g cooperation between p53 and TGFb signaling. Thus, TGFb-dependent phosphorylation of BIX02189 web 14-3-3s is a feedforward mechanism for TGFb/Smad3 transcriptional regulation. The feed-forward tuning includes a 14-3-3s-dependent recruitment of p53 to a Smad3-initiated transcriptional complex, which led to restriction of ligand-independent transcription and to enhancement of the ligand-induced effect. This indicates that p53 is an enhancer of bi-stability for Smad3-dependent transcriptional activation, e.g. on-off accentuation. Taken together, these results suggest that TGFb dependent 14-3-3s phosphorylation plays an important role in regulation of tumorigenicity and radioresistance that can be mediated by the integrated TGFb and p53/21 signaling pathways. Discussion Signaling by a network, as compared to the model of unidirectional signaling pathways, is required for coordination of various functions in cells undergoing significant changes, e.g. proliferation or carcinogenic transformation. Our study identified proteins which may mediate coordinated regulation of cell signaling and metabolism, proliferation, death and differentiation of human breast epithelial cells. The network built with TGFb-regulated phosphoproteins showed characteristics of a scale-free network. However, the frequency of highly-connected hubs is higher than would be expected according to a power law distribution of connections in an ideal scale-free network. The presence of such hubs indicates the key points of convergence for Phosphoproteomics of TGFb1 Signaling various signals. In the TGFb phosphoprotein-network, 
these hubs represent signaling activities initiated and/or mediated by EGF, TNF, IGF, AKT, Src, H-Ras, CDK2 and NF-kB. Therefore the status of these hubs may dictate the output of TGFb action on cells. 1417812 For many of the above mentioned hubs, functional crosstalks with TGFb have been reported in model systems specific for each of the hubs,,. Phosphoproteome profiling of TGFb1 in MCF7 cells using in vivo metabolic labeling with 32P was the first step in an exploration of TGFb phosphoproteome. Despite use of different cell lines and different technical approaches, we identified in MCF10A cells some of the proteins which were regulated by TGFb1 in MCF7 cells, e.g. keratin 10, enolase-1 and HSP70. IMAC-enrichment of phosphoproteins showed capability to enrich for low abundance proteins, which explains high representation of regulatory proteins. Thus, our approach provided the most comprehensive description of phosphorylation events initiated by TGFb1. To confirm that our network-based approach unveils novel crucial regulatory mechanisms, we explored the role of TGFb1dependent phosphorylation of 14-3-3s. Some phosphorylation sites in 14-3-3s important for its function were identified earlier, e.g. Thr198, Ser216, Thr291, Ser428, Ser642,. In this report, we identified two novel and TGFb-dependent phosphorylation sites, i.e. Ser69 and Ser74. Identified by us network suggested that phosphorylation of 14-3-3s at Ser69 and Ser74 may play crucial role in TGFb signaling. Indeed, we observed that 14-3-3s phosphorylation is a feed-forward mechanism 11404282 in TGFb/ Smad3-dependent transcription. Our data suggest that overexpression of the wild type or single Ser74 mutant 14-3-3s proteins could significantly increase TGFb and Smad3-dependent transcription activity of the reporter. In addition to the regulation of Smad3-specific transcription, 14-3-3s phosphorylation at Ser69 and Ser74 regula
