Characterization of the specificity and functions of the protein phosphatase Cdc14

Protein phosphorylation is perhaps the most ubiquitous posttranslational modification in eukaryotes and recent studies suggest that upwards of 75% of human proteins are phosphorylated. Many proteins are phosphorylated at multiple sites, often controlled by multiple kinases and phosphatases. Multisite phosphorylation can differentially affect the functional and regulatory cellular outcomes. For example, dephosphorylation of a protein at a particular site may inhibit nuclear localization of a protein while dephosphorylation of a different site may be necessary for enzymatic activation of a protein. Thus, multisite protein phosphorylation can complicate our understanding of the biological significance and the functional consequences of protein phosphorylation. A clearer understanding of the functional consequences associated with an individual proteins phosphorylation status requires methods that can quantitatively monitor each phosphorylation site on a protein independent of one another. Here, I present a general method for quantitatively studying multisite protein phosphorylation. I demonstrate that label-free selected reaction monitoring (SRM) mass spectrometry is comparable to conventional assays for measuring reaction rates and steady-state kinetic parameters of phosphatases and kinases. Furthermore, I demonstrate that this method can be employed to measure the activity of an enzyme towards multiple substrates in a single reaction, suggesting that this approach is a useful tool for studying substrate specificity of kinases and phosphatases. I also demonstrate that this method can be used to simultaneously measure relative rates of dephosphorylation at individual phosphorylation sites on intact protein substrates in the context of a whole cell extract. Failure to properly regulate protein phosphorylation during the cell cycle compromises genome integrity and can lead to cancer and other disease states. The protein phosphatase Cdc14 has been implicated in the general reversal of cyclin dependent kinase (Cdk) phosphorylation at the end of cell division. However, the molecular mechanisms responsible for ordered Cdk substrate dephosphorylation are poorly understood. The protein Fin1 is a multisite phosphorylated protein and is a known substrate of Cdk and Cdc14. The SRM assay affords one the ability to examine the dephosphorylation of individual phosphorylation sites on an intact protein substrate, a task that cannot be accomplished using a conventional phosphatase assay. Therefore, using the SRM assay, I examined the dephosphorylation of Fin1 in response to Cdc14 treatment. This approach to studying protein dephosphorylation revealed that Cdc14 differentially dephosphorylates Fin1 such that the three phosphoserine sites that were monitored were readily dephosphorylated while the phosphothreonine site was resistant to Cdc14 activity. Importantly, these results suggested that Cdc14 may specifically reverse only a subset of Cdk-dependent phosphorylation events. These findings sparked an investigation into the specificity of Cdc14 and eventually lead to the identification of the first experimentally defined consensus sequence for Cdc14. Using our experimentally defined consensus sequence for Cdc14, we searched the yeast proteome for potential Cdc14 substrates. From this list, I identified the protein Yen1, a Holliday junction (HJ) resolvase, as a potential Cdc14 substrate. HJs are DNA intermediates that form during homologous recombination (HR) in response to repair of DNA double strand breaks. Yen1 has been shown to resolve HJs in a cell cycle and phosphorylation dependent manner; however, the protein kinase and phosphatase responsible for modulating the resolvase activity of Yen1 in vivo were previously unidentified. Here I demonstrate that Yen1 is indeed a bona fide physiological Cdc14 substrate and that dephosphorylation of distinct clusters of Cdk sites on Yen1 by Cdc14 is important for 1) modulating the nucleocytoplasmic localization of Yen1 and 2) triggering enzymatic activation of Yen1. I show that dephosphorylation of Yen1 is essential for its biological function in recombination based repair of DNA damage. These findings suggest that Cdc14 plays a previously unknown role in DNA repair and maintenance of genome stability

