Ten intermediate models were generated and energy minimization was performed for each model using the Amber force field ff99SB48 and implicit solvation. cells. The dynamic interplay between the ubiquitin system and the kinase scaffold of the Ras pathway shapes the activation profile of the mitogenic cascade. By controlling KSR1 levels, praja2 directly affects compartmentalized ERK activities, impacting on physiological events required for cell proliferation and maintenance of embryonic stem cell pluripotency. The Ras-Raf-ERK protein kinase cascade constitutes a central signaling mechanism that controls important cell functions, such as differentiation, metabolism and cell growth. Activation of Ras by growth factors or G protein-coupled receptor (GPCR) ligands promotes a kinase suppressor of Ras 1 (KSR1)-mediated formation of a tripartite kinase complex, which compartmentalizes Raf, MEK and ERK.1, 2, 3 By juxtaposing upstream and downstream signaling kinases, KSR1 optimally couples stimulation of membrane receptors to the propagation of the signals to a variety of ERK substrates controlling multiple biological activities, such as cell proliferation, metabolism and synaptic activity.4, 5, 6, 7, 8, 9 Dysregulation or mutations in the genes encoding components of this transduction pathway are frequently found in several human cancers.10, 11, 12 Interfering with KSR1 function reduces Ras signaling and cancer cell growth.13, 14, 15, 16, 17 Distinct Guanosine 5′-diphosphate disodium salt attenuation mechanisms of signaling cascade have been identified.18, 19, 20 As for Guanosine 5′-diphosphate disodium salt mitogenic pathway, a negative loop between ERK1/2 and KSR1 ensures an efficient and tightly controlled cycle of activation/de-activation process that limits unrestrained and widespread activation of mitogenic signaling. Phosphorylation of KSR1 and B-Raf by locally activated ERK1 dissociates the KSR1 multi-kinase complex, turning-off the ERK cascade.15, 20 The mitogenic cascade could also be firmly regulated through phosphorylation of Raf and KSR1 by cAMP-dependent protein kinase (PKA).21 The bi-directional regulation of KSR1 BII and ERK cascade, and the integration of the Ras pathway with signals carried out by the GPCR?cAMP signaling axis control the rate, magnitude and persistence of the downstream mitogenic pathway. The ubiquitinCproteasome system (UPS) emerged as an important posttranslational mechanism that controls cell growth, differentiation, metabolism and survival. The UPS couples ubiquitylation of a target protein to its proteolytic cleavage, eliminating unneeded or damaged proteins and contributing to essential aspects of cell signaling and homeostasis.22 The process involves the sequential action of ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2) and ubiquitin ligases (E3), where each enzyme transfers ubiquitin molecules from one enzyme to the next and eventually to the target protein. praja2 belongs to a growing family of widely expressed mammalian RING-H2 proteins with intrinsic E3 ubiquitin-ligase activity.23, 24, 25, 26 During GPCR?cAMP stimulation, praja2 ubiquitylates and degrades the inhibitory (R) subunits of PKA, sustaining downstream signals carried out by cAMP.27 In proliferating cells, praja2 promotes ubiquitin-dependent proteolysis of MOB1, a core component the tumor-suppressor Hippo cascade. Degradation of MOB1 through the UPS attenuates the Hippo cascade and sustains tumor growth.28 A Guanosine 5′-diphosphate disodium salt role of praja2?UPS in neuronal differentiation and glucose homeostasis has also been recently described.29, 30 However, the impact of praja2 in the control of ERK signaling was unknown. Here we report that the KSR1 abundance is controlled by specific components of the ubiquitin pathway. We identified praja2 as the E3 ligase that ubiquitylates KSR1 in response to growth factor or cAMP stimulation. Ubiquitination of KSR1 eventually attenuates the ERK1/2 cascade. By modulating KSR1?ERK signaling, praja2 profoundly impacts on essential aspects of embryonic stem cell (ESC) differentiation. Results Identification of KSR1 as novel praja2 interactor By controlling the stability of protein kinases (PKA and Lats/Mob1), praja2 integrates signals carried out by two evolutionary conserved transduction cascades, having a major role in cell proliferation and tumor growth.28 Large-scale proteomic analyses revealed that praja2 is a component of a macromolecular complex that includes the ERK scaffold KSR1.31 Based on this finding, we tested whether praja2 interacts with and regulates the stability KSR1. First, we demonstrated the interaction by isolating an endogenous praja2/KSR1 complex from cell lysates (Figure 1a). Similarly, exogenous flag-praja2 and co-expressed myc-KSR1 formed a stable complex in cell lysates (Figure 1b). The core domain of praja2 (residues 401C530) was required for KSR1 binding (Figure 1b). Next we confirmed a direct interaction between KSR1 and praja2 (Figure 1e). The potential binding site is, indeed, located at.