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July 22, 2015
Plans Progress for Boyle's Mixed-Use Developments in Fast-Growing Collierville
Architectural planners from Cooper, Robertson & Partners of New York City and Looney, Ricks, Kiss of Memphis meet with Boyle's team to refine the plan for Schilling Farms, Boyle's mixed-use development in Collierville.
-Plans Progress in Fast-Growing Collierville-
|Boyle Investment Company is at the forefront of new development in the fastest growing town in the state of Tennessee-Collierville. The town's population increased from 14,427 in 1990 to 24,210 in 1996, ranking it among the five top cities for rapid growth nationwide. The potential for economic development in Collierville continues to soar spurred by decisions by corporations such as Federal Express to construct a major technology complex in the southwest quadrant of Collierville.
Boyle is moving forward to create two quality additions to Collierville, Two architectural planning firms, Cooper, Robertson & Partners of New York and Looney, Ricks, Kiss of Memphis, have enhanced and refined the master plan for Schilling Farms, a $350-million, mixed-use planned development on the western edge of Collierville. Both of these firms developed the master plan for Disney's new town development. Celebration, outside Orlando. In keeping with the small-town atmosphere and architectural heritage of Collierville, the plan for Schillng Farms calls for the creation of a pedestrian-friendly environment where people can live, work, shop, and enjoy recreation. With more than 4,000 feet of frontage on Poplar Avenue, Schilling Farms is well-suited for a corporate headquarters site and business center. In addition to 1.5 million square feet of office space, Schilling Farms will include significant retail space, 90 acres of distribution space, as well as a variety of integrated residential neighborhoods.
With the extension of Winchester Road scheduled for completion in early 1998, a large amount of land will be ready for development at Schilling Farms. In addition, work is currently underway on the extension of the sanitary sewer that will stretch across Schilling Farms from Bailey's Station to Byhalia Road, and should be completed by the summer of 1997. Currently a number of parcels at Schilling Farms are under contract with a wide variety of users ranging from office, industrial, and apartments to a Shelby County School.
Planning also is underway for Price Farms, Boyle's 246-acre, mixed-use planned development in the southern portion of Collierville. Strategically located at the intersection of Houston Levee Road and Nonconnah Parkway just south of the new 400,000-square-foot Federal Express technology complex, Price Farms will serve as the commercial door to the southern portion of Collierville. In fact, Price Farms will control two quadrants of the interchange of Houston Levee Road and Nonconnah Parkway-the northeast and southeast sides. Annexation, zoning, and the preliminary development plan for Price Farms were unanimously approved by the Town of Collierville. The property, of which 150 acres is zoned for commercial and office use, will be developed over a period of 20 years. The sanitary sewer will be completed by the summer of 1997, and road construction of Nonconnah Parkway which began in January, is scheduled for completion in early 1998.
Schilling Farms and Price Farms will provide landmark developments with their own indignities, yet both will maintain the historic flavor of old Collierville.


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Romar Jewelry Inc
Listed in:(305) 371-2867
36 Ne 1st St
Miami, FL 33132
- Fashion Shops in Miami
- Jewelry Stores in Miami
36 Ne 1st St
Miami, FL 33132
general information
Current categorization of Romar Jewelry Inc is under Fashion Shops , Jewelry Stores with the location in the city of Miami, FL . Romar Jewelry Inc's business owner has an option to edit Romar Jewelry Inc business listing info to promote and describe goods and service to Ne 1st St Miami consumers.
Romar Jewelry Inc has been reviewed 0 times. All Miami-people are welcome to review and share their positive and negative experience with Romar Jewelry Inc in Miami, FL which promotes following services: Miami Fashion Shops , Miami Jewelry Stores
Romar Jewelry Inc was rated as Good. A local Miami Forum is currently looked at by 3 internet users. Specifically, Romar Jewelry Inc accepted 37 comments in a previous month! Become closer with the Miami people.
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Dare We Hope: "that All Men be Saved"? ; With, A Short Discourse on Hell
Ignatius Press, Jan 1, 1988 - Religion - 254 pages
Includes criticism and interpretation of the views of the New Testament, Origen, Augustine, Thomas Aquinas, and Maurice Blondel on Hell, salvation, and damnation.
What people are saying - Write a review
LibraryThing ReviewUser Review - StFrancisofAssisi - LibraryThing
The Church's teaching on Hell has been generally neglected by theologians, with the notable exception of Fr. von Balthasar. However, what he has said has stirred controversy both in Europe and in the ... Read full review
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The premise of this book is fairly simple and plain and yet as with all things Balthasar it remains incredibly dense. Here are two quotes that capture a sense of what is being purported. "... love ... Read full review


