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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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mSpry-1 and -2 are palmitoylated. HUVECs infected with AdmSpry-1 (lanes 1 and 3) or -2 (lanes 2 and 4) were labeled with [3H]palmitate (lanes 1 and 2) or [35S]methionine/cysteine (lanes 3 and 4) for 2 h. mSpry-1 and -2 were immunoprecipitated with affinity-purified antibodies, and radiolabeled mSpry-1 and -2 were resolved by SDS-PAGE and visualized by fluorography.
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Figure 10: mSpry-1 and -2 are palmitoylated. HUVECs infected with AdmSpry-1 (lanes 1 and 3) or -2 (lanes 2 and 4) were labeled with [3H]palmitate (lanes 1 and 2) or [35S]methionine/cysteine (lanes 3 and 4) for 2 h. mSpry-1 and -2 were immunoprecipitated with affinity-purified antibodies, and radiolabeled mSpry-1 and -2 were resolved by SDS-PAGE and visualized by fluorography.

Mentions: Next, we analyzed how Sprys associate with membranes. Extensive protein sequence analysis did not reveal any apparent signal peptide, transmembrane domain, or conserved motifs known to be modified by prenylation or myristylation (Veit and Schmidt 1998). In contrast, Sprys contain an unusually high number of internal cysteine residues, amino acids that, via a labile thioester bond, can be modified by palmitoylation (Veit and Schmidt 1998). To investigate the possibility that Sprys were palmitoylated, HUVECs were infected with AdmSpry-1 and -2, respectively, and metabolically labeled with either [3H]palmitate or [35S]methionine/cysteine. Subsequently, mSpry-1 and -2 were immunoprecipitated, and palmitoylation of the proteins was analyzed by SDS-PAGE under nonreducing conditions and fluorography (Fig. 10). Both mSpry-1 and -2 were specifically labeled by the incorporation of [3H]palmitate. Specific palmitoylation was further confirmed by soaking the SDS-PAGE gels in hydroxylamine, a treatment that is known to specifically hydrolyze thioester bonds, such as palmitoylation, but not amide bonds, such as myristylation (Linder et al. 1995). Such treatment specifically removed incorporated hydogen-3 from mSpry-1 and -2 (data not shown). These results demonstrate that both mSpry-1 and -2 are palmitoylated, most likely the molecular basis for their integral membrane localization.


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)

mSpry-1 and -2 are palmitoylated. HUVECs infected with AdmSpry-1 (lanes 1 and 3) or -2 (lanes 2 and 4) were labeled with [3H]palmitate (lanes 1 and 2) or [35S]methionine/cysteine (lanes 3 and 4) for 2 h. mSpry-1 and -2 were immunoprecipitated with affinity-purified antibodies, and radiolabeled mSpry-1 and -2 were resolved by SDS-PAGE and visualized by fluorography.
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Related In: Results  -  Collection

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Figure 10: mSpry-1 and -2 are palmitoylated. HUVECs infected with AdmSpry-1 (lanes 1 and 3) or -2 (lanes 2 and 4) were labeled with [3H]palmitate (lanes 1 and 2) or [35S]methionine/cysteine (lanes 3 and 4) for 2 h. mSpry-1 and -2 were immunoprecipitated with affinity-purified antibodies, and radiolabeled mSpry-1 and -2 were resolved by SDS-PAGE and visualized by fluorography.
Mentions: Next, we analyzed how Sprys associate with membranes. Extensive protein sequence analysis did not reveal any apparent signal peptide, transmembrane domain, or conserved motifs known to be modified by prenylation or myristylation (Veit and Schmidt 1998). In contrast, Sprys contain an unusually high number of internal cysteine residues, amino acids that, via a labile thioester bond, can be modified by palmitoylation (Veit and Schmidt 1998). To investigate the possibility that Sprys were palmitoylated, HUVECs were infected with AdmSpry-1 and -2, respectively, and metabolically labeled with either [3H]palmitate or [35S]methionine/cysteine. Subsequently, mSpry-1 and -2 were immunoprecipitated, and palmitoylation of the proteins was analyzed by SDS-PAGE under nonreducing conditions and fluorography (Fig. 10). Both mSpry-1 and -2 were specifically labeled by the incorporation of [3H]palmitate. Specific palmitoylation was further confirmed by soaking the SDS-PAGE gels in hydroxylamine, a treatment that is known to specifically hydrolyze thioester bonds, such as palmitoylation, but not amide bonds, such as myristylation (Linder et al. 1995). Such treatment specifically removed incorporated hydogen-3 from mSpry-1 and -2 (data not shown). These results demonstrate that both mSpry-1 and -2 are palmitoylated, most likely the molecular basis for their integral membrane localization.

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