Endogenous transforming growth factor-beta receptor-mediated Smad signaling complexes analyzed by mass spectrometry.
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.
Laboratory for Macromolecular Analysis and Proteomics, Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Qilie
Q
Luo
Edward
E
Nieves
Julia
J
Kzhyshkowska
Ruth Hogue
RH
Angeletti
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D047468:Immunoprecipitation; D013058:Mass Spectrometry / Q000379:methods*; D008907:Mink; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry*; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D040901:Proteomics / Q000379:methods; D018125:Receptors, Transforming Growth Factor beta / Q000737:chemistry* / Q000378:metabolism*; D015203:Reproducibility of Results; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D051785:Smad Proteins / Q000737:chemistry* / Q000378:metabolism*; D051899:Smad2 Protein / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013997:Time Factors
D046912:Multiprotein Complexes; D019151:Peptide Library; D010455:Peptides; D018125:Receptors, Transforming Growth Factor beta; D051785:Smad Proteins; D051899:Smad2 Protein