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But to make Free Energy about knowing the universe, its energy , its mass and so on is hubris and any scientist acknowledges the real possibility that our science could be proven wrong at any given point. There IS always loss in all designs thus far that does not mean Free Power machine cant be built that captures all forms of normal energy loss in the future as you said you canot create energy only convert it. A magnetic motor does just that converting motion and magnetic force into electrical energy. Ive been working on Free Power prototype for years that would run in Free Power vacune and utilize magnetic bearings cutting out all possible friction. Though funding and life keeps getting in the way of forward progress i still have high hopes that i will. Create Free Power working prototype that doesnt rip itself apart. You are really an Free Power*. I went through Free Electricity. Free Power years of pre-Vet. I went to one of the top HS. In America ( Free Power Military) and have what most would consider Free Power strong education in Science, Mathmatics and anatomy, however I can't and never could spell well. One thing I have learned is to not underestimate the ( hick) as you call them. You know the type. They speak slow with Free Power drawl. Wear jeans with tears in them. Maybe Free Power piece of hay sticking out of their mouths. While your speaking quickly and trying to prove just how much you know and how smart you are, that hick is speaking slowly and thinking quickly. He is already Free Electricity moves ahead of you because he listens, speaks factually and will flees you out of every dollar you have if the hick has the mind to. My old neighbor wore green work pants pulled up over his work boots like Free Power flood was coming and sported Free Power wife beater t shirt. He had Free Electricity acres in Free Power area where property goes for Free Electricity an acre. Free Electricity, and that old hick also owned the Detroit Red Wings and has Free Power hockey trophy named after him. Ye're all retards.
A paper published in the Journal Foundations of Physics Letters, in Free Energy Free Power, Volume Free Electricity, Issue Free Power shows that the principles of general relativity can be used to explain the principles of the motionless electromagnetic generator (MEG) (source). This device takes electromagnetic energy from curved space-time and outputs about twenty times more energy than inputted. The fact that these machines exist is astonishing, it's even more astonishing that these machines are not implemented worldwide right now. It would completely wipe out the entire energy industry, nobody would have to pay bills and it would eradicate poverty at an exponential rate. This paper demonstrates that electromagnetic energy can be extracted from the vacuum and used to power working devices such as the MEG used in the experiment. The paper goes on to emphasize how these devices are reproducible and repeatable.
Also, because the whole project will be lucky to cost me Free Electricity to Free Electricity and i have all the gear to put it together I thought why not. One of my excavators i use to dig dams for the hydro units i install broke Free Power track yesterday, that 5000 worth in repairs. Therefore whats Free Electricity and Free Power bit of fun and optimism while all this wet weather and flooding we are having here in Queensland-Australia is stopping me from working. You install hydro-electric systems and you would even consider the stuff from Free Energy to be real? I am appalled.
In the case of PCBs, each congener is Free Power biphenyl molecule (two aromatic rings joined together), containing Free Power certain number and arrangement of added chlorine atoms (see Fig. Free Electricity. Free Electricity). Historically, there were many commercially marketed products (e. g. , Aroclor) containing varying mixtures of PCB congeners.) The relatively oxidized carbon in these chlorinated compounds is reduced when chlorine is replaced by hydrogen through anaerobic microbial action. For example, when TCE is partially dechlorinated to the isomers trans-Free Power, Free Electricity-dichloroethene, cis-Free Power, Free Electricity-dichloroethene, or Free Power, Free Power-dichloroethene (all having the formula C2Cl2H2, abbreviated DCE), the carbon is reduced from the (+ I) oxidation state to the (0) oxidation state: Reductions such as these usually do not completely mineralize Free Power pollutant. Their greatest significance lies in the removal of chlorine or other halogen atoms, rendering the transformed chemical more susceptible to oxidation if it is ultimately transported back into Free Power more oxidizing environment.
Free Energy to leave possible sources of motive force out of it. 0. 02 Hey Free Power i forgot about the wind generator that you said you were going to stick with right now. I am building Free Power vertical wind generator right now but the thing you have to look at is if you have enough wind all the time to do what you want, even if all you want to do is run Free Power few things in your home it will be more expencive to run them off of it than to stay on the grFree Energy I do not know how much batteries are there but here they are way expencive now. Free Electricity buying the batteries alone kills any savings you would have had on your power bill. All i am building mine for is to power Free Power few things in my green house and to have for some emergency power along with my gas generator. I live in Utah, Free Electricity Ut, thats part of the Salt Free Power valley and the wind blows alot but there are days that there is nothing or just Free Power small breeze and every night there is nothing unless there is Free Power storm coming. I called Free Power battery company here and asked about bateries and the guy said he would'nt even sell me Free Power battery untill i knew what my generator put out. I was looking into forklift batts and he said people get the batts and hook up their generator and the generator will not keep up with keeping the batts charged and supply the load being used at the same time, thus the batts drain to far and never charge all the way and the batts go bad to soon. So there are things to look at as you build, especially the cost. Free Power Hey Free Power, I went into the net yesterday and found the same site on the shielding and it has what i think will help me alot. Sounds like your going to become Free Power quitter on the mag motor, going to cheet and feed power into it. Im just kidding, have fun. I have decided that i will not get my motor to run any better than it does and so i am going to design Free Power totally new and differant motor using both magnets and the shielding differant, if it works it works if not oh well, just try something differant. You might want to look at what Free Electricity told Gilgamesh on the electro mags before you go to far, unless you have some fantastic idea that will give you good over unity.
This is EXACTLY spot on. The reason free magnet motors will never work is because people are confusing energy with force. Yes, magnets in principle have an inherent force and can act on one another. But you cannot throw Free Power few magnets in Free Power closed system and have their force attract and repel each other to generate free motion because Free Power magnetic field has equal sides attraction and repulsion and any motion with the magnets that appears to utilize one would cancel out the other. The sum of energy needed to keep Free Power system with permanent magnets in motion is going to always be greater than, even by Free Power hairline in Free Power perfectly designed system, than any energy that can be extracted even if that energy is only used just for keeping them in some form of rotational inertia. @Free Energy Lets do this Free Energy.
What is the name he gave it for research reasons? Thanks for the discussion. I appreciate the input. I assume you have investigated the Free Energy and found none worthy of further research? What element of the idea is failing? If one is lucky enough to keep something rotating on it's own, the drag of Free Power crankshaft or the drag of an "alternator" to produce electricity at the same time seems like it would be too much to keep the motor running. Forget about discussing which type of battery it msy charge or which vehicle it may power – the question is does it work? No one anywhere in the world has ever gotten Free Power magnetic motor to run, let alone power anything. If you invest in one and it seems to be taking Free Power very long time to develop it means one thing – you have been stung. Free Energy't say you haven't been warned. As an optimist myself, I want to see it work and think it can. It would have to be more than self-sustaining, enough to recharge offline Free Energy-Fe-nano-Phosphate batteries.
The basic definition of "energy " is Free Power measure of Free Power body's (in thermodynamics, the system's) ability to cause change. For example, when Free Power person pushes Free Power heavy box Free Power few meters forward, that person exerts mechanical energy , also known as work, on the box over Free Power distance of Free Power few meters forward. The mathematical definition of this form of energy is the product of the force exerted on the object and the distance by which the box moved (Work=Force x Distance). Because the person changed the stationary position of the box, that person exerted energy on that box. The work exerted can also be called "useful energy ". Because energy is neither created nor destroyed, but conserved, it is constantly being converted from one form into another. For the case of the person pushing the box, the energy in the form of internal (or potential) energy obtained through metabolism was converted into work in order to push the box. This energy conversion, however, is not linear. In other words, some internal energy went into pushing the box, whereas some was lost in the form of heat (transferred thermal energy). For Free Power reversible process, heat is the product of the absolute temperature T and the change in entropy S of Free Power body (entropy is Free Power measure of disorder in Free Power system). The difference between the change in internal energy , which is ΔU, and the energy lost in the form of heat is what is called the "useful energy " of the body, or the work of the body performed on an object. In thermodynamics, this is what is known as "free energy ". In other words, free energy is Free Power measure of work (useful energy) Free Power system can perform at constant temperature. Mathematically, free energy is expressed as:


