C) Transfected HEK293T cells were treated with 4 ng/ml of TGF- for 30 minutes before cell lysis. Smad pathway however, accumulating evidence indicate that alternative signaling routes are also employed by this pleiotropic cytokine. For instance recently, we have demonstrated that ligand occupied TGF- receptors can directly trigger the TRAF6-TAK1 signaling module, resulting in MAP kinase activation. Here we report identification of the adaptor molecule TTRAP as a novel component of this non-canonical TGF- pathway. We show that the protein associates with TGF- Radiprodil receptors and components of the TRAF6-TAK1 signaling module, resulting in differential regulation of TGF- activated p38 and NF-B responses. Modulation of APT1 cellular TTRAP level affects cell viability in the presence of TGF-, suggesting that the protein is an important component of the TGF- induced apoptotic process. Introduction TGF- has pervasive and diverse effects on cell physiology as well as it acts as a potent anticancer agent that prohibits uncontrolled cell proliferation [1]C[3]. The most accepted model for the signaling mechanism of TGF- family cytokines portrays a relatively simple pathway, in which ligand binding to a membrane bound receptor complex induces a conformational change, resulting in phosphorylation and activation of the type I receptor (TRI) by the type II receptor kinase (TRII). Through its own kinase activity, TRI then phosphorylates the appropriate receptor Smads (R-Smads, Smad2/3). Once phosphorylated, R-Smads can form complexes with the common Smad (Smad4), whereupon they translocate to the nucleus to initiate specific transcriptional programs [4], [5]. It is becoming increasingly apparent however, that the picture depicted above is significantly more complex. TGF- can mobilize several intracellular signal transducers in Smad-independent manner as well [6]C[8]. These non-canonical, non-Smad pathways are also activated directly by ligand-occupied receptors to reinforce, attenuate or otherwise modulate downstream cellular responses. The non-Smad pathways include various branches of MAP kinase pathways, Rho-like GTPase signaling pathways, the phosphatidylinositol-3-kinase/AKT pathway and more. Such alternative signal transducers often regulate the Smad pathway itself and represent extensive opportunities for crosstalk with other signaling routes, contributing to the surprising diversity of TGF- responses. Perhaps one of the most important non-Smad pathways is the p38/JNK MAP kinase cascade [9]C[12]. This signaling route functions in conjunction with the Smad pathway to regulate such cellular responses as apoptosis and eptithelial-to-mesenchymal transition (EMT). Despite their obvious biological significance however, we still have serious caveats in understanding the mechanisms by which TGF- governs them. The need to fill out these gaps is further underscored by Radiprodil several recent observations, suggesting that imbalances arising between the Smad-pathway and the p38/JNK MAPK signaling branches during tumorigenesis may contribute to the conversion of TGF- from a suppressor to a Radiprodil promoter of cancer growth [13]C[19]. Previous genetic studies placed TGF–activated kinase 1 (TAK1) in the TGF- mediated p38/JNK activation pathway however, the link between TAK1 and the activated receptor complex had been lacking [20]C[22]. Recently, we and others have demonstrated that the E3 ubiquitin ligase, TRAF6 is one of the missing pieces [23], [24]. The molecule physically interacts with the TGF- receptor complex and is required for Smad-independent activation of the JNK and p38 kinases. TGF- promotes association between TRAF6 and TAK1, resulting in lysine 63-linked (K63) ubiquitylation and subsequent activation of TAK1. Interestingly, the TRAF6-TAK1 signaling module is also employed by a number of different signaling routes such as those emanating from the IL-1 receptor, Toll-like receptors, T-cell receptor etc. and cellular processes.