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Mammalian sprouty-1 and -2 are membrane-anchored phosphoprotein inhibitors of growth factor signaling in endothelial cells.

Impagnatiello MA, Weitzer S, Gannon G, Compagni A, Cotten M, Christofori G - J. Cell Biol. (2001)

Bottom Line: Recently, Sprouty, an inhibitor of Drosophila development-associated RTK signaling, has been discovered.They are phosphorylated on serine residues and, upon growth factor stimulation, a subset is recruited to the leading edge of the plasma membrane.The data indicate that mammalian Spry-1 and -2 are membrane-anchored proteins that negatively regulate angiogenesis-associated RTK signaling, possibly in a RTK-specific fashion.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria.

ABSTRACT
Growth factor-induced signaling by receptor tyrosine kinases (RTKs) plays a central role in embryonic development and in pathogenesis and, hence, is tightly controlled by several regulatory proteins. Recently, Sprouty, an inhibitor of Drosophila development-associated RTK signaling, has been discovered. Subsequently, four mammalian Sprouty homologues (Spry-1-4) have been identified. Here, we report the functional characterization of two of them, Spry-1 and -2, in endothelial cells. Overexpressed Spry-1 and -2 inhibit fibroblast growth factor- and vascular endothelial growth factor-induced proliferation and differentiation by repressing pathways leading to p42/44 mitogen-activating protein (MAP) kinase activation. In contrast, although epidermal growth factor-induced proliferation of endothelial cells was also inhibited by Spry-1 and -2, activation of p42/44 MAP kinase was not affected. Biochemical and immunofluorescence analysis of endogenous and overexpressed Spry-1 and -2 reveal that both Spry-1 and -2 are anchored to membranes by palmitoylation and associate with caveolin-1 in perinuclear and vesicular structures. They are phosphorylated on serine residues and, upon growth factor stimulation, a subset is recruited to the leading edge of the plasma membrane. The data indicate that mammalian Spry-1 and -2 are membrane-anchored proteins that negatively regulate angiogenesis-associated RTK signaling, possibly in a RTK-specific fashion.

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Growth factor–induced recruitment to the plasma membrane. The subcellular localization of endogenous hSpry-1 was determined in HUVECs by immunofluorescence microscopy using an affinity-purified antibody that specifically recognized mSpry-1 and hSpry-1 (green) and Hoechst staining of nuclei (blue). In actively proliferating endothelial cells (exponential growth), Spry-1 is localized to the perinuclear region, vesicular structures, and lamellipodia in the leading edge of the plasma membrane. In serum-starved cells, Spry-1 is not found in the plasma membrane (serum-starved), but a subset of it is recruited to the plasma membrane 30 min after stimulation with 10 ng/ml FGF2 (serum-starved + FGF2). Preabsorption of the affinity-purified antibodies with corresponding antigenic peptide (1 μg/ml) resulted in a complete abrogation of the immunofluorescence signal (peptide competition). Bars, 10 μm.
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Figure 5: Growth factor–induced recruitment to the plasma membrane. The subcellular localization of endogenous hSpry-1 was determined in HUVECs by immunofluorescence microscopy using an affinity-purified antibody that specifically recognized mSpry-1 and hSpry-1 (green) and Hoechst staining of nuclei (blue). In actively proliferating endothelial cells (exponential growth), Spry-1 is localized to the perinuclear region, vesicular structures, and lamellipodia in the leading edge of the plasma membrane. In serum-starved cells, Spry-1 is not found in the plasma membrane (serum-starved), but a subset of it is recruited to the plasma membrane 30 min after stimulation with 10 ng/ml FGF2 (serum-starved + FGF2). Preabsorption of the affinity-purified antibodies with corresponding antigenic peptide (1 μg/ml) resulted in a complete abrogation of the immunofluorescence signal (peptide competition). Bars, 10 μm.

Mentions: To determine the subcellular localization of Spry-1 and -2 in mammalian endothelial cells, we performed immunofluorescence experiments using affinity-purified polyclonal antibodies that specifically recognized mSpry-1 and -2 hSpry-1 and -2. In actively proliferating HUVECs, endogenous (human) hSpry-1 was widely distributed throughout the cells; it was found predominantly in perinuclear regions, in vesicular structures, and in the plasma membrane at the leading edge of the cells (Fig. 5, exponential growth). Preabsorption of the affinity-purified antibodies with the corresponding Spry-specific antigenic peptide completely abrogated the immunofluorescence signal, indicating that the antibodies specifically visualized hSpry-1 (Fig. 5, peptide competition). Upon serum-starvation of HUVECs, hSpry-1 was found to be absent from the plasma membrane (Fig. 5, serum-starved). In contrast, upon stimulation with FGF2 (Fig. 5, serum-starved + FGF2) or VEGF (data not shown), a small subset of hSpry-1 was recruited within 30 min to the plasma membrane, mainly to the lamellipodia of the leading edge of the cells. Identical results were obtained for endogenous mSpry-1 in 1G11 cells (data not shown). Expression levels of Spry-2 in endothelial cells were too low to be detected by immunofluorescence; however, a subcellular localization similar to endogenous hSpry-1 was observed when either mSpry-1 or mSpry-2 were overexpressed in the cells by transient transfection or adenoviral gene transfer (data not shown). Thus, not only Spry gene expression but also the subcellular localization of Spry proteins is directly modulated by growth factors.


