T.Kawakami and T.Chiba contributed equally to this workNEDD8/Rub1 is a ubiquitin (Ub)-like post-translational modi®er that is covalently linked to cullin (Cul)-family proteins in a manner analogous to ubiquitylation. NEDD8 is known to enhance the ubiquitylating activity of the SCF complex (composed of Skp1, Cul-1, ROC1 and F-box protein), but the mechanistic role is largely unknown. Using an in vitro reconstituted system, we report here that NEDD8 modi®cation of Cul-1 enhances recruitment of Ub-conjugating enzyme Ubc4 (E2) to the SCF complex (E3). This recruitment requires thioester linkage of Ub to Ubc4. Our ®ndings indicate that the NEDD8-modifying system accelerates the formation of the E2±E3 complex, which stimulates protein polyubiquitylation.

PKA phosphorylates multiple molecules involved in calcium (Ca 2+ ) handling in cardiac myocytes and is considered to be the predominant regulator of β-adrenergic receptor-mediated enhancement of cardiac contractility; however, recent identification of exchange protein activated by cAMP (EPAC), which is independently activated by cAMP, has challenged this paradigm. Mice lacking Epac1 (Epac1 KO) exhibited decreased cardiac contractility with reduced phospholamban (PLN) phosphorylation at serine-16, the major PKA-mediated phosphorylation site. In Epac1 KO mice, intracellular Ca 2+ storage and the magnitude of Ca 2+ movement were decreased; however, PKA expression remained unchanged, and activation of PKA with isoproterenol improved cardiac contractility. In contrast, direct activation of EPAC in cardiomyocytes led to increased PLN phosphorylation at serine-16, which was dependent on PLC and PKCε. Importantly, Epac1 deletion protected the heart from various stresses, while Epac2 deletion was not protective. Compared with WT mice, aortic banding induced a similar degree of cardiac hypertrophy in Epac1 KO; however, lack of Epac1 prevented subsequent cardiac dysfunction as a result of decreased cardiac myocyte apoptosis and fibrosis. Similarly, Epac1 KO animals showed resistance to isoproterenol-and aging-induced cardiomyopathy and attenuation of arrhythmogenic activity. These data support Epac1 as an important regulator of PKA-independent PLN phosphorylation and indicate that Epac1 regulates cardiac responsiveness to various stresses.

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The photolyase gene from Thermus thermophilus was cloned and sequenced. The characteristic absorption and fluorescence spectra of the purified T. thermophilus photolyase suggested that the protein has flavin adenine dinucleotide as a chromophore. The second chromophore binding site was not conserved in T. thermophilus photolyase. The purified enzyme showed light-dependent photoreactivation activity in vitro at 35 and 65°C and was stable when subjected to heat and acidic pH.UV irradiation, including sunlight, induces damage to DNA. One of the most lethal types of damage is thymine-thyminetype pyrimidine dimerization. DNA photolyase catalyzes the photoreversal of pyrimidine dimers with near-UV or visible light as a source of energy (21,22). All known DNA photolyases contain (reduced) flavin adenine dinucleotide (FAD), which is the photochemically active chromophore. It has been shown in experiments with Escherichia coli and Anacystis nidulans photolyases that electron transfer from reduced FAD to a pyrimidine dimer splits and restores the pyrimidine dimer (22).It is still unclear whether extremely thermophilic bacterial cells have photoreactivation activity. In the present study, we demonstrate such activity and characterize for the first time a thermophilic class I photolyase gene from an aerobic, rodshaped, nonsporulating, gram-negative eubacterium, Thermus thermophilus HB27, which can grow at temperatures over 75°C (15,19). Another thermophilic photolyase from the archaebacterium Methanobacterium thermoautotrophicum belongs to class II, and the gene product has not yet been studied in detail (9). Although recent X-ray crystallographic studies of the E. coli photolyase (16) have provided data useful for elucidating the photoreactivation reaction, the three-dimensional structure of the enzyme-substrate complex has not been determined. In order to study the molecular mechanism of substrate recognition and to carry out more detailed physicochemical analysis including nuclear magnetic resonance (NMR), an enzyme that is more easily crystallized and more stable than that of E. coli is required. Many proteins from T. thermophilus are heat stable and easily crystallized (6, 12, 13), making them ideal materials for such purposes.Photoreactivation activity in T. thermophilus cells. To clarify whether T. thermophilus cells have photoreactivation activity, the UV sensitivity of wild-type cells was measured in the dark or under illumination. As shown in Fig. 1, T. thermophilus cells illuminated with visible light were more UV resistant than those in the dark, indicating that the cells have photoreactivation activity. This suggested that T. thermophilus cells possess both functional photolyase and a photolyase gene.Cloning, sequencing, and primary structure of the T. thermophilus photolyase gene. The carotenoid biosynthesis gene, crtB, has been cloned from T. thermophilus, and a truncated open reading frame (ORF) with an unknown function, named ORF-B, has been found neighboring the gene (5). In order to elucidate the function...