A single-force model for the 1975 Kalapana, Hawaii, Earthquake

A single force mechanism is investigated as the source of long-period seismic radiation from the 1975 Kalapana, Hawaii, earthquake (M_S = 7.1). The observed Love wave radiation pattern determined from the spectra of World-Wide Standard Seismograph Network and High Gain Long Period records at 100 s is two-lobed with azimuth, consistent with a near-horizontal single force acting opposite (strike ∼330°) to the observed displacement direction of the earthquake; this pattern is inconsistent with the expected double-couple pattern. Assuming a form of the force time history of a one-cycle sinusoid, the total duration of the event estimated from Rayleigh waves at two International Deployment of Accelerometers stations is approximately 180 s. The peak amplitude f_o of the time function is 1 × 10^(15) N from amplitudes of Love and Rayleigh waves. The interpretation is that the bulk of the seismic radiation was produced by large-scale slumping of a large area of the south flank of Kilauea volcano. The single force is a crude representation of the effect on the earth of the motion of a partially decoupled large slide mass. Using the mass estimated from the tsunami generation area (∼ 10^(15)–10^(16) kg), the peak acceleration of the slide block (0.1–1 m s^(−2)) inferred from the seismic force is comparable with the acceleration due to gravity on a gently inclined plane. The slump model for the Kalapana earthquake is also more qualitatively consistent with the large horizontal deformation (8 m on land) and tsunami associated with the earthquake, which are difficult to explain with the conventional double-couple source model. The single-force source has been used previously to model the long-period seismic waves from the landslide accompanying the eruption of Mount St. Helens volcano, and may explain other anomalous seismic events as being due to massive slumping of sediments or unconsolidated material and not to elastic dislocation

Depth estimates of large earthquakes on the Island of Hawaii since 1940

Although hypocenters of earthquakes on the island of Hawaii are now routinely assigned to within 5 km, depth was a poorly determined parameter until the early 1960's. However, the 1950–1960 period was very active both in volcanic eruptions and large earthquakes. Source depths for the 12 largest Hawaiian earthquakes (magnitude 6 or greater) since 1940 are estimated from the ratios of body and surface wave amplitudes recorded at Pasadena, California. Excitation functions for Rayleigh waves are calculated as a function of source depth for the two dominant periods in the Pasadena records, 8s and 20s. Theoretical body wave amplitudes are determined from synthetic seismograms. Calculated ratios are very sensitive to source depth; for example, amplitudes of 8-s Rayleigh waves diminish by a factor of 300 between depths of 10 km and 50 km. This is a much larger effect than the fault geometry, which we estimate to be a factor of 4 between representative focal mechanisms. Estimated depths for post-1960 earthquakes agree fairly well with the instrumental depths. In general, large earthquakes near the volcanic flanks and fault systems are shallow (≤20 km). Two earthquakes of magnitude 6 occurred under the volcanoes Mauna Loa (in 1950) and Kilauea (in 1951); they preceded major eruptions by 3 days and 14 months, respectively, and had the largest depth estimates at 40–55 km and 35–50 km. MS values assigned from global amplitudes are compared with those assigned from Pasadena amplitudes alone, for 70 events in 1973–1974 with 5.1≤ M_S ≤ 6.0. The global values are only slightly larger (0.05 magnitude units) than the Pasadena values, indicating that Pasadena amplitudes are on the average representative of the event magnitude

Bacterial Active Community Cycling in Response to Solar Radiation and Their Influence on Nutrient Changes in a High-Altitude Wetland

Indexación: Web of Science; Scopus.Microbial communities inhabiting high-altitude spring ecosystems are subjected to extreme changes in solar irradiance and temperature throughout the diel cycle. Here, using 16S rRNA gene tag pyrosequencing (cDNA) we determined the composition of actively transcribing bacteria from spring waters experimentally exposed through the day (morning, noon, and afternoon) to variable levels of solar radiation and light quality, and evaluated their influence on nutrient recycling. Solar irradiance, temperature, and changes in nutrient dynamics were associated with changes in the active bacterial community structure, predominantly by Cyanobacteria, Verrucomicrobia, Proteobacteria, and 35 other Phyla, including the recently described Candidate Phyla Radiation (e.g., Parcubacteria, Gracilibacteria, OP3, TM6, SR1). Diversity increased at noon, when the highest irradiances were measured (3.3-3.9 H', 1125 W m(-2)) compared to morning and afternoon (0.6-2.8 H'). This shift was associated with a decrease in the contribution to pyrolibraries by Cyanobacteria and an increase of Proteobacteria and other initially low frequently and rare bacteria phyla (< 0.5%) in the pyrolibraries. A potential increase in the activity of Cyanobacteria and other phototrophic groups, e.g., Rhodobacterales, was observed and associated with UVR, suggesting the presence of photo activated repair mechanisms to resist high levels of solar radiation. In addition, the percentage contribution of cyanobacterial sequences in the afternoon was similar to those recorded in the morning. The shifts in the contribution by Cyanobacteria also influenced the rate of change in nitrate, nitrite, and phosphate, highlighted by a high level of nitrate accumulation during hours of high radiation and temperature associated with nitrifying bacteria activity. We did not detect ammonia or nitrite oxidizing bacteria in situ, but both functional groups (Nitrosomona and Nitrospira) appeared mainly in pyrolibraries generated from dark incubations. In total, our results reveal that both the structure and the diversity of the active bacteria community was extremely dynamic through the day, and showed marked shifts in composition that influenced nutrient recycling, highlighting how abiotic variation affects potential ecosystem functioning.http://journal.frontiersin.org/article/10.3389/fmicb.2016.01823/ful

