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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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Sprys are tightly associated with caveolin-1 and membranes. (A) mSpry-1 associates with caveolin-1. HUVECs infected with AdmSpry-1 or AdeGFP were lysed in digitonin (D) or Triton X-100 (T) buffers, as described in Materials and Methods and indicated below the lanes. Aliquots of the cell lysates were subjected to immunoprecipitations with antibodies specific for mSpry-1 (top) or unrelated rabbit immunoglobulins, as negative control (bottom), in the respective buffers. Precipitated proteins and nonprecipitated proteins (Unbound) were resolved by SDS-PAGE, and caveolin-1, mSpry-1, and annexin II were detected by immunoblotting as indicated. (B) mSpry-1 is tightly associated with membranes. HUVECs infected with AdmSpry-1 or AdeGFP were subjected to a subcellular fractionation protocol, as described in Materials and Methods. Lanes 1 and 2 (Cyto) show cytoplasmatic extract obtained by permeabilizing the cells with digitonin (0.003%). Lanes 3 and 4 (Peri) show peripheral membrane proteins washed from partially purified membranes with sodium carbonate (Na2HCO3). Lanes 5 and 6 (Int) show integral membrane proteins that were not removed from the membrane fraction by sodium carbonate treatment. Protein levels of mSpry-1, LDH, transferrin receptor (Tf-R), caveolin-1, and annexin II in the various fractions were determined by immunoblotting as indicated. (C) The majority of mSpry-1 and caveolin-1 are not in detergent-resistant lipid rafts. HUVECs infected with AdmSpry-1 or AdeGFP were lysed in a Triton X-100 buffer and sedimented on Optiprep gradients as described in Materials and Methods. mSpry-1, caveolin-1, and annexin II in the gradient fractions were visualized by immunoblotting as indicated. The Optiprep concentrations of the fractions are given below the lanes. *Indicates that half of the fractions has been loaded in these lanes.
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Figure 8: Sprys are tightly associated with caveolin-1 and membranes. (A) mSpry-1 associates with caveolin-1. HUVECs infected with AdmSpry-1 or AdeGFP were lysed in digitonin (D) or Triton X-100 (T) buffers, as described in Materials and Methods and indicated below the lanes. Aliquots of the cell lysates were subjected to immunoprecipitations with antibodies specific for mSpry-1 (top) or unrelated rabbit immunoglobulins, as negative control (bottom), in the respective buffers. Precipitated proteins and nonprecipitated proteins (Unbound) were resolved by SDS-PAGE, and caveolin-1, mSpry-1, and annexin II were detected by immunoblotting as indicated. (B) mSpry-1 is tightly associated with membranes. HUVECs infected with AdmSpry-1 or AdeGFP were subjected to a subcellular fractionation protocol, as described in Materials and Methods. Lanes 1 and 2 (Cyto) show cytoplasmatic extract obtained by permeabilizing the cells with digitonin (0.003%). Lanes 3 and 4 (Peri) show peripheral membrane proteins washed from partially purified membranes with sodium carbonate (Na2HCO3). Lanes 5 and 6 (Int) show integral membrane proteins that were not removed from the membrane fraction by sodium carbonate treatment. Protein levels of mSpry-1, LDH, transferrin receptor (Tf-R), caveolin-1, and annexin II in the various fractions were determined by immunoblotting as indicated. (C) The majority of mSpry-1 and caveolin-1 are not in detergent-resistant lipid rafts. HUVECs infected with AdmSpry-1 or AdeGFP were lysed in a Triton X-100 buffer and sedimented on Optiprep gradients as described in Materials and Methods. mSpry-1, caveolin-1, and annexin II in the gradient fractions were visualized by immunoblotting as indicated. The Optiprep concentrations of the fractions are given below the lanes. *Indicates that half of the fractions has been loaded in these lanes.

Mentions: Next, we performed coimmunoprecipitation experiments to assess whether Sprys could directly associate with caveolin-1. HUVECs infected with AdmSpry-1 or AdeGFP were lysed in buffers that differed in their strength of membrane disaggregation: 1% Triton X-100 for solubilization of conventional membranes, and 1% digitonin for the quantitative depletion of cholesterol from cholesterol-rich membrane domains (rafts). Under these different buffer conditons, mSpry-1 was immunoprecipitated with antibodies specific for Spry-1, and precipitated mSpry-1 and coprecipitated caveolin-1, respectively, were analyzed by immunoblotting (Fig. 8 A). These experiments revealed that overexpressed mSpry-1 was tightly associated with caveolin-1, even under conditions known to disrupt lipid rafts (Fig. 8 A). A similar caveolin-1 association was also observed for overexpressed mSpry-2 (data not shown). Control immunoprecipitations using unrelated rabbit immunoglobulins did not precipitate caveolin-1 (Fig. 8 A, right). Moreover, annexin II, a peripheral membrane protein, did not coprecipitate with mSpry-1, confirming a specific association between overexpressed mSpry-1 and caveolin-1. In AdeGFP-infected HUVECs, precipitation of endogenous hSpry-1 did not recover significant amounts of hSpry-1 and caveolin-1, most likely due to the low levels of endogenous hSpry-1 in HUVECs (Fig. 8 A). Notably, immunoprecipitations of caveolin-1 did not recover detectable amounts of overexpressed mSpry-1, raising the possibility that only a subset of caveolin-1 is associated with mSpry-1 (data not shown).