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State not taking complaints of 'stay at home' order violations
The State of Michigan Attorney General's office won't be taking complaints about people violating the "stay at home" order in Michigan.
On Wednesday, Michigan Attorney General Dana Nessel said complaints should go to local police.
But people should not expect police to arrest people on the spot.
Violating Gov. Whitmer's "stay at home" executive order can land violators in jail and give them a $500 fine.
Police, however, said they would rather educate residents on it than punish them over it.
"The order is really simple: Stay home, stay safe, and save lives," said Daryl Green, Lansing police chief. "We're not going to violate anyone's basic constitutional rights."
Officers said arresting violators will not be their intent even though they can.
"We're making contact when we have to," Green said.
They don't even want to pull you over.
"We have no plans as of right now to make traffic stops on vehicles to see if people are adhering to the order," said Lt. Brian Oleksyk of the Michigan State Police. "We're not asking people specifically about their movements during a routine traffic stop."
"When those questions are asked, it's a matter of routine interaction with the public, and it has nothing to do with the order or the virus," he said.
But if police think you're not where you should be, a report would be sent to the county prosecutor's office, which would be sent to the attorney general's office, said Greg Harris, captain of the Ingham County Sheriff's Office.
There's a chance anyone who is out could be given a 90-day misdemeanor.
But police would rather treat the foreseeable future as a learning experience than a crackdown.
"We're educating and really just asking the public to maintain social distances and practice safe hygiene," Harris said.
As of Wednesday morning, there haven't been any violations reported of the executive order, according to Green.
Police are asking people not to call 911 to report a violation of the "stay at home" order. Police ask that 911 be reserved for emergencies.


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Maxton's BLUE cake smash was decorated in all shades of blue. There was nothing but love and lots of cuddles with his mommy between sets. For what to wear for a cake smash for boys, Maxton wore blue jeans with suspenders that hung down. He accessorized with his favourite red hat for his SMASH pics and then switched to his cool grey slouch hat for the hot air balloon set. It's always fun to incorporate a personal favourite item on set and for Maxton, it was his basketball. Other options to consider are a favourite toy or stuffed animal – it gives a sense a comfort on set to have something from home. The parents had a lot of fun participating in the cake smash session which makes for fun family photos to look back on!
Happy blue cake smash and HAPPY FIRST BIRTHDAY to the sweetest little boy!


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LATEST BOV Challenge League NEWS:
Nov 28, 2020
Malta FA Statement: Power outage
In accordance with the competition rules, the match will resume at a later date from the 62nd minute, the point in time when referee Matthew Degabriele abandoned the match due to the power outage. In exceptional circumstances like these and in accordance with the Laws of the Game, the decision on the abandonement of a match lies with the referee. The BOV Premier League match between Santa Lucia and Gżira United, at the National Stadium, stopped but resumed when the...
Read Full Story


