cAMP (3′,5′ cyclic adenosine monophosphate) is a second messenger that in eukaryotic cells induces physiological responses ranging from growth, differentiation, and gene expression to secretion and neurotransmission. Most of these effects have been attributed to the binding of cAMP to cAMP-dependent protein kinase A (PKA). Here, a family of cAMP-binding proteins that are differentially distributed in the mammalian brain and body organs and that exhibit both cAMP-binding and guanine nucleotide exchange factor (GEF) domains is reported. These cAMP-regulated GEFs (cAMP-GEFs) bind cAMP and selectively activate the Ras superfamily guanine nucleotide binding protein Rap1A in a cAMP-dependent but PKA-independent manner. Our findings suggest the need to reformulate concepts of cAMP-mediated signaling to include direct coupling to Ras superfamily signaling.

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.

We report an optimized backbone for the rapid development of a highly sensitive intramolecular fluorescence resonance energy transfer (FRET) biosensor, which includes an optimized pair of fluorescent proteins and a long flexible linker ranging from 116 to 244 amino acids in length. With this backbone system, we developed FRET biosensors of PKA, ERK, JNK, EGFR, RSK, S6K, Akt, PKC, Ras, and Rac1.

Effects of the crystallization temperature on the crystal structure and its melting behavior of poly (l-lactic acid) (PLLA) have been investigated by means of wide-angle (WAXS) and small-angle (SAXS) X-ray scattering, optical microscopy, and differential scanning calorimetory (DSC). PLLA was found to crystallize as the α form when the crystallization temperature T c was higher than 120 °C, while significant change in lattice parameters was seen for T c's below 120 °C. The ratio of the a- and b-axis lengths begins to decrease with T c below 120 °C and is 31/2 below 90 °C, which suggests a new crystalline form with hexagonal packing, namely, the α‘ form. The possible reason for α‘ formation is discussed. High-temperature WAXS and SAXS measurements showed that α‘ crystal transforms into ordered a form during heating. The transition takes place at 150 °C without a decrease in scattering intensity and without heating rate dependence. The mechanism for the transition is discussed.

Rho family G proteins, including Rac and Cdc42, regulate a variety of cellular functions such as morphology, motility, and gene expression. We developed fluorescent resonance energy transfer-based probes which monitored the local balance between the activities of guanine nucleotide exchange factors and GTPase-activating proteins for Rac1 and Cdc42 at the membrane. These probes, named Raichu-Rac and Raichu-Cdc42, consisted of a Cdc42-and Rac-binding domain of Pak, Rac1 or Cdc42, a pair of green fluorescent protein mutants, and a CAAX box of Ki-Ras. With these probes, we video imaged the Rac and Cdc42 activities. In motile HT1080 cells, activities of both Rac and Cdc42 gradually increased toward the leading edge and decreased rapidly when cells changed direction. Under a higher magnification, we observed that Rac activity was highest immediately behind the leading edge, whereas Cdc42 activity was most prominent at the tip of the leading edge. Raichu-Rac and Raichu-Cdc42 were also applied to a rapid and simple assay for the analysis of putative guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) in living cells. Among six putative GEFs and GAPs, we identified KIAA0362/DBS as a GEF for Rac and Cdc42, KIAA1256 as a GEF for Cdc42, KIAA0053 as a GAP for Rac and Cdc42, and KIAA1204 as a GAP for Cdc42. In conclusion, use of these single-molecule probes to determine Rac and Cdc42 activity will accelerate the analysis of the spatiotemporal regulation of Rac and Cdc42 in a living cell.Ras superfamily G proteins function as molecular switches in a variety of signaling cascades (51). Among them, Rho family G proteins, including Rho, Rac, and Cdc42, are involved in the regulation of a variety of cellular processes, probably through actin cytoskeleton reorganization (1, 9, 13, 48). In a pioneering work by Nobes and Hall, it was shown that Rho regulates the assembly of the actin stress fiber, that Rac induces lamellipodia and membrane ruffles, and that Cdc42 triggers filopodium formation (41).Rho family G proteins are regulated by three classes of protein, guanine nucleotide exchange factor (GEF), GTPaseactivating protein (GAP), and guanine nucleotide dissociation inhibitor (GDI) (51). GEF promotes the exchange of GDP with GTP, which results in the binding of the G proteins to their effector proteins. A typical GEF protein of the Rho family of G proteins consists of a Dbl homology (DH) domain, which exhibits GEF activity, and additional domains that mediate interactions with peptides or lipids. DOCK180, originally isolated as a protein bound to adapter protein Crk (14), also promotes guanine nucleotide exchange of Rac, although it does not contain the DH domain (20). The GTP on the activated Rho family G protein is hydrolyzed in the presence of GAP to resume the GDP-bound inactive state. GDI not only competes with GEF but also holds the Rho family G proteins in the cytoplasm (43). Therefore, the dissociation of GDI is a prerequisite for the membrane association and activation of the Rho family G prot...

SummaryIntravital imaging of BRAF-mutant melanoma cells containing an ERK/MAPK biosensor reveals how the tumor microenvironment affects response to BRAF inhibition by PLX4720. Initially, melanoma cells respond to PLX4720, but rapid reactivation of ERK/MAPK is observed in areas of high stromal density. This is linked to “paradoxical” activation of melanoma-associated fibroblasts by PLX4720 and the promotion of matrix production and remodeling leading to elevated integrin β1/FAK/Src signaling in melanoma cells. Fibronectin-rich matrices with 3–12 kPa elastic modulus are sufficient to provide PLX4720 tolerance. Co-inhibition of BRAF and FAK abolished ERK reactivation and led to more effective control of BRAF-mutant melanoma. We propose that paradoxically activated MAFs provide a “safe haven” for melanoma cells to tolerate BRAF inhibition.

Rho-family GTPases regulate many cellular functions. To visualize the activity of Rho-family GTPases in living cells, we developed fluorescence resonance energy transfer (FRET)–based probes for Rac1 and Cdc42 previously (Itoh, R.E., K. Kurokawa, Y. Ohba, H. Yoshizaki, N. Mochizuki, and M. Matsuda. 2002. Mol. Cell. Biol. 22:6582–6591). Here, we added two types of probes for RhoA. One is to monitor the activity balance between guanine nucleotide exchange factors and GTPase-activating proteins, and another is to monitor the level of GTP-RhoA. Using these FRET probes, we imaged the activities of Rho-family GTPases during the cell division of HeLa cells. The activities of RhoA, Rac1, and Cdc42 were high at the plasma membrane in interphase, and decreased rapidly on entry into M phase. From after anaphase, the RhoA activity increased at the plasma membrane including cleavage furrow. Rac1 activity was suppressed at the spindle midzone and increased at the plasma membrane of polar sides after telophase. Cdc42 activity was suppressed at the plasma membrane and was high at the intracellular membrane compartments during cytokinesis. In conclusion, we could use the FRET-based probes to visualize the complex spatio-temporal regulation of Rho-family GTPases during cell division.

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...