bovine GAP at one major site and one minor site in vitro. Significantly, the major site of GAP phosphorylation in vitro is also the major site of in vivo tyrosine phosphorylation of GAP in rat fibroblasts transformed by v-Src. Analyses of GAP deletion mutants and TrpE-GAP fusion proteins established that Tyr-457 of bovine GAP (and the corresponding residue of rat and human GAP) is the major site of tyrosine phosphorylation. Our results demonstrate that the v-Src kinase induces phosphorylation of the same tyrosine residue of GAP in vitro and in vivo, suggesting that GAP is a direct substrate of activated Src kinases in vivo. Because epidermal growth factor receptor phosphorylates the equivalent tyrosine residue in human GAP (Tyr-460), these findings are consistent with the hypothesis that specific phosphorylation of GAP at this site may have a physiologically important role in regulating mitogenic Ras signaling pathways.     

Characterization of a novel Xenopus tropicalis cell line as a model for in vitro studies

Cell lines are useful tools to facilitate in vitro studies of many biological and molecular processes. We describe a new permanent fibroblast-type cell line obtained from disaggregated Xenopus tropicalis limb bud. The cell line population doubling time was ~24 h. Its karyotype was genetically stable with a chromosome number of 2n = 21 and a chromosome 10 trisomy. These cells could be readily transfected and expressed transgenes faithfully. We obtained stable transformants using transposon-based gene transfer technology. These cells responded to thyroid hormone and thus can provide a complementary research tool to study thyroid hormone signaling events. In conclusion, this cell line baptized "Speedy" should prove useful to couple in vitro and in vivo biological studies in the X. tropicalis frog model.     

BMP signaling is required for heart formation in vertebrates

In these studies, we have taken advantage of a transient transgenic strategy in Xenopus embryos to demonstrate that BMP signaling is required in vivo for heart formation in vertebrates. Ectopic expression of dominant negative Type I (tALK3) or Type II (tBMPRII) BMP receptors in developing Xenopus embryos results in reduction or absence of heart formation. Additionally, blocking BMP signaling in this manner downregulates expression of XNkx2-5, a homeobox gene required for cardiac specification, prior to differentiation. Notably, however, initial expression of XNkx2-5 is not affected. Mutant phenotypes can be rescued by co-injection of mutant with wild-type receptors or co-injection of mutant receptors with XSmad1, a downstream mediator of BMP signaling. Whole-mount in situ analyses indicate that ALK3 and XSmad1 are coexpressed in cardiogenic regions. Together, our results demonstrate that BMP signaling is required for maintenance of XNkx2-5 expression and heart formation and suggest that ALK3, BMPRII, and XSmad1 may mediate this signaling.     

Characterization of a Rac1- and RhoGDI-Associated Lipid Kinase Signaling Complex

Rho family GTPases regulate a number of cellular processes, including actin cytoskeletal organization, cellular proliferation, and NADPH oxidase activation. The mechanisms by which these G proteins mediate their effects are unclear, although a number of downstream targets have been identified. The interaction of most of these target proteins with Rho GTPases is GTP dependent and requires the effector domain. The activation of the NADPH oxidase also depends on the C terminus of Rac, but no effector molecules that bind to this region have yet been identified. We previously showed that Rac interacts with a type I phosphatidylinositol-4-phosphate (PtdInsP) 5-kinase, independent of GTP. Here we report the identification of a diacylglycerol kinase (DGK) which also associates with both GTP- and GDP-bound Rac1. In vitro binding analysis using chimeric proteins, peptides, and a truncation mutant demonstrated that the C terminus of Rac is necessary and sufficient for binding to both lipid kinases. The Rac-associated PtdInsP 5-kinase and DGK copurify by liquid chromatography, suggesting that they bind as a complex to Rac. RhoGDI also associates with this lipid kinase complex both in vivo and in vitro, primarily via its interaction with Rac. The interaction between Rac and the lipid kinases was enhanced by specific phospholipids, indicating a possible mechanism of regulation in vivo. Given that the products of the PtdInsP 5-kinase and the DGK have been implicated in several Rac-regulated processes, and they bind to the Rac C terminus, these lipid kinases may play important roles in Rac activation of the NADPH oxidase, actin polymerization, and other signaling pathways.     