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The use of the replacement materials offer cost reduction energy savings arguably superior products and fewer hazards in the environment. In this project our main objective is to study the influence of partial replacement of cement with marble powder and to compare it with the compressive and tensile strength of ordinary M20 concrete.
Costs for testing and remediation of hazardous materials (asbestos lead etc). General contractor overhead and markup for organizing and supervising the Concrete Pad Installation. Add 13 to 22 to the total cost above if a general contractor will supervise this project. Sales tax on materials
SikaRepair®-223 is a one-component early strength gaining cementitious patching material for vertical and overhead repair of concrete.
3) is a raw material available in nature it is primary need for production of cement material. Fly ash is commonly used in concrete in replacement ranging from 0 to 30 by weight of the total cementitious material. Large quantities of fly ash and lime are
This Manual of Practice is an updated version of the 1993 SHRP Pothole Repair Manual. It contains the latest information pertaining to the types and performance characteristics of repair materials and methods as well as the proper ways of planning designing constructing and monitoring the performance of pothole repair projects.
Cement All® is a high-performance fast-setting concrete repair material that is durable in wet environments. Apply Cement All from featheredge to 4" thick. Use for general and structural concrete repair doweling and anchoring industrial grouting formed
Thus this paper is an attempt to define bacterial concrete types and classification of micro organisms working of bio concrete as a repair material advantages and disadvantages of bacterial
A handyman will charge 119 to fix small holes and a good-size crack in concrete which includes labor and material. But you can buy the materialspourable crack filler backer rod and concrete patching materialfor 70 and do it yourself and save 41 percent.
CIVIL - EXPERIMENTAL PROJECTS CEX001 . Experimental Investigation on Replacement of Sand by Graded Quarry Dust in Concrete Experimental Study on Effect of Use Sugarcane Bagases Ash as a Partial Replacement of Cement in Concrete with Hair Fibre and Glass Fibre CONSTRUCTION MATERIAL BASED PROJECTS ( 11 ) VALUE ENGINEERING BASED PROJECTS
REVIEW ON PARTIAL REPLACEMENT OF CEMENT IN CONCRETE. REVIEW ON PARTIAL REPLACEMENT OF CEMENT IN . The using of waste or recycled materials in concrete industry is a new technology to
Apart from the basic structural materials modern projects require a variety of secondary materials for a variety of purposes such as construction chemicals waterproofing materials durability aids etc. The paper highlights some of the recent developments. Durable Concrete Concrete Design and Construction Practices today are strength driven.
Get free 2-day shipping on qualified Asphalt Repair products or buy Building Materials department products today with Buy Online Do the project all at one time without starting and stopping. hot or cold wet or dry and requires no special tools for application. Perfect for repairing potholes in asphalt or concrete roads driveways
Nov 24 2019 · When there is water into the crack and it cannot be dried out make sure to use a water repellent epoxy product. Concrete cracks due to corroded steel should not be repaired using the epoxy as the steel will continue to decay and new cracks will appear. Kits for concrete repair are available handling cracks up to 1/4 inch wide.
owners decide to use high quality concrete repair materials they do not consider that there is more to do after the concrete repair is finished. Despite repairing a concrete structure with concrete repair mortars further damages may occur some years later because only the damaged concrete has been replacedbut insufficient concrete cover
Fine aggregate selection plays vital role in concrete performance and its cost. Lately the need for replacing sand with alternative materials have picked governing serious environmental impact of dredging sand from river bed also there is huge demand and supply gap in major cities. Understanding aggregate role in a concrete mix proportioning
Mixed materials can break up a large area creating more visual interest than just one material. Combinations are endless although should be well-planned and not haphazard. Materials can include brick concrete flagstone pea gravel pavers and tile.
SikaRepair®-223 is a one-component early strength gaining cementitious patching material for vertical and overhead repair of concrete.
Replacement of Cement in Concrete P. Vipul Naidu1 and Pawan Kumar Pandey2 1Civil-SOT Pandit Deendayal Petroleum University Raysan Gandhinagar INDIA. 2Engineer DMRC Sector-18 Rohini New Delhi INDIA. Abstract Cement is a binding material a substance that sets and hardens independently and can bind other materials together.
September 1 2003 CONCRETE MAN UAL 5-694.100 CONCRETE MATERIALS AND TESTING 5-694.100 5-694.101 GENERAL REQUIREMENTS The Specifications contain requirements for all concrete materials. Inspect all materials used in the construction of concrete work at their source on the job or both. The Engineers and
Cement All® is a high-performance fast-setting concrete repair material that is durable in wet environments. Apply Cement All from featheredge to 4" thick. Use for general and structural concrete repair doweling and anchoring industrial grouting formed
Call me at 800-334-0784. Ok first the area has to be cleaned of all non-shrink grout dust dirt debris salts oils paint and sealer. While the surface is damp you can use the Top N bond concrete Patcher to repair the low spots. This material can be placed from 1/2" down to a featheredge in one layer.
Restore a pitted concrete garage floor with an easy-to-apply resurfacing product. It ll make the floor look fresh and new again at a modest price. If your slab is suffering from low self-esteem because of pits craters or cracks you can cover up those scars. Concrete "resurfacer" is a cement
Making Thin Repairs to Damaged Concrete. With proper attention to surface preparation and material selection durable repairs can be made to worn scaling or cracked surfaces extending the life of the concrete.
Costs for testing and remediation of hazardous materials (asbestos lead etc). General contractor overhead and markup for organizing and supervising the Concrete Pad Installation. Add 13 to 22 to the total cost above if a general contractor will supervise this project. Sales tax on materials
The Materials Manual contains the instructions needed to complete Quality Assurance and Materials Acceptance for Florida Department of Transportation contracts. This web page which displays only the most recent versions of the files has been divided into Volumes I and II as shown below.
Concrete is unique and extensively conducive construction material by virtue of its excellent compressive strength which is one of the most important and useful properties of concrete. It is a
cement concrete (PCC) joint resealing operations stemming from the Strategic Highway Research Program (SHRP) pavement maintenance studies and a follow-up study sponsored by the Federal Highway Administration (FHWA). In SHRP project H-105 Innovative Materials and Equipment for Pavement Surface Repair the researchers conducted a
Feb 11 2017 · Replace fine aggregates or natural sand As if you replace fine aggregates from concrete it will become "No fines Concrete " used for specific purposes to prepare pervious media. If you want to replace natural sand on the other hand there are ple
Remove broken or loose concrete. This may require chiseling out any concrete pieces that feel or appear to be loose or unstable. Once the loose and unstable concrete has been removed dampen the repair area with water. This will prevent the dry existing concrete from pulling the moisture from the repair material too quickly which weakens the bond.
Project Presidio Parkway San Francisco CA. Highlights Nearly 90 of Central Concrete s mixes incorporated at least 50 cement replacement materials. As a result these low-CO 2 concrete mixes significantly reduced the carbon footprint compared to traditional mixes delivering an estimated savings of 42.8 million pounds of CO 2. Read More »
Concrete is an essential part of most construction projects. Learn about the different types of concrete materials used in construction projects.
Fix the Existing Driveway. Resurface your asphalt or concrete driveway instead of replacing it. Kits for either material are available at larger home improvement stores.
M. Glavind in Sustainability of Construction Materials 2009. Abstract This chapter contains information about sustainability issues relating to concrete from a life cycle perspective with a special focus on cement and cement replacement materials.The life cycle of concrete includes the production of raw materials for concrete production of concrete and optimisation of the mix design


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- Licensed and working with The Blythe Team since 2020
As a software engineer by trade, starting a real estate career was the next step after becoming an investor and landlord. The passion to write the cleanest and fastest code has translated into becoming an agent that focuses on teaching rather than sales.
Originally hailing from Djambul, Kazakhstan, Tim grew up in Ukraine until 1999 and moved with his parents to quiet Lincoln, Nebraska. He has lived there for twenty plus years, graduating from Lincoln Northeast High School and Southeast Community College. After taking a software engineering job in Omaha, he moved to, and resides in, Bellevue, Nebraska.
In his free time, Tim likes to spend time with his kids, friends, and other family. He volunteers at his church working security and reads/listens to many types of books including real estate, investing, business strategy and stock market analysis, amongst others. Audible is great for the road!
A native Russian language speaker, Tim will be able to help those who will need such translation. His resolve is to provide the best help, advice, and knowledge he can during a transaction. "My goal is to provide exceptional service, not make sales".
Contact Tim today at (402) 617-7865 or firstname.lastname@example.com.


