Proteomics-based identification of proteins interacting with Smad3: SREBP-2 forms a complex with Smad3 and inhibits its transcriptional activity.
Smad3 is an important component of transforming growth factor-beta (TGFbeta) intracellular signalling. To identify novel interacting proteins of Smad3, we performed pull-down assays with Smad3 constructs fused to glutathione-S-transferase. Proteins which formed complexes with these constructs were analyzed by two-dimensional gel electrophoresis, and were identified by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. We identified 14 proteins interacting with the Smad3 construct lacking the N-terminal Mad homology domain 1 (MH1), and 12 proteins interacting with the construct lacking the C-terminal MH2 domain. Proteins involved in signalling processes, in metabolism regulation, novel proteins, and components of cytoskeleton form four groups of interacting proteins. Interactions of AGP7, sex-determining region Y protein, actin beta and sterol regulatory element binding protein-2 (SREBP-2) proteins with Smad3 constructs were confirmed by immunoblotting with specific antibodies. Interaction of Smad3 with SREBP-2 was also confirmed by co-immunoprecipitation of myc-Smad3 and Flag-SREBP-2 upon expression in mammalian cells. We found that SREBP-2 inhibited the transcriptional activity of Smad3 in luciferase reporter assays.
Grimsby
Susanne
S
Ludwig Institute for Cancer Research, Box 595, BMC, SE-751 24, Uppsala, Sweden.
Jaensson
Hanna
H
Dubrovska
Anna
A
Lomnytska
Marta
M
Hellman
Ulf
U
Souchelnytskyi
Serhiy
S
D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D011485:Protein Binding; D020543:Proteome; D051900:Smad3 Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D051782:Sterol Regulatory Element Binding Protein 2; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D020543:Proteome; C497790:SMAD3 protein, human; C497390:SREBF2 protein, human; D051900:Smad3 Protein; D051782:Sterol Regulatory Element Binding Protein 2; D015534:Trans-Activators; D014157:Transcription Factors