Source characteristics of earthquakes in the Michoacan seismic gap in Mexico

We investigated the source characteristics of large earthquakes which occurred in the Michoacan, Mexico, seismic gap during the period from 1981 to 1986 in relation to historical seismicity in the region. The rupture pattern of the Michoacan gap during this period can be characterized by a sequential failure of five distinct asperities. Before 1981, the Michoacan gap had not experienced a large earthquake since 1911 when an M_S = 7.8 earthquake occurred. The recent sequence started in October 1981 with the Playa Azul earthquake which broke the central part of the gap. Body-wave modeling indicates that the Playa Azul earthquake is 27 km deep with a seismic moment of 7.2 × 10^(27) dyne-cm. It is slightly deeper than the recent Michoacan earthquakes, and its stress drop is higher, suggesting a higher stress level at depths in the Michoacan gap. The seismic moment of the 19 September 1985 (M_w = 8.1) earthquake was released in two distinct events, with the rupture starting in the northern portion of the seismic gap and propagating to the southeast with low moment release through the area already broken by the 1981 Playa Azul earthquake. The rupture propagated further southeast with an M_w = 7.5 event on 21 September 1985. Another aftershock occurred on 30 April 1986 to the northwest of the 19 September main shock. Body-wave modeling indicates that this event has a simple source 10 sec long at 21 km depth, and fault parameters consistent with subduction of the Cocos plate (ϑ = 280°, δ = 12°, and λ = 70°) and M_0 = 2.0 to 3.1 × 10^(26) dyne-cm (M_w = 6.8 to 6.9). Although this distribution of asperities is considered characteristic of the Michoacan gap, whether the temporal sequence exhibited by the 1981 to 1986 sequence is also characteristic of this gap or not is unclear. It is probable that, depending on the state of stress in each asperity, the entire gap may fail in either a single large event with a complex time history or a sequence of moderate to large events spread over a few years. The seismic moment and the time since the last earthquake in Michoacan (in 1911) fit an empirical relation between moment and recurrence time found for the Guerrero-Oaxaca region of the Mexico subduction zone

Reply [to “Comment on ‘A single-force model for the 1975 Kalapana, Hawaii, earthquake’ by Holly K. Eissler and Hiroo Kanamori”]

We showed that the long-period seismic radiation from the November 29, 1975, Kalapana Hawaii earthquake, which involved seaward displacement of the south flank of Kilauea volcano, was best explained by invoking a near-horizontal single force as the kinematic source of the earthquake [Eissler and Kanamori, 1987]. In particular, the azimuthal dependence of 100-s Love surface waves is difficult to explain by a conventional double-couple source. The unusual Love wave pattern was noted by Ando [1979] but not explained until our suggestion of the single-force source model

Tectonic setting and source parameters of the September 19, 1985 Michoacan, Mexico earthquake

Analysis of body waves and long-period surface waves from the September 1985 earthquake in coastal Michoacan, Mexico shows that the event was an interplate subduction event with a low dip angle fault plane (δ=9°) striking parallel to the Mid-America trench (ϕ=288°) and a small component of left lateral motion (λ=72°) with a point source depth of 17 km, and a seismic moment in excess of 1 × 10^(28) dyn cm. The earthquake was a multiple event, with a second source of identical moment, fault geometry, and depth occurring approximately 26 s after the first. Directivity in the body wave time function indicates that the second event occurred roughly 100 km to the southeast of the first. This suggests that the earthquake first broke the northern portion of the Michoacan gap, propagated with low moment release through the rupture zone of the 1981 Playa Azul earthquake, and then broke the remaining asperity in the southern section of the gap. The seismic moment determined from Rayleigh and Love waves is between 1.0 - 1.7 × 10^(28) dyn cm (M_W = 7.9 - 8.1), the largest moment determined to date for a Mexico subduction earthquake. Comparison of seismograms at Pasadena with records of other large Mexico events shows that the Michoacan earthquake is basically the same size as the 1932 Jalisco, Mexico earthquake, and clearly larger than other significant events in Mexico since 1932. The seismic moment and the time since the last large earth-quake in Michoacan (in 1911) fit an empirical relation between moment and recurrence time found for the Guerrero-Oaxaca region of the subduction zone. The large aftershock on September 21 (M_s=7.5) has the same geometry as the mainshock, a somewhat larger source depth (22 km), a simple time function, and a seismic moment between 2.9 - 4.7 × 10^(27) dyn cm (M_w = 7.6 - 7.7)