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)

Sprys are tightly associated with caveolin-1 and membranes. (A) mSpry-1 associates with caveolin-1. HUVECs infected with AdmSpry-1 or AdeGFP were lysed in digitonin (D) or Triton X-100 (T) buffers, as described in Materials and Methods and indicated below the lanes. Aliquots of the cell lysates were subjected to immunoprecipitations with antibodies specific for mSpry-1 (top) or unrelated rabbit immunoglobulins, as negative control (bottom), in the respective buffers. Precipitated proteins and nonprecipitated proteins (Unbound) were resolved by SDS-PAGE, and caveolin-1, mSpry-1, and annexin II were detected by immunoblotting as indicated. (B) mSpry-1 is tightly associated with membranes. HUVECs infected with AdmSpry-1 or AdeGFP were subjected to a subcellular fractionation protocol, as described in Materials and Methods. Lanes 1 and 2 (Cyto) show cytoplasmatic extract obtained by permeabilizing the cells with digitonin (0.003%). Lanes 3 and 4 (Peri) show peripheral membrane proteins washed from partially purified membranes with sodium carbonate (Na2HCO3). Lanes 5 and 6 (Int) show integral membrane proteins that were not removed from the membrane fraction by sodium carbonate treatment. Protein levels of mSpry-1, LDH, transferrin receptor (Tf-R), caveolin-1, and annexin II in the various fractions were determined by immunoblotting as indicated. (C) The majority of mSpry-1 and caveolin-1 are not in detergent-resistant lipid rafts. HUVECs infected with AdmSpry-1 or AdeGFP were lysed in a Triton X-100 buffer and sedimented on Optiprep gradients as described in Materials and Methods. mSpry-1, caveolin-1, and annexin II in the gradient fractions were visualized by immunoblotting as indicated. The Optiprep concentrations of the fractions are given below the lanes. *Indicates that half of the fractions has been loaded in these lanes.
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Figure 8: Sprys are tightly associated with caveolin-1 and membranes. (A) mSpry-1 associates with caveolin-1. HUVECs infected with AdmSpry-1 or AdeGFP were lysed in digitonin (D) or Triton X-100 (T) buffers, as described in Materials and Methods and indicated below the lanes. Aliquots of the cell lysates were subjected to immunoprecipitations with antibodies specific for mSpry-1 (top) or unrelated rabbit immunoglobulins, as negative control (bottom), in the respective buffers. Precipitated proteins and nonprecipitated proteins (Unbound) were resolved by SDS-PAGE, and caveolin-1, mSpry-1, and annexin II were detected by immunoblotting as indicated. (B) mSpry-1 is tightly associated with membranes. HUVECs infected with AdmSpry-1 or AdeGFP were subjected to a subcellular fractionation protocol, as described in Materials and Methods. Lanes 1 and 2 (Cyto) show cytoplasmatic extract obtained by permeabilizing the cells with digitonin (0.003%). Lanes 3 and 4 (Peri) show peripheral membrane proteins washed from partially purified membranes with sodium carbonate (Na2HCO3). Lanes 5 and 6 (Int) show integral membrane proteins that were not removed from the membrane fraction by sodium carbonate treatment. Protein levels of mSpry-1, LDH, transferrin receptor (Tf-R), caveolin-1, and annexin II in the various fractions were determined by immunoblotting as indicated. (C) The majority of mSpry-1 and caveolin-1 are not in detergent-resistant lipid rafts. HUVECs infected with AdmSpry-1 or AdeGFP were lysed in a Triton X-100 buffer and sedimented on Optiprep gradients as described in Materials and Methods. mSpry-1, caveolin-1, and annexin II in the gradient fractions were visualized by immunoblotting as indicated. The Optiprep concentrations of the fractions are given below the lanes. *Indicates that half of the fractions has been loaded in these lanes.
Mentions: Next, we performed coimmunoprecipitation experiments to assess whether Sprys could directly associate with caveolin-1. HUVECs infected with AdmSpry-1 or AdeGFP were lysed in buffers that differed in their strength of membrane disaggregation: 1% Triton X-100 for solubilization of conventional membranes, and 1% digitonin for the quantitative depletion of cholesterol from cholesterol-rich membrane domains (rafts). Under these different buffer conditons, mSpry-1 was immunoprecipitated with antibodies specific for Spry-1, and precipitated mSpry-1 and coprecipitated caveolin-1, respectively, were analyzed by immunoblotting (Fig. 8 A). These experiments revealed that overexpressed mSpry-1 was tightly associated with caveolin-1, even under conditions known to disrupt lipid rafts (Fig. 8 A). A similar caveolin-1 association was also observed for overexpressed mSpry-2 (data not shown). Control immunoprecipitations using unrelated rabbit immunoglobulins did not precipitate caveolin-1 (Fig. 8 A, right). Moreover, annexin II, a peripheral membrane protein, did not coprecipitate with mSpry-1, confirming a specific association between overexpressed mSpry-1 and caveolin-1. In AdeGFP-infected HUVECs, precipitation of endogenous hSpry-1 did not recover significant amounts of hSpry-1 and caveolin-1, most likely due to the low levels of endogenous hSpry-1 in HUVECs (Fig. 8 A). Notably, immunoprecipitations of caveolin-1 did not recover detectable amounts of overexpressed mSpry-1, raising the possibility that only a subset of caveolin-1 is associated with mSpry-1 (data not shown).

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