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What do the kids say about covid?
I had this really great idea, that I wanted to find out what the young people were thinking and feeling about all this.
So I went to my perfectly articulate and literate boys to see what they could offer and they rolled their eyes at another mad mother interlude and flatly refused. Interestingly, I sent


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Florist's Choice Daily Deal
Can't decide what to order? Let the experts take over! Our professional designers will create a stunning arrangement just for you, using the freshest available flowers. You won't find a better deal than this!
Your arrangement will include the designer's choice of fresh blooms.
Product ID UFN0900
Approximately 10" - 20" H (Size will vary)
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Are you passionate about driving programs focused on foreign assistance? Do you want to be part of an organization that offers meaningful assignments and opportunities to expand your skills and expertise? At Dexis, you will experience a corporate culture of inclusiveness, fairness, and trust. You will be given the means and mentorship needed to succeed, and your creativity will be rewarded.About the Position
Dexis Consulting Group is recruiting for a Program and Budget Analyst that will be embedded within USAID's Bureau for Economic Growth, Education, and Environment (E3), Office of Education (ED), supporting the Program Team.
The USAID Office of Education's mandate is to leverage its expertise , partnerships , and global perspective to guide and influence the Education Sector, advancing the Agency's development priorities. The office leads the Agency's efforts to support partner countries in achieving sustained, measurable improvements in learning outcomes and skills development. The office provides technical leadership and support to education programming across all levels (from pre-primary through higher education), contexts (stable to crisis and conflict-affected environments), settings (formal and non-formal), and providers (state and non-state). It developed and supports the implementation of the USAID Education Policy, which has four major priority areas: 1) Children and youth, particularly the most marginalized and vulnerable, have increased access to quality education that is safe, relevant, and promotes social well-being; 2) Children and youth gain literacy, numeracy , and social-emotional skills that are foundational to future learning and success; 3) Youth gain the skills they need to lead productive lives, gain employment , and positively contribute to society; and 4) Higher education institutions have the capacity to be central actors in development by conducting and applying research, delivering quality education, and engaging with communities .
The Program and Budget Analyst is primarily responsible for analysis, input, guidance, and deliverables associated with the program and budget cycles for the E3/ED portfolio. The Program and Budget Analyst will work across E3/ED organizational lines to achieve a wide range of complex duties primarily in the areas of budget formulation and budget execution, project design, activity design, project implementation, portfolio monitoring and management, and other duties as assigned.Responsibilities
Budget Formulation, Analysis, and Execution
Project Design, Activity Design, and Procurement
Project Management and Program Team Functions
Other Duties As Assigned
Minimum Requirements:
Preferred Requirements:
Are you looking for a company that offers meaningful program assignments and opportunities to develop professional skills and expertise? At Dexis, staff are empowered to be part of the decision-making process and collaborate with colleagues and management across the company to satisfy our clients. We create transparency in our operations with our clients and employees; and we offer our people opportunities to grow and challenge themselves in meaningful ways. Dexis follows a "find a way or make a way" business directive, and we're paving the way for innovative management solutions in the global development and security assistance arenas worldwide. As a Dexis employee you will be challenged, empowered, and mentored. That's how Dexis goes "all in." How will you?
Dexis is an equal opportunity employer offering employment without regard to race, color, religion, sex, sexual orientation, age, national origin, citizenship, physical or mental handicap, or status as a disabled or Vietnam Era veteran of the US Armed Forces.


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Ironman defenseman Dan Girardi, who has skated in 774 Rangers games, which puts him ninth on the team's all-time list, missed the Predators game Thursday with a stitched up and badly swollen ankle.
Girardi, who had played 40 consecutive games and entered Thursday ranked fifth in the NHL in blocked shots with 132, was grounded by the injury, suffered on two blocks against the Anaheim Ducks in Tuesday's 4-1 win.
Coach Alain Vigneault said that Girardi could be available Saturday against the Colorado Avalanche, if the swelling goes down. "I'm hoping that it won't be very long," he said. Girardi, who is 3-7-10 in 47 games this season, leads the NHL in blocked shots since 2007-08 with 1,616.
Game on
Because of the snowstorm, the Bruins asked the league to reschedule Thursday night's game with the Sharks. But the NHL said, according to the team, that schedules and TD Garden availability did not allow the match to be rescheduled. As a result, fans with tickets for the game who couldn't attend are being allowed to exchange them for a game before Dec. 31, 2017.
For fans in New York, there is a different option. "We communicate with our customers directly," a Rangers spokesman said Thursday, "and will handle it on a case-by-case basis."
Blue notes
On a side-by-side race to the puck on the first shift of the game, Michael Grabner and Colin Wilson collided and crashed into the corner boards. Both stayed face down on the ice and trainers ran out. Each player went off gingerly, but both returned. Grabner scored midway through the second period . . . Forwards Brandon Pirri and Matt Puempel were healthy scratches . . . The referees were Gord Dwyer and Jean Hebert. Because of weather issues, linesmen Tony Sericolo and Kory Nagy replaced the original duo.


