Endogenous Transforming Growth Factor-β Receptor-mediated Smad Signaling Complexes Analyzed by Mass Spectrometry

Abstract

ASmad proteins are the central feature of the transforming growth factor-beta (TGF-beta) intracellular signaling cascade. They function by carrying signals from the cell surface to the nucleus through the formation of a series of signaling complexes. Changes in Smad proteins and their complexes upon treatment with TGF-beta were studied in mink lung epithelial (Mv1Lu) cell cultures. A time course of incubation with TGF-beta was carried out to determine the peak of appearance of phosphorylated Smad2. Immobilized monoclonal antibody against Smad2 was then used to isolate the naturally occurring complexes. Three strategies were used to identify changes in proteins partnering with Smad2: separation by one-dimensional SDS-PAGE followed by MALDI peptide mass fingerprinting, cleavable ICAT labeling of the protein mixtures analyzed by LC-MS/MS, and nano-LC followed by MALDI MS TOF/TOF. Smad2 forms complexes with many other polypeptides both in the presence and absence of TGF-beta. Some of the classes of proteins identified include: transcription regulators, proteins of the cytoskeletal scaffold and other tethering proteins, motility proteins, proteins involved in transport between the cytoplasm and nucleus, and a group of membrane adaptor proteins. Although some of these have been reported in the literature, most have not been reported previously. This work expands the repertoire of proteins known to participate in the TGF-beta signal transduction processes.

A tale of two proteins: Differential roles and regulation of Smad2 and Smad3 in TGF‐β signaling

“…One approach to further understand the selectivity is to identify proteins that uniquely interact with Smad2 or Smad3. Previous reports have identified novel Smad2 and/ or Smad3 protein-protein interactions with techniques including yeast two-hybrid assays [Akiyoshi et al, 1999;Verschueren et al, 1999;Wotton et al, 1999;Lin et al, 2000;Chen et al, 2002b;Ellis et al, 2003;Warner et al, 2003aWarner et al, ,b, 2004Colland et al, 2004;Wicks et al, 2005;Richard et al, 2006], luminescence-based mammalian interactome mapping (LUMIER) [Barrios-Rodiles et al, 2005], and immunoprecipitation of endogenous [Luo et al, 2006] and epitope-tagged Smad proteins Knuesel et al, 2003;Grimsby et al, 2004] combined with mass spectrometry. These studies identified proteins that play important roles in Smad-mediated TGF-b signaling such as c-SKI, SMURF2, SNON.…”

Altered protein expression at early-stage rat hepatic neoplasia

“…The whole protocol of protein in-gel digestion is the same as performed before (45) except that two steps, protein reduction and alkylation, were excluded in this experiment. The matrix-assisted laser desorption ionization (MALDI) mass spectrometric analysis of supernatants from in-gel digestion, and database searches were performed as before (45). Some identifications were checked with mass spectrometry (MS)/MS to screen the case, more than one protein in one spot.…”

“…Table 1 lists proteins identified using PMF and LC-MS/MS. All listed proteins meet the current stringent criteria for positive identifications (45,81). The proteins in the table are identified as upregulated or downregulated from sham-operated control to nodule-enriched neoplastic liver.…”

Adenovirus E1B 55-Kilodalton Protein: Multiple Roles in Viral Infection and Cell Transformation

“…E1B-AP5 is a multifunctional protein; in addition to its role in mRNA metabolism, it may play a part in transcriptional regulation via interactions with transcription factors such as bromodomain-containing protein 7 (BRD7), p53, and small and mothers against decaplentaplegic-related protein 2 (SMAD2) (7,85,96). By using transient reporter assays, it was shown that E1B-AP5 is a repressor of basal transcription driven by several cellular and viral promoters (85).…”