Phosphoproteome profiling of transforming growth factor (TGF)-beta signaling: abrogation of TGFbeta1-dependent phosphorylation of transcription factor-II-I (TFII-I) enhances cooperation of TFII-I and Smad3 in transcription.
Transforming growth factor-beta (TGFbeta) 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 TGFbeta1. We identified 32 proteins that change their phosphorylation upon treatment with TGFbeta1; 26 of these proteins are novel targets of TGFbeta1. We show that Smad2 and Smad3 have different effects on the dynamics of TGFbeta1-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 TGFbeta1-dependent phosphorylation of one of the targets, i.e., transcription factor-II-I (TFII-I). We confirmed that TGFbeta1 stimulated TFII-I phosphorylation at serine residues 371 and 743. Abrogation of the phosphorylation by replacement of Ser371 and Ser743 with alanine residues resulted in enhanced complex formation between TFII-I and Smad3, and enhanced cooperation between TFII-I and Smad3 in transcriptional regulation, as evaluated by a microarray-based measurement of expression of endogenous cyclin D2, cyclin D3, and E2F2 genes, and by a luciferase reporter assay. Thus, TGFbeta1-dependent phosphorylation of TFII-I may modulate TGFbeta signaling at the transcriptional level.
Stasyk
Taras
T
Ludwig Institute for Cancer Research, Uppsala University, SE-751 24 Uppsala, Sweden.
Dubrovska
Anna
A
Lomnytska
Marta
M
Yakymovych
Ihor
I
Wernstedt
Christer
C
Heldin
Carl-Henrik
CH
Hellman
Ulf
U
Souchelnytskyi
Serhiy
S
D019943:Amino Acid Substitution; D045744:Cell Line, Tumor; D056742:Cyclin D2; D056743:Cyclin D3; D016213:Cyclins / Q000378:metabolism; D050703:E2F2 Transcription Factor / Q000378:metabolism; D006801:Humans; D010766:Phosphorylation; D011485:Protein Binding; D020543:Proteome / Q000378:metabolism*; D015398:Signal Transduction; D051899:Smad2 Protein / Q000378:metabolism; D051900:Smad3 Protein / Q000378:metabolism*; D019473:Transcription Factors, TFII / Q000502:physiology*; D014158:Transcription, Genetic; D016212:Transforming Growth Factor beta / Q000502:physiology*; D053773:Transforming Growth Factor beta1
C080336:CCND2 protein, human; C534696:CCND3 protein, human; D056742:Cyclin D2; D056743:Cyclin D3; D016213:Cyclins; D050703:E2F2 Transcription Factor; C493761:E2F2 protein, human; C104259:GTF2I protein, human; D020543:Proteome; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; C508960:TGFB1 protein, human; D019473:Transcription Factors, TFII; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1