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Somehow in the pandemic crisis, I remembered the last time I saw U.S. Rep Dina Titus before the entertainment shutdown. It was at the Composers Showcase of Las Vegas at Myron's Cabaret Jazz, a very Vegas hang teeming with the city's best artists.
Yes, Titus is a live entertainment fan and supporter. I caught up with her this week as federal assistance to help the entertainment industry is still stalled like a sidelined Cirque show.
"The Senate hasn't passed anything, and the president's been all over the map, 'Send me a package; no, don't sent me anything; send me piecemeal; no, wait till after the election,' " Titus said. "So, they're negotiating, but right now it's not making much progress."
Titus, whose 1st Congressional District covers the entertainment capital of the world, says support from the federal level is overdue. It's a $10 billion aid package with nowhere to go at the moment. Titus rolled back the history of what became of the Save Our Stages Act — beginning with the Cares Act — targeted at unemployed entertainment workers.
"In the Cares Act, the first bill that we passed, there was a provision for unemployment for people who are independent contractors, and you know they don't traditionally qualify for unemployment," Titus said. "It was a loan program that was set up for small businesses, but it didn't really accommodate entertainment venues.
"So we went back with the Save Our Stages Act, rolled in to the Heroes Act, out of the House and trying to get it included in some kind of compromise package."
Save Our Stages is the $10 billion grant program to help offset the the economic impact that COVID-19 has had on independent live venue operators, producers, promoters or talent representatives. The legislation passed in the House on Oct. 1 but is stalled in the Senate.
A fundraiser for the campaign is set for Friday through Sunday at SaveOurStages.com/sos. The event is raising awareness, advocacy and donations for the National Independent Venue Association. G-Eazy, Marshmello with Demi Lovato, Jason Mraz, Kelsea Ballerini, Miley Cyrus, Foo Fighters, The Roots, Little Big Town, Dave Matthews, The Lumineers and Reba McEntire are in the lineup.
Titus reiterated Las Vegas' position, chiseled over decades, as one of the world's leading tourism destinations. Those who are out of work in the entertainment industry, and across the hospitality culture, need to be sustained until the city returns to full tourism and convention business.
"Our reputation, the reputation of Las Vegas, has gone so far beyond just being a gambling town," Titus said. "It is an entertainment town, it's a sports town, it's a fine dining town. We've got all the chefs here, and we just want to be sure that we don't lose that image."Titus reps the megaresorts on the Strip, and many small businesses in downtown Las Vegas.
"All of the Arts District, The Smith Center, the small restaurant venues where you've got a little jazz combo playing," Titus said. "So it really has hit District 1 harder than anyplace else."
Titus says in the extended timeline, presenting Las Vegas as a safe place to return to business and vacations is paramount to recovery. She chairs the subcommittee that oversees economic development, which funds tourism agencies to help promote such venues as the Las Vegas Convention Center.
"You saw this in the Gulf (of Mexico), after the big oil spill (in 2010), where the Gulf put out advertisements nationally and even internationally, 'Hey, we're back in business. Come back to see us,' " she said. "We will need that for places like Las Vegas, 'Hey, we're back in business' and 'What happens here,' that sort of thing."
Titus says that Las Vegas resorts have emphasized safety to those visiting the city.
"One thing that I have heard is that there was a fear that people wouldn't want to come to Las Vegas and that the casinos didn't want to have a lot of sanitary things around or masks or temperatures or all of that," Titus said. "How would that affect people's idea of fun? But it's turned out just the opposite, people feel more reassured by seeing all of that. It makes a difference, and people feel like they're safe and they can have fun."


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nonnative Drops All-Weather Dweller Trainer Hi
Four military-inspired colorways equipped with Cordura and GORE-TEX 2L.
nonnative has weatherized its Dweller Trainer with hi-tech materials to keep your toes dry during rainy days. The ankle hugging silhouette debuted a little over a year ago and is produced by SPRINGLE COMPANY, one of Japan's last manufacturers that still have the machinery to make vulcanized soles. This release comes with four military-inspired colorways that have been equipped with both Cordura and GORE-TEX fabrics.
Most of the shoe's uppers are made up of a sleek cotton-nylon Cordura ripstop blend, offering optimal durability and abrasion resistance. The materials have been reinforced with a batted weave to lessen wear-and-tear, while the interior walls boast breathable, waterproof GORE-TEX 2L lining. Each shoe comes with flat laces, metal eyelets, a woven backstay and a thick, high-cushioned Japan-made vulcanized midsole, rounding up the details of the understated sneaker.
nonnative's Dweller Trainers are currently available on COVERCHORD's website for $304 USD.
In case you missed it, check out nonnative and Suicoke's collaborative leather-equipped sandals.


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Ashlee Bond is a 34-year-old international show jumper competing for Israel. Raised in Hidden Hills, Calif., outside of Los Angeles, she first sat on a horse at 6 months of age. Her father, Steve Bond, originally hails from Israel and was instrumental in instilling a love of horses in his daughter.
Bond quickly rose through the ranks in competition at a young age, winning five nationals championships in the pony divisions by the age of 11. By 16, she earned the title of Pacific Coast Horse Association Grand Prix Rookie Rider of the Year. Bond made her senior international debut for Team USA in 2008 at Spruce Meadows.
The 2009 season was a banner year for Bond: she won eight grand prix events, broke into the top 50 in the FEI/Rolex World Rankings, and reached the no. 2 position on the Rolex/USEF Show Jumping Ranking List. The year ended with the honor of the Show Jumping Horseman of the Year title, presented by The Chronicle of the Horse.
Bond continued the momentum from there, winning in Europe and in Florida with her top horse Cadett 7 leading the charge. In 2014, Bond won the AIG $1 Million Grand Prix at HITS Desert Circuit with Chela LS, the fiery chestnut mare that Bond currently campaigns at the CSI5* level.
In 2018, on her 33rd birthday, Bond became an Israeli citizen and began to ride for Team Israel.
Fun Facts:
- Ashlee operates Little Valley Farms alongside her father, Steve.
- Her mother, Cindy Bond, is a movie producer.
- Ashlee is married to Roy (a former professional soccer player) and together they have a daughter, Scottie Meeus Bond, born October 10, 2016.
- Ashlee can act and sing: she has film credits and even recorded a few songs years ago.
- She is a foodie and loves to watch the cooking channels.
Career Highlights:


