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1.
Mol Endocrinol ; 26(6): 1056-73, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22570334

ABSTRACT

GH and GH receptors are expressed throughout life, and GH elicits a diverse range of responses, including growth and altered metabolism. It is therefore important to understand the full spectrum of GH signaling pathways and cellular responses. We applied mass spectrometry-based phosphoproteomics combined with stable isotope labeling with amino acids in cell culture to identify proteins rapidly phosphorylated in response to GH in 3T3-F442A preadipocytes. We identified 132 phosphosites in 95 proteins that exhibited rapid (5 or 15 min) GH-dependent statistically significant increases in phosphorylation by more than or equal to 50% and 96 phosphosites in 46 proteins that were down-regulated by GH by more than or equal to 30%. Several of the GH-stimulated phosphorylation sites were known (e.g. regulatory Thr/Tyr in Erks 1 and 2, Tyr in signal transducers and activators of transcription (Stat) 5a and 5b, Ser939 in tuberous sclerosis protein (TSC) 2 or tuberin). The remaining 126 GH-stimulated sites were not previously associated with GH. Kyoto Encyclopedia of Genes and Genomes pathway analysis of GH-stimulated sites indicated enrichment in proteins associated with the insulin and mammalian target of rapamycin (mTOR) pathways, regulation of the actin cytoskeleton, and focal adhesions. Akt/protein kinase A consensus sites (RXRXXS/T) were the most commonly phosphorylated consensus sites. Immunoblotting confirmed GH-stimulated phosphorylation of all seven novel GH-dependent sites tested [regulatory sites in proline-rich Akt substrate, 40 kDA (PRAS40), regulatory associated protein of mTOR, ATP-citrate lyase, Na(+)/H(+) exchanger-1, N-myc downstream regulated gene 1, and Shc]). The immunoblot results suggest that many, if not most, of the GH-stimulated phosphosites identified in this large-scale quantitative phosphoproteomics analysis, including sites in multiple proteins in the Akt/ mTOR complex 1 pathway, are phosphorylated in response to GH. Their identification significantly broadens our thinking of GH-regulated cell functions.


Subject(s)
Growth Hormone/physiology , Phosphoproteins/metabolism , Protein Processing, Post-Translational , Proteome/metabolism , 3T3 Cells , Amino Acid Motifs , Animals , Chromatography, Ion Exchange , Consensus Sequence , Mice , Peptide Fragments/chemistry , Peptide Fragments/isolation & purification , Phosphatidylinositol 3-Kinases/metabolism , Phosphoproteins/chemistry , Phosphoproteins/isolation & purification , Proteome/chemistry , Proteome/isolation & purification , Proteomics , Proto-Oncogene Proteins c-akt/metabolism , Receptors, Somatotropin/metabolism , Signal Transduction , Tandem Mass Spectrometry
2.
Carcinogenesis ; 31(3): 435-41, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20042635

ABSTRACT

Endoglin, an endothelial cell-specific transforming growth factor-beta (TGF-beta) superfamily coreceptor, has an essential role in angiogenesis. Endoglin-null mice have an embryonic lethal phenotype due to defects in angiogenesis and mutations in endoglin result in the vascular disease hereditary hemorrhagic telangiectasia type I. Increased endoglin expression in the proliferating endothelium of tumors has been correlated with metastasis, tumor grade and decreased survival. Although endoglin is thought to regulate TGF-beta superfamily signaling in endothelial cells through regulating the balance between two TGF-beta-responsive pathways, the activin receptor-like kinase 5 (ALK5)/Smad2/3 pathway and the activin receptor-like kinase 1 (ALK1)/Smad1/5/8 pathway, the mechanism by which endoglin regulates angiogenesis has not been defined. Here, we investigate the role of the cytoplasmic domain of endoglin and its phosphorylation by ALK5 in regulating endoglin function in endothelial cells. We demonstrate that the cytoplasmic domain of endoglin is basally phosphorylated by ALK5, primarily on serines 646 and 649, in endothelial cells. Functionally, the loss of phosphorylation at serine 646 resulted in a loss of endoglin-mediated inhibition of Smad1/5/8 signaling in response to TGF-beta and endothelial cell migration, whereas loss of phosphorylation at both serines 646 and 649 resulted in a loss of endoglin-mediated inhibition of Smad1/5/8 signaling in response to bone morphogenetic protein-9. Taken together, these results support endoglin phosphorylation by ALK5 as an important mechanism for regulating TGF-beta superfamily signaling and migration in endothelial cells.


Subject(s)
Endothelial Cells/metabolism , Intracellular Signaling Peptides and Proteins/physiology , Protein Serine-Threonine Kinases/physiology , Receptors, Transforming Growth Factor beta/physiology , Smad1 Protein/physiology , Smad5 Protein/physiology , Smad8 Protein/physiology , Animals , COS Cells , Cell Movement , Chlorocebus aethiops , Endoglin , Growth Differentiation Factor 2/physiology , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/deficiency , Intracellular Signaling Peptides and Proteins/genetics , Mice , Mice, Knockout , Mink , Phosphorylation , Phosphoserine/metabolism , Protein Processing, Post-Translational , Protein Structure, Tertiary , Receptor, Transforming Growth Factor-beta Type I , Signal Transduction/drug effects , Transforming Growth Factor beta/physiology
3.
Trends Biochem Sci ; 30(11): 611-21, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16185874

ABSTRACT

Extracellular signals are transmitted to cells through two classes of cell-surface receptors: signaling receptors that directly transduce signals and signaling co-receptors that bind ligand but that, traditionally, have not been thought to signal directly. Signaling co-receptors modulate the ligand binding and signaling of their respective signaling receptors. In recent years, roles for co-receptors have expanded to include essential functions in morphogen gradient formation, localizing signaling, signaling independently, regulating cell adhesion and orchestrating the signaling of several pathways. The importance of signaling co-receptors is demonstrated by their ubiquitous expression, their conservation during evolution, their prominent role in signaling cascades, their indispensable role during development and their frequent mutation or altered expression in human disease.


Subject(s)
Genetic Diseases, Inborn/genetics , Receptors, Cell Surface/physiology , Signal Transduction , Animals , Cell Adhesion , Embryonic Development , Humans , Ligands , Mice , Models, Biological , Mutation , Receptors, Cell Surface/metabolism
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