Toll-like receptors (TLRs) are involved in the recognition of microbial pathogens. A subset of TLRs, TLR7, TLR8 and TLR9, induces antiviral responses by producing interferon-alpha (IFN-alpha). Production of IFN-alpha is dependent on the Toll-interleukin-1 receptor domain-containing adaptor MyD88. Here we show that MyD88 formed a complex with the transcription factor IRF7 but not with IRF3. The death domain of MyD88 interacted with an inhibitory domain of IRF7, and this interaction resulted in activation of the IFN-alpha-dependent promoters. Furthermore, the adaptor molecule TRAF6 also bound and activated IRF7. Ubiquitin ligase activity of TRAF6 was required for IRF7 activation. These results indicate that TLR-mediated IFN-alpha induction requires the formation of a complex consisting of MyD88, TRAF6 and IRF7 as well as TRAF6-dependent ubiquitination.

[Keywords: CRL4; DDB1; PCNA; p21 ubiquitylation; Cdt2; DCAFs] Supplemental material is available at http://www.genesdev.org.

To comprehend the Ras/ERK MAPK cascade, which comprises Ras, Raf, MEK, and ERK, several kinetic simulation models have been developed. However, a large number of parameters that are essential for the development of these models are still missing and need to be set arbitrarily. Here, we aimed at collecting these missing parameters using fluorescent probes. First, the levels of the signaling molecules were quantitated. Second, to monitor both the activation and nuclear translocation of ERK, we developed probes based on the principle of fluorescence resonance energy transfer. Third, the dissociation constants of Ras⅐Raf, Raf⅐MEK, and MEK⅐ERK complexes were estimated using a fluorescent tag that can be highlighted very rapidly. Finally, the same fluorescent tag was used to measure the nucleocytoplasmic shuttling rates of ERK and MEK. Using these parameters, we developed a kinetic simulation model consisting of the minimum essential members of the Ras/ERK MAPK cascade. This simple model reproduced essential features of the observed activation and nuclear translocation of ERK. In this model, the concentration of Raf significantly affected the levels of phospho-MEK and phospho-ERK upon stimulation. This prediction was confirmed experimentally by decreasing the level of Raf using the small interfering RNA technique. This observation verified the usefulness of the parameters collected in this study.The Ras/ERK 2 MAPK cascade has been highly conserved throughout evolution and plays a pivotal role in many aspects of cellular events, including proliferation, differentiation, and survival (reviewed in Refs. 1-5). The principal components of this Ras/ERK MAPK cascade include H-Ras, N-Ras, K-Ras, c-Raf, B-Raf, A-Raf, MEK1, MEK2, ERK1, and ERK2. In addition to these, a number of proteins regulate this signaling pathway either positively or negatively: A few examples include phosphatases and scaffold proteins that have been shown to play critical roles in the spatiotemporal regulation of ERK MAPK (reviewed in Refs. 6 -9). Furthermore, there are many positive and negative feedback loops that modulate the activity of each signaling component, rendering this signal transduction cascade ever more complicated.To comprehend the Ras/ERK MAPK cascade, many research groups have attempted to reconstruct this cascade in silico (10 -17). These systems analyses are used to propose mechanisms to explain the ultrasensitivity of ERK to the input signal (18), the stable response of the Ras/ERK signaling cascade to a wide range of epidermal growth factor (EGF) concentrations (15), and the different responses of PC12 pheochromocytoma cells upon stimulation by EGF or nerve growth factor (17). Each kinetic simulation model reported previously recapitulates the stimulus-induced ERK activation very nicely. Nevertheless, the parameters used therein are sometimes astonishingly different from each other. One apparent reason for this discrepancy is that many studies set parameters to fit experimental data using different algorithms (11-13). Another reason...

Endothelial cells (ECs) line the inside of blood vessels and respond to mechanical cues generated by blood flow. Mechanical stimuli regulate the localization of YAP by reorganizing the actin cytoskeleton. Here we demonstrate blood-flow-mediated regulation of endothelial YAP in vivo. We indirectly monitored transcriptional activity of Yap1 (zebrafish YAP) and its spatiotemporal localization in living zebrafish and found that Yap1 entered the nucleus and promoted transcription in response to blood flow. In cultured human ECs, laminar shear stress induced nuclear import of YAP and its transcriptional activity in a manner independent of Hippo signaling. We uncovered a molecular mechanism by which flow induced the nuclear translocation of YAP through the regulation of filamentous actin and angiomotin. Yap1 mutant zebrafish showed a defect in vascular stability, indicating an essential role for Yap1 in blood vessels. Our data imply that endothelial Yap1 functions in response to flow to maintain blood vessels.

Semaphorins and their receptors have diverse functions in axon guidance, organogenesis, vascularization and/or angiogenesis, oncogenesis and regulation of immune responses. The primary receptors for semaphorins are members of the plexin family. In particular, plexin-A1, together with ligand-binding neuropilins, transduces repulsive axon guidance signals for soluble class III semaphorins, whereas plexin-A1 has multiple functions in chick cardiogenesis as a receptor for the transmembrane semaphorin, Sema6D, independent of neuropilins. Additionally, plexin-A1 has been implicated in dendritic cell function in the immune system. However, the role of plexin-A1 in vivo, and the mechanisms underlying its pleiotropic functions, remain unclear. Here, we generated plexin-A1-deficient (plexin-A1(-/-)) mice and identified its important roles, not only in immune responses, but also in bone homeostasis. Furthermore, we show that plexin-A1 associates with the triggering receptor expressed on myeloid cells-2 (Trem-2), linking semaphorin-signalling to the immuno-receptor tyrosine-based activation motif (ITAM)-bearing adaptor protein, DAP12. These findings reveal an unexpected role for plexin-A1 and present a novel signalling mechanism for exerting the pleiotropic functions of semaphorins.