Phosphorylation-dependent inhibition of Akt1

Protein kinase B (Akt1) is a proto-oncogene that is overactive in most cancers. Akt1 activation requires phosphorylation at Thr308; phosphorylation at Ser473 further enhances catalytic activity. Akt1 activity is also regulated via interactions between the kinase domain and the N-terminal auto-inhibitory pleckstrin homology (PH) domain. As it was previously difficult to produce Akt1 in site-specific phosphorylated forms, the contribution of each activating phosphorylation site to auto-inhibition was unknown. Using a combination of genetic code expansion and in vivo enzymatic phosphorylation, we produced Akt1 variants containing programmed phosphorylation to probe the interplay between Akt1 phosphorylation status and the auto-inhibitory function of the PH domain. Deletion of the PH domain increased the enzyme activity for all three phosphorylated Akt1 variants. For the doubly phosphorylated enzyme, deletion of the PH domain relieved auto-inhibition by 295-fold. We next found that phosphorylation at Ser473 provided resistance to chemical inhibition by Akti-1/2 inhibitor VIII. The Akti-1/2 inhibitor was most effective against pAkt1T308 and showed four-fold decreased potency with Akt1 variants phosphorylated at Ser473. The data highlight the need to design more potent Akt1 inhibitors that are effective against the doubly phosphorylated and most pathogenic form of Akt1

The amino-terminal domain of pyrrolysyl-tRNA synthetase is dispensable in vitro but required for in vivo activity

AbstractPyrrolysine (Pyl) is co-translationally inserted into a subset of proteins in the Methanosarcinaceae and in Desulfitobacterium hafniense programmed by an in-frame UAG stop codon. Suppression of this UAG codon is mediated by the Pyl amber suppressor tRNA, tRNAPyl, which is aminoacylated with Pyl by pyrrolysyl-tRNA synthetase (PylRS). We compared the behavior of several archaeal and bacterial PylRS enzymes towards tRNAPyl. Equilibrium binding analysis revealed that archaeal PylRS proteins bind tRNAPyl with higher affinity (KD=0.1–1.0μM) than D. hafniense PylRS (KD=5.3–6.9μM). In aminoacylation the archaeal PylRS enzymes did not distinguish between archaeal and bacterial tRNAPyl species, while the bacterial PylRS displays a clear preference for the homologous cognate tRNA. We also show that the amino-terminal extension present in archaeal PylRSs is dispensable for in vitro activity, but required for PylRS function in vivo

The Helium-Rich Cataclysmic Variable ES Ceti

We report photometry of the helium-rich cataclysmic variable ES Ceti during 2001-2004. The star is roughly stable at V ~ 17.0 and has a light curve dominated by a single period of 620 s, which remains measurably constant over the 3 year baseline. The weight of evidence suggests that this is the true orbital period of the underlying binary, not a "superhump" as initially assumed. We report GALEX ultraviolet magnitudes, which establish a very blue flux distribution (F_nu ~ nu^1.3), and therefore a large bolometric correction. Other evidence (the very strong He II 4686 emission, and a ROSAT detection in soft X-rays) also indicates a strong EUV source, and comparison to helium-atmosphere models suggests a temperature of 130+-10 kK. For a distance of 350 pc, we estimate a luminosity of (0.8-1.7)x10^34 erg/s, yielding a mass accretion rate of (2-4)x10^-9 M_sol/yr onto an assumed 0.7 M_sol white dwarf. This appears to be about as expected for white dwarfs orbiting each other in a 10 minute binary, assuming that mass transfer is powered by gravitational radiation losses. We estimate mean accretion rates for other helium-rich cataclysmic variables, and find that they also follow the expected M-dot ~ P_o^-5 relation. There is some evidence (the lack of superhumps, and the small apparent size of the luminous region) that the mass transfer stream in ES Cet directly strikes the white dwarf, rather than circularizing to form an accretion disk.Comment: PDF, 26 pages, 3 tables, 9 figures; accepted, in press, to appear February 2005, PASP; more info at http://cba.phys.columbia.edu

Gld2 activity is regulated by phosphorylation in the N-terminal domain

The de-regulation of microRNAs (miRNAs) is associated with multiple human diseases, yet cellular mechanisms governing miRNA abundance remain largely elusive. Human miR-122 is required for Hepatitis C proliferation, and low miR-122 abundance is associated with hepatic cancer. The adenylyltransferase Gld2 catalyses the post-transcriptional addition of a single adenine residue (A + 1) to the 3ʹ-end of miR-122, enhancing its stability. Gld2 activity is inhibited by binding to the Hepatitis C virus core protein during HepC infection, but no other mechanisms of Gld2 regulation are known. We found that Gld2 activity is regulated by site-specific phosphorylation in its disordered N-terminal domain. We identified two phosphorylation sites (S62, S110) where phosphomimetic substitutions increased Gld2 activity and one site (S116) that markedly reduced activity. Using mass spectrometry, we confirmed that HEK 293 cells readily phosphorylate the N-terminus of Gld2. We identified protein kinase A (PKA) and protein kinase B (Akt1) as the kinases that site-specifically phosphorylate Gld2 at S116, abolishing Gld2-mediated nucleotide addition. The data demonstrate a novel phosphorylation-dependent mechanism to regulate Gld2 activity, revealing tumour suppressor miRNAs as a previously unknown target of Akt1-dependent signalling