Mammalian sprouty-1 and -2 are membrane-anchored phosphoprotein inhibitors of growth factor signaling in endothelial cells.

Impagnatiello MA, Weitzer S, Gannon G, Compagni A, Cotten M, Christofori G - J. Cell Biol. (2001)

Growth factor–induced recruitment to the plasma membrane. The subcellular localization of endogenous hSpry-1 was determined in HUVECs by immunofluorescence microscopy using an affinity-purified antibody that specifically recognized mSpry-1 and hSpry-1 (green) and Hoechst staining of nuclei (blue). In actively proliferating endothelial cells (exponential growth), Spry-1 is localized to the perinuclear region, vesicular structures, and lamellipodia in the leading edge of the plasma membrane. In serum-starved cells, Spry-1 is not found in the plasma membrane (serum-starved), but a subset of it is recruited to the plasma membrane 30 min after stimulation with 10 ng/ml FGF2 (serum-starved + FGF2). Preabsorption of the affinity-purified antibodies with corresponding antigenic peptide (1 μg/ml) resulted in a complete abrogation of the immunofluorescence signal (peptide competition). Bars, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2198812&req=5

Figure 5: Growth factor–induced recruitment to the plasma membrane. The subcellular localization of endogenous hSpry-1 was determined in HUVECs by immunofluorescence microscopy using an affinity-purified antibody that specifically recognized mSpry-1 and hSpry-1 (green) and Hoechst staining of nuclei (blue). In actively proliferating endothelial cells (exponential growth), Spry-1 is localized to the perinuclear region, vesicular structures, and lamellipodia in the leading edge of the plasma membrane. In serum-starved cells, Spry-1 is not found in the plasma membrane (serum-starved), but a subset of it is recruited to the plasma membrane 30 min after stimulation with 10 ng/ml FGF2 (serum-starved + FGF2). Preabsorption of the affinity-purified antibodies with corresponding antigenic peptide (1 μg/ml) resulted in a complete abrogation of the immunofluorescence signal (peptide competition). Bars, 10 μm.
Mentions: To determine the subcellular localization of Spry-1 and -2 in mammalian endothelial cells, we performed immunofluorescence experiments using affinity-purified polyclonal antibodies that specifically recognized mSpry-1 and -2 hSpry-1 and -2. In actively proliferating HUVECs, endogenous (human) hSpry-1 was widely distributed throughout the cells; it was found predominantly in perinuclear regions, in vesicular structures, and in the plasma membrane at the leading edge of the cells (Fig. 5, exponential growth). Preabsorption of the affinity-purified antibodies with the corresponding Spry-specific antigenic peptide completely abrogated the immunofluorescence signal, indicating that the antibodies specifically visualized hSpry-1 (Fig. 5, peptide competition). Upon serum-starvation of HUVECs, hSpry-1 was found to be absent from the plasma membrane (Fig. 5, serum-starved). In contrast, upon stimulation with FGF2 (Fig. 5, serum-starved + FGF2) or VEGF (data not shown), a small subset of hSpry-1 was recruited within 30 min to the plasma membrane, mainly to the lamellipodia of the leading edge of the cells. Identical results were obtained for endogenous mSpry-1 in 1G11 cells (data not shown). Expression levels of Spry-2 in endothelial cells were too low to be detected by immunofluorescence; however, a subcellular localization similar to endogenous hSpry-1 was observed when either mSpry-1 or mSpry-2 were overexpressed in the cells by transient transfection or adenoviral gene transfer (data not shown). Thus, not only Spry gene expression but also the subcellular localization of Spry proteins is directly modulated by growth factors.

Bottom Line: Recently, Sprouty, an inhibitor of Drosophila development-associated RTK signaling, has been discovered.They are phosphorylated on serine residues and, upon growth factor stimulation, a subset is recruited to the leading edge of the plasma membrane.The data indicate that mammalian Spry-1 and -2 are membrane-anchored proteins that negatively regulate angiogenesis-associated RTK signaling, possibly in a RTK-specific fashion.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria.

ABSTRACT
Growth factor-induced signaling by receptor tyrosine kinases (RTKs) plays a central role in embryonic development and in pathogenesis and, hence, is tightly controlled by several regulatory proteins. Recently, Sprouty, an inhibitor of Drosophila development-associated RTK signaling, has been discovered. Subsequently, four mammalian Sprouty homologues (Spry-1-4) have been identified. Here, we report the functional characterization of two of them, Spry-1 and -2, in endothelial cells. Overexpressed Spry-1 and -2 inhibit fibroblast growth factor- and vascular endothelial growth factor-induced proliferation and differentiation by repressing pathways leading to p42/44 mitogen-activating protein (MAP) kinase activation. In contrast, although epidermal growth factor-induced proliferation of endothelial cells was also inhibited by Spry-1 and -2, activation of p42/44 MAP kinase was not affected. Biochemical and immunofluorescence analysis of endogenous and overexpressed Spry-1 and -2 reveal that both Spry-1 and -2 are anchored to membranes by palmitoylation and associate with caveolin-1 in perinuclear and vesicular structures. They are phosphorylated on serine residues and, upon growth factor stimulation, a subset is recruited to the leading edge of the plasma membrane. The data indicate that mammalian Spry-1 and -2 are membrane-anchored proteins that negatively regulate angiogenesis-associated RTK signaling, possibly in a RTK-specific fashion.

Show MeSH
Related in: MedlinePlus