Prioritizer-in-chief : the role of the president in the policy process from Reagan to Obama

This dissertation sets out a fresh approach to understanding presidential decision-making by connecting the presidency to information processing theories. This approach to behavioral choice highlights how the structure of the presidency creates a decision-making process that relies on the cognitive and emotional capacities of the individuals in the office, while the political and policy environment put pressures on their choices. Once presidents have decided to get involved in policy making, they have to process information about the responsibilities of the office, the policy and political environment, as well as their own political strength, to make decisions about what policy areas to prioritize and what strategies they should use to pursue those policy goals. To examine those decisions and understand the forces that shape them, I analyze ten datasets of presidential actions, seven of which are original to this project: presidential press conferences, budget messages, State of the Union addresses, major televised addresses, addresses to a joint session of Congress, proclamations, memoranda, signing statements, executive orders, and veto threats. By examining these datasets of presidential policy action, from Ronald Reagan (1981) to Barack Obama (2014), we gain a clearer insight into the decisions that presidents make about the policy process, their strategies, and the factors that affect their abilities to make trade-offs between their policy priorities and strategies. This dissertation makes a contribution to the presidency and policy process literatures by moving towards a empirically-grounded study of the presidency, one which relies on the combination of theory and data to better understand the decisions that presidents make and the factors that shape those decisions.Governmen

Investigations of Earthquakes and Other Seismic Sources in Regions of Volcanism

Source properties of earthquakes in Hawaii and seismological aspects of explosive volcanic eruptions are examined in three chapters. In Chapter 1, source depths are estimated for all earthquakes larger than magnitude 6 on the island of Hawaii since 1940 by comparing relative amplitudes of short-period surface waves to body waves. Rayleigh wave excitation functions are calculated versus source depth, and the calculation is compared with observed data and calibrated using known depths of recent earthquakes. In general, results show that large earthquakes near the volcanic flanks and fault systems are shallow (≤ 20 km), but those near active volcanic centers can be deeper (~ 50 km). Two earthquakes with the largest depth estimates (40-55 km and 35-50 km) occurred under the active volcanoes Mauna Loa and Kilauea, preceding eruptions by three days and 14 months respectively. As a check on the data set, which consisted of Pasadena seismograms alone, M_s values assigned from many global amplitude readings were compared with those from Pasadena amplitudes for worldwide earthquakes. Global M_s values on the average are 0.05 magnitude units larger than M_s values from Pasadena amplitudes. In Chapter 2, the horizontal single-force source used to model seismic radiation from the Mt. St. Helens landslide is investigated as the source of the M_s = 7.1 Kalapana, Hawaii earthquake. The azimuthal radiation pattern of 100 s Love waves is two-lobed, consistent with a horizontal single-force source. The observed surface deformation is also more consistent with the single force than the conventional double-couple shear dislocation source. The single force is a crude representation of motion of a large slide mass that is partially decoupled from the Earth. The interpretation is that the bulk of seismic radiation from the Kalapana earthquake was produced by large-scale slumping of the south flank of Kilauea volcano. The peak amplitude f₀ of the force time function is estimated at 1 x 10²⁰ dyne from Love and Rayleigh surface waves. The peak acceleration inferred from the seismic force is 10 - 100 cm s⁻², comparable to that of gravity on a gently inclined plane. In Chapter 3, far-field seismograms were searched for signals associated with recent large volcanic eruptions to examine whether models of the volcano as a seismic source derived for Mt. St. Helens are applicable to other explosive volcanoes. The 1982 eruption of El Chich6n in Mexico produced Rayleigh waves and body waves that were marginally recorded at IDA and SRO stations less than 40° away; still, several characteristics of the eruption can be inferred from the seismic waves. Near-field seismograms of smaller eruptions at Mt. Asama, Japan, were found to be comparable in size to smaller secondary eruptions of Mt. St. Helens, and appear to have a more complicated source. Atmospheric pressure waves recorded on barographic instruments from several large explosive eruptions are compared and show differences in signal duration, amplitude, and characteristic period that are indicative of the overall size of the eruption.</p

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