Discovery of a Nova-Like Cataclysmic Variable in the Kepler Mission Field

We announce the identification of a new cataclysmic variable star in the field of the Kepler Mission, KIC J192410.81+445934.9. This system was identified during a search for compact pulsators in the Kepler field. High-speed photometry reveals coherent large-amplitude variability with a period of 2.94 h. Rapid, large-amplitude quasi-periodic variations are also detected on time scales of ~1200 s and ~650 s. Time-resolved spectroscopy covering one half photometric period shows shallow, broad Balmer and He I absorption lines with bright emission cores as well as strong He II and Bowen blend emission. Radial velocity variations are also observed in the Balmer and He I emission lines that are consistent with the photometric period. We therefore conclude that KIC J192410.81+445934.9 is a nova-like variable of the UX UMa class in or near the period gap, and it may belong to the rapidly growing subclass of SW Sex systems. Based on 2MASS photometry and companion star models, we place a lower limit on the distance to the system of ~500 pc. Due to limitations of our discovery data, additional observations including spectroscopy and polarimetry are needed to confirm the nature of this object. Such data will help to further understanding of the behavior of nova-like variables in the critical period range of 3-4 h, where standard cataclysmic variable evolutionary theory finds major problems. The presence of this system in the Kepler mission field-of-view also presents a unique opportunity to obtain a continuous photometric data stream of unparalleled length and precision on a cataclysmic variable system.Comment: Accepted for publication in the Astronomical Journal. 8 pages, 7 figures, uses emulateapj

Structural insights into RNA-dependent eukaryal and archaeal selenocysteine formation

The micronutrient selenium is present in proteins as selenocysteine (Sec). In eukaryotes and archaea, Sec is formed in a tRNA-dependent conversion of O-phosphoserine (Sep) by O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase (SepSecS). Here, we present the crystal structure of Methanococcus maripaludis SepSecS complexed with PLP at 2.5 Å resolution. SepSecS, a member of the Fold Type I PLP enzyme family, forms an (α2)2 homotetramer through its N-terminal extension. The active site lies on the dimer interface with each monomer contributing essential residues. In contrast to other Fold Type I PLP enzymes, Asn247 in SepSecS replaces the conserved Asp in binding the pyridinium nitrogen of PLP. A structural comparison with Escherichia coli selenocysteine lyase allowed construction of a model of Sep binding to the SepSecS catalytic site. Mutations of three conserved active site arginines (Arg72, Arg94, Arg307), protruding from the neighboring subunit, led to loss of in vivo and in vitro activity. The lack of active site cysteines demonstrates that a perselenide is not involved in SepSecS-catalyzed Sec formation; instead, the conserved arginines may facilitate the selenation reaction. Structural phylogeny shows that SepSecS evolved early in the history of PLP enzymes, and indicates that tRNA-dependent Sec formation is a primordial process

High speed photometry of faint Cataclysmic Variables - VII. Targets selected from the Sloan Digital Sky Survey and the Catalina Real-time Transient Survey

We present high speed photometric observations of 20 faint cataclysmic variables, selected from the Sloan Digital Sky Survey and Catalina catalogues. Measurements are given of 15 new directly measured orbital periods, including four eclipsing dwarf novae (SDSS0904+03, CSS0826-00, CSS1404-10 and CSS1626-12), two new polars (CSS0810+00 and CSS1503-22) and two dwarf novae with superhumps in quiescence (CSS0322+02 and CSS0826-00). Whilst most of the dwarf novae presented here have periods below 2 h, SDSS0805+07 and SSS0617-36 have relatively long orbital periods of 5.489 and 3.440 h, respectively. The double humped orbital modulations observed in SSS0221-26, CSS0345-01, CSS1300+11 and CSS1443-17 are typical of low mass transfer rate dwarf novae. The white dwarf primary of SDSS0919+08 is confirmed to have non-radial oscillations and quasi-periodic oscillations were observed in the short-period dwarf nova CSS1028-08 during outburst. We further report the detection of a new nova-like variable (SDSS1519+06). The frequency distribution of orbital periods of CVs in the Catalina survey has a high peak near ~80 min orbital period, independently confirming that found by Gaensicke et al (2009) from SDSS sources. We also observe a marked correlation between the median in the orbital period distribution and the outburst class, in the sense that dwarf novae with a single observed outburst (over the 5-year baseline of the CRTS coverage) occur predominantly at shortest orbital period.Comment: 17 pages, 38 figures, accepted for publication in MNRA