Cyclic AMP-responsive element (CRE)-binding protein (CREB) is a transcription factor that plays an important role in numerous physiological events, such as cell proliferation, survival, tumorigenesis, glucose metabolism and memory, in a phosphorylation-dependent manner [1,2] Cyclic AMP responsive element (CRE)-binding protein (CREB) is known to activate transcription when its Ser133 is phosphorylated. Two independent investigations have suggested the presence of Ser133-independent activation. One study identified a kinase, salt-inducible kinase (SIK), which repressed CREB; the other isolated a novel CREB-specific coactivator, transducer of regulated CREB activity (TORC), which upregulated CREB activity. These two opposing signals are connected by the fact that SIK phosphorylates TORC and induces its nuclear export. Because LKB1 has been reported to be an upstream kinase of SIK, we used LKB1-defective HeLa cells to further elucidate TORC-dependent CREB activation. In the absence of LKB1, SIK was unable to phosphorylate TORC, which led to constitutive activation of CRE activity. Overexpression of LKB1 in HeLa cells improved the CRE-dependent transcription in a regulated manner. The inactivation of kinase cascades by 10 nm staurosporine in LKB1-positive HEK293 cells also induced unregulated, constitutively activated, CRE activity. Treatment with staurosporine completely inhibited SIK kinase activity without any significant effect on the phosphorylation level at the LKB1-phosphorylatable site in SIK or the activity of AMPK, another target of LKB1. Constitutive activation of CREB in LKB1-defective cells or in staurosporine-treated cells was not accompanied by CREB phosphorylation at Ser133. The results suggest that LKB1 and its downstream SIK play an important role in silencing CREB activity via the phosphorylation of TORC, and such silencing may be indispensable for the regulated activation of CREB.Abbreviations A-loop, activation loop; AMPK, AMP-activated protein kinase; bZIP, basic leucine zipper domain; CRE, cAMP-response element; CREB, CRE-binding protein; DAPI, 4¢,6-diamidino-2-phenylindole; GFP, green fluorescent protein; GST, glutathione-S-transferase; HA, hemagglutinin; KID, kinase-inducible domain; moi, multiplicities of infection; PKA, protein kinase A; RT, reverse transcription; SIK, salt-inducible kinase; TORC, transducer of regulated CREB activity.

The CAPRI and CASP prediction experiments have demonstrated the power of community wide tests of methodology in assessing the current state of the art and spurring progress in the very challenging areas of protein docking and structure prediction. We sought to bring the power of community wide experiments to bear on a very challenging protein design problem that provides a complementary but equally fundamental test of current understanding of protein-binding thermodynamics. We have generated a number of designed protein-protein interfaces with very favorable computed binding energies but which do not appear to be formed in experiments, suggesting there may be important physical chemistry missing in the energy calculations. 28 research groups took up the challenge of determining what is missing: we provided structures of 87 designed complexes and 120 naturally occurring complexes and asked participants to identify energetic contributions and/or structural features that distinguish between the two sets. The community found that electrostatics and solvation terms partially distinguish the designs from the natural complexes, largely due to the non-polar character of the designed interactions. Beyond this polarity difference, the community found that the designed binding surfaces were on average structurally less embedded in the designed monomers, suggesting that backbone conformational rigidity at the designed surface is important for realization of the designed function. These results can be used to improve computational design strategies, but there is still much to be learned; for example, one designed complex, which does form in experiments, was classified by all metrics as a non-binder.

The fluorescence excitation and dispersed fluorescence spectra of jet-cooled uracil, thymine, and their derivatives have been observed. Two band systems having well-resolved vibrational structures were found for uracil and thymine in the frequency region from 31 000 to 38 000 cm"1, which corresponds to the region of the long tail in the vapor absorption spectrum. The shorter wavelength band system (system I) was identified as the 8,( , *) *-S0 transition of the diketo tautomer, while the longer wavelength system (system II) was assigned to the Si(n,ir*) *-S0 transition of one of the keto-enol tautomers. Successful detection of a very small amount of the keto-enol tautomer is due to a high fluorescence yield of the keto-enol tautomer.The nature of the ^ , *) states of the tautomers and the hydrogen-bonded complexes with water are also discussed.

Community-wide blind prediction experiments such as CAPRI and CASP provide an objective measure of the current state of predictive methodology. Here we describe a community-wide assessment of methods to predict the effects of mutations on protein-protein interactions. Twenty-two groups predicted the effects of comprehensive saturation mutagenesis for two designed influenza hemagglutinin binders and the results were compared with experimental yeast display enrichment data obtained using deep sequencing. The most successful methods explicitly considered the effects of mutation on monomer stability in addition to binding affinity, carried out explicit side chain sampling and backbone relaxation, and evaluated packing, electrostatic and solvation effects, and correctly identified around a third of the beneficial mutations. Much room for improvement remains for even the best techniques, and large-scale fitness landscapes should continue to provide an excellent test bed for continued evaluation of methodological improvement.