Ectopic EphA4 receptor induces posterior protrusions via FGF signaling in Xenopus embryos

The Eph family of receptor tyrosine kinases regulates numerous biological processes. To examine the biochemical and developmental contributions of specific structural motifs within Eph receptors, wild-type or mutant forms of the EphA4 receptor were ectopically expressed in developing Xenopus embryos. Wild-type EphA4 and a mutant lacking both the SAM domain and PDZ binding motif were constitutively tyrosine phosphorylated in vivo and catalytically active in vitro. EphA4 induced loss of cell adhesion, ventro-lateral protrusions, and severely expanded posterior structures in Xenopus embryos. Moreover, mutation of a conserved SAM domain tyrosine to phenylalanine (Y928F) enhanced the ability of EphA4 to induce these phenotypes, suggesting that the SAM domain may negatively regulate some aspects of EphA4 activity in Xenopus. Analysis of double mutants revealed that the Y928F EphA4 phenotypes were dependent on kinase activity; juxtamembrane sites of tyrosine phosphorylation and SH2 domain-binding were required for cell dissociation, but not for posterior protrusions. The induction of protrusions and expansion of posterior structures is similar to phenotypic effects observed in Xenopus embryos expressing activated FGFR1. Furthermore, the budding ectopic protrusions induced by EphA4 express FGF-8, FGFR1, and FGFR4a. In addition, antisense morpholino oligonucleotide-mediated loss of FGF-8 expression in vivo substantially reduced the phenotypic effects in EphA4Y928F expressing embryos, suggesting a connection between Eph and FGF signaling.     

Signaling initiated by overexpression of the fibroblast growth factor receptor-1 investigated by mass spectrometry

Overexpression of the fibroblast growth factor receptor-1 (FGFR-1), a prototypic receptor tyrosine kinase, is a feature of several human tumors. In human 293 cells overexpression of the FGFR-1 leads to constitutive activation of the receptor with concomitant sustained high increase in the cellular level of phosphotyrosine-containing proteins. Here we use mass spectrometry to study the tyrosine-phosphorylated proteins induced by overexpression of the FGFR-1. Several well known components of FGFR-1 signaling were identified along with two novel candidates: NS-1-associated protein-1 and target of Myb 1-like protein. We subsequently applied mass spectrometry precursor ion scanning to identify 22 tyrosine phosphorylation sites distributed on six substrate proteins of the FGFR-1 or downstream tyrosine kinases. Novel in vivo tyrosine phosphorylation sites were found in the FGFR-1, phospholipase Cgamma, p90 ribosomal S6 kinase, cortactin, and NS-1-associated protein-1 as a result of sustained FGFR-1 signaling, and we propose these as functional links to downstream molecular and cellular processes.     

VEGF receptor signaling in vertebrate development

The secreted glycoprotein vascular endothelial growth factor A (VEGF or VEGFA) affects many different cell types and modifies a wide spectrum of cellular behaviors in tissue culture models, including proliferation, migration, differentiation and survival. The versatility of VEGF signaling is reflected in the complex composition of its cell surface receptors and their ability to activate a variety of different downstream signaling molecules. A major challenge for VEGF research is to determine which of the specific signaling pathways identified in vitro control development and homeostasis of tissues containing VEGF-responsive cell types in vivo.

Note: Previously published in VEGF in Development, edited by Christiana Ruhrberg. Landes Bioscience and Springer Science+Business Media 2008; pp. 14-29.     

Reconstitution of Src-dependent phospholipase Cgamma phosphorylation and transient calcium release by using membrane rafts and cell-free extracts from Xenopus eggs

We reported previously that egg membrane rafts serve as a subcellular microdomain for sperm-dependent tyrosine kinase signaling in Xenopus fertilization. Moreover, we demonstrated that raft-associated Src tyrosine kinase was activated by sperm in vitro. Here we show that egg rafts incubated with sperm or hydrogen peroxide (H2O2) can promote Src-dependent phosphorylation of phospholipase Cgamma (PLCgamma) and transient calcium release in the extracts of unfertilized Xenopus eggs. In vivo egg activation by sperm or H2O2 also promotes tyrosine phosphorylation and raft-translocalization of PLCgamma. Immunodepletion of PLCgamma from the egg extracts inhibits the raft-dependent calcium release. Rafts prepared from H2O2-activated eggs also promote Src-dependent dephosphorylation of p42 mitogen-activated protein kinase and cell cycle transition from metaphase II to interphase in egg extracts. PLCgamma phosphorylation and calcium release in egg extracts can be promoted by rafts prepared from COS-7 cells expressing the Xenopus Src gene. These results demonstrate that the signaling events elicited by fertilization in Xenopus eggs can be reconstituted in vitro. The development of such experimental platforms will allow us to dissect the molecular mechanism of sperm-dependent activation of raft-associated Src and subsequent up-regulation of PLCgamma and egg activation machinery in Xenopus eggs.