Phosphoproteome Profiling of Transforming Growth Factor (TGF)-β Signaling: Abrogation of TGFβ1-dependent Phosphorylation of Transcription Factor-II-I (TFII-I) Enhances Cooperation of TFII-I and Smad3 in Transcription

Abstract

Transforming growth factor-␤ (TGF␤) signaling involves activation of a number of signaling pathways, several of which are controlled by phosphorylation events. Here, we describe a phosphoproteome profiling of MCF-7 human breast epithelial cells treated with TGF␤1. We identified 32 proteins that change their phosphorylation upon treatment with TGF␤1; 26 of these proteins are novel targets of TGF␤1. We show that Smad2 and Smad3 have different effects on the dynamics of TGF␤1-induced protein phosphorylation. The identified proteins belong to nine functional groups, e.g., proteins regulating RNA processing, cytoskeletal rearrangements, and proteasomal degradation. To evaluate the proteomics findings, we explored the functional importance of TGF␤1-dependent phosphorylation of one of the targets, i.e.,

Identification of the TFII-I family target genes in the vertebrate genome

“…TFII-I but not BEN recruits to the promoter of Hdac1. It is well known that the TGF␤ signaling pathway plays an important role in the activity of TFII-I proteins (17,18). We identified Tgfb2 as a potential downstream target of BEN in MEFs.…”

A Crystal Structure of the Cyclic GMP-dependent Protein Kinase Iβ Dimerization/Docking Domain Reveals Molecular Details of Isoform-specific Anchoring*

“…Phosphorylation of IRAG by PKG I␤ inhibits 1,4,5-inositol triphosphate receptordependent calcium release from the endoplasmic reticulum, contributing to smooth muscle relaxation (7). Phosphorylation of TFII-I by PKG I␤ modulates the co-activator functions of TFII-I for serum response factor and Smad transcription factors (6,8).The physiological importance of GKAP interactions with PKG dimers is illustrated by the cardiovascular abnormalities found in transgenic mice expressing a dimerization-deficient form of PKG I␣ (9). These mice show a complex cardiovascular phenotype, which includes impaired vasodilatation with systemic hypertension and cardiac hypertrophy despite high basal PKG activity.…”

“…Phosphorylation of IRAG by PKG I␤ inhibits 1,4,5-inositol triphosphate receptordependent calcium release from the endoplasmic reticulum, contributing to smooth muscle relaxation (7). Phosphorylation of TFII-I by PKG I␤ modulates the co-activator functions of TFII-I for serum response factor and Smad transcription factors (6,8).…”

Temporal and spatial profiling of nuclei-associated proteins upon TNF-α/NF-κB signaling

“…Although large-scale high-throughput experimental techniques have greatly increased our knowledge of signal transduction pathways [10][11][12][13][14][15], our understanding of the signaling process is nevertheless incomplete. Our study here represents the thus far largestscale, dynamic and systematic analysis of TNF-α/NF-κB signaling.…”

“…Using mass-spectrometry-based proteomics and SILAC method [8,9], it is possible to delineate the global events of a signaling pathway. Several signaling pathways including those of EGF, FGF, Wnt, interferon- alpha, insulin and TGF-β, have been systematically analyzed by this mass-spectrometry-based proteomics method [10][11][12][13][14][15]. These studies facilitate functional characterization of protein complexes and signaling pathways, and help to provide a more global understanding of those complicated biological processes.…”