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The SkepticMembers
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Modernized Experiment
The Skeptic posted a topic in ExperimentsUrey-Miller Experiment The URL below has a diagram that displays the Urey-Miller experiment. This experiment's purpose was to simulate pre-biotic soup and the generation of early life. The experiment used water (H20), methane (CH4), ammonia (NH3), and hydrogen (H2). Looking at the diagram will explain this a lot better. Miller and Urey believed the experiment was successful. I have my doubts. Just producing 11 out of 20 amino acids is not close to a cell. Wikipedia will have much more information if you would like to know more about the original experiment. http://en.wikipedia....Urey_experiment Modernizing the Experiment I am wishing to perform a modernized Urey-Miller experiment for a local science fair. I need to perform research on what fossils and rocks can tell us about earth's early atmosphere. Some evidence points towards a much different atmosphere but I believe if I could get some input then this will turn out to be a much more thorough experiment. I would prefer direct evidence such as diagnosis of air bubbles in amber rather than speculation. Also I would like to see if any changes of the setup to the experiment could be made to reflect a more accurate representation. I would also love advice on how to put this together, sterilizing the equipment, tips, and how to analyze the chemicals. Any input would be great! Hope this interests many people, I will publish my results when I am done.


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Appraisal: 1892 Alexander III Russian Imperial Plate
Clip: Season 19 Episode 11 | 2m 34s Video has closed captioning.
Check out David Lackey's appraisal of an 1892 Alexander III Russian Imperial Plate, in Birmingham, Hour 2.
Aired: 04/05/15
The full episode is no longer available for online streaming. Why?
Please continue to enjoy extra(s) from this episode.
Problems Playing Video? | Closed Captioning


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International rights watchdog, Human Rights Watch (HRW), has warned that Zimbabwe could face another violent and flawed election in next year's harmonised polls as the military gets more active to support Vice President Mnangagwa's push for office. Commander Defence Forces General Constantino Chiwenga has publicly threatened Prof. Jonathan Moyo with unspecified action for his constant criticism of Mnangagwa. Moyo is one of the key members of the G40 Faction which is opposed to Mnangagwa taking over from President Mugabe.
In a statement HRW said:
Zimbabwe also faces serious threats to its ability to hold credible elections, including the involvement of security forces in political affairs and the ruling Zanu PF party's endorsement of 93-year-old President Robert Mugabe as its presidential candidate amid rumours about his health.
The involvement of security forces in Zimbabwe's political and electoral affairs could, as in the past, severely undermine the credibility of the elections and raise the risk of political violence within political parties and across the country.
…non-governmental organisations have reported that the police use outdated and abusive laws to violate basic rights such as freedom of expression and assembly, and harass activists, human rights defenders, and LGBT people.
There has been no progress toward justice for human rights violations and past political violence.
More: New Zimbabwe


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The stability of cell cycle checkpoint and regulatory proteins is controlled by the ubiquitin-proteasome degradation machinery. A critical regulator of cell cycle molecules is the E3 ubiquitin ligase SCFSkp2, known to facilitate the polyubiquitination and degradation of p27, E2F, and c-myc. SCFSkp2 is frequently deregulated in human cancers. In this study, we have revealed a novel link between the essential Epstein-Barr virus (EBV) nuclear antigen EBNA3C and the SCFSkp2 complex, providing a mechanism for cell cycle regulation by EBV. EBNA3C associates with cyclin A/cdk2 complexes, disrupting the kinase inhibitor p27 and enhancing kinase activity. The recruitment of SCFSkp2 activity to cyclin A complexes by EBNA3C results in ubiquitination and SCFSkp2-dependent degradation of p27. This is the first report of a viral protein usurping the function of the SCFSkp2 cell cycle regulatory machinery to regulate p27 stability, establishing the foundation for a mechanism by which EBV regulates cyclin/cdk activity in human cancers.
Epstein-Barr virus (EBV) is the etiologic agent of most cases of infectious mononucleosis, a syndrome resulting from a dramatic increase in CD8 T-cell activation in response to EBV infection and expansion of B lymphocytes (17, 37). This clinical manifestation of primary EBV infection highlights the ability of the virus not only to establish latent infection in B lymphocytes but also to drive lymphocyte proliferation in vivo. In addition, EBV potently transforms B cells in vitro and contributes to numerous lymphoid malignancies in humans, including Burkitt's lymphoma, Hodgkin's lymphoma, and posttransplant lymphoproliferative disease (17, 37). Transformation of B lymphocytes by EBV has been attributed to the expression of a growth stimulatory program by the virus. Four viral antigens have been shown to be absolutely essential for B-cell transformation: EBNA2, LMP1, EBNA3A, and EBNA3C (8, 13, 16, 41). EBNA1 and EBNA-LP are critical but not essential for the immortalization process (25).
EBNA3C was initially described as a transcriptional regulator targeting RBP-Jκ and Spi-1/Spi-B transcription factors to regulate viral promoters such as Cp and LMP1 (2, 3, 46). EBNA3C has also been shown to play a role in histone acetylation as it apparently targets and regulates both histone acetyltransferase activity and histone deacetylase complexes, including HDAC1, HDAC2, and the corepressors mSin3a and NcoR (19, 36, 40). More recently, it has become apparent that EBNA3C plays a role in deregulation of the cell cycle by EBV. EBNA3C regulates transcription of the EBV oncogene LMP1 in a cell cycle-dependent manner and targets Rb-E2F regulatory pathways leading to the accumulation of cells in the S/G2 phase of the cell cycle (3, 32, 33). Recently, we demonstrated that EBNA3C targets cyclin A complexes in cells and enhances cyclin A-dependent kinase activity by disrupting the cdk inhibitor p27 from kinase complexes (20).
In this report, we demonstrate that, in addition to targeting cyclin A, EBNA3C targets the SCFSkp2 E3 ubiquitin ligase complex. Components of SCFSkp2, Skp1 and Skp2, were initially identified as S-phase kinase-associated proteins that complex with cyclin A in tumor cells but not primary cells (45). Subsequently, it was demonstrated that the SCFSkp2 complex consists of additional core components, Cul1 and Roc1, and that this complex plays a critical role in regulating the stability of diverse cell cycle proteins which include p27, E2F, and c-myc (6, 18, 26, 44). SCFSkp2 is an E3 ubiquitin ligase functionally linking specific substrates to the ubiquitin-activating and ubiquitin-conjugating machinery (5). This link targets substrates for polyubiquitination and ultimately degradation by the 26S proteasome (5). SCFSkp2 is perhaps the best-studied member of the group of SCF E3 ligases, each with a unique F-box protein that confers substrate specificity (47). Skp2, containing a so-called F-box domain that facilitates its interaction with Skp1, is overexpressed in many diverse classes of human lymphomas and tumors and presumably contributes to the destabilization of p27 in these cancers (7, 12, 22, 23, 39). Here we demonstrate that EBNA3C regulates p27 stability by manipulating the oncoprotein Skp2 as well as other SCF components. Regulation of p27 stability by EBNA3C is most likely at the level of its interaction with cyclin A complexes, providing a potential mechanism by which EBNA3C disrupts p27 from cyclin A complexes and ultimately stimulates cyclin A-dependent kinase activity.
Plasmids, antibodies, and cell lines.pA3M-EBNAC constructs express either full-length EBNA3C or EBNA3C truncations with a carboxy-terminal myc tag and have been described previously (9). Glutathione S-transferase (GST)-EBNA3C truncation mutants and pSG5-EBNA3C have been previously described (21). pCMV-Cdk2, RC-cyclin A, and RC-cyclin E were kindly provided by Philip Hinds (14). The construct expressing GST-cyclin A was provided by Maria Mudryj (10). pCDNA3-p27 and pCDNA3-Cks1-FL were provided by Michele Pagano (11, 31). pCDNA-myc-Skp2, pCDNA3-HA-Cul1, and pCDNA3-HA-Roc1 were provided by Yue Xiong (28, 30). Skp1 cDNA was provided by Stephen Elledge (5). pCDNA3-HA-Ub was provided by George Mosialos (42). Constructs expressing myc-tagged cyclin A, Skp1, p27, and p27 amino acids 1 to 185 were prepared by cloning PCR-amplified cDNAs into the previously described pA3M vector (4). Constructs expressing untagged Skp1 and Skp2 and hemagglutinin (HA)-tagged Roc1 amino acids 36 to 108 were prepared by cloning PCR-amplified cDNAs into the pCDNA3.1 vector (Invitrogen Corporation, Carlsbad, Calif.). C53A/C56A point mutations in the Roc1 gene were prepared by a standard PCR primer mutagenesis method. Rabbit polyclonal antibodies reactive to cyclin A, cdk2, Skp2, Cul1, and p27 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.). A10 monoclonal antibody reactive to EBNA3C has been previously described (9). Mouse monoclonal antibody reactive to the HA tag was purchased from Covance Research Products, Inc. (Berkeley, Calif.). HEK 293 cells are human embryonic kidney cells transformed by adenovirus type 5 DNA; HEK 293T cells stably express the simian virus 40 large-T antigen (1). U2OS is a human osteosarcoma cell line (34). HEK 293, 293T, U2OS, and HeLa cells were grown in Dulbecco's modified Eagle's medium (Invitrogen Corporation) supplemented with 10% fetal bovine serum unless otherwise indicated. HEK 293T cells were transfected by electroporation at 210 V and 975 μF in a 0.4-μm gap cuvette. Lymphoblastoid cell lines (LCLs) were maintained in RPMI (Invitrogen Corporation) supplemented the same as Dulbecco's modified Eagle's medium.
GST pull-down assays.GST fusion proteins were purified from bulk Escherichia coli cultures following induction with isopropyl-β-d-thiogalactopyranoside (IPTG) as described previously (9). For pull-down assays from cell lysates, lysates were prepared in radioimmunoprecipitation (RIPA) buffer (0.5% NP-40, 10 mM Tris [pH 7.5], 2 mM EDTA, 150 mM NaCl, supplemented with protease inhibitors). Lysates were precleared and then rotated with either GST control or the appropriate GST fusion protein bound to glutathione-Sepharose beads. For in vitro binding experiments, GST fusion proteins were incubated with 35S-labeled, in vitro-translated protein in binding buffer (1× phosphate-buffered saline [PBS], 0.1% NP-40, 0.5 mM dithiothreitol [DTT], 10% glycerol, supplemented with protease inhibitors). In vitro translation was done with the TNT T7 quick-coupled transcription/translation system (Promega Corporation, Madison, Wis.) according to the manufacturer's instructions.
Immunoprecipitation and immunofluorescence.For transfected HEK 293T samples, cells were lysed on ice in 500 μl of RIPA buffer (0.5% NP-40, 10 mM Tris [pH 7.5], 2 mM EDTA, 150 mM NaCl, supplemented with protease inhibitors). For LCLs, 100 million viable cells were lysed in 1 ml of RIPA buffer. Lysates were precleared with either normal rabbit or normal mouse serum and then rotated with 1 μg of specific antibody for 4 h at 4°C. Immune complexes were precipitated with a 1:1 mixture of protein A- and protein G-Sepharose beads. Samples were washed, fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to a 0.45-μm nitrocellulose membrane for Western blotting. Detection was done according to a standard chemiluminescence protocol unless otherwise indicated.
HeLa cells were transfected by Lipofectamine 2000 reagent (Invitrogen Corporation) with pA3M-EBNA3C and pCDNA3-Skp2. Cells were harvested at 24 h, trypsinized, and allowed to adhere to glass slides overnight. Cells were fixed and permeabilized in methanol at −20°C for 10 min followed by acetone at room temperature for 30 s. Slides were blocked with 5% goat serum and incubated at 4°C overnight with primary antibodies (1:100 9E10 myc ascites fluid, 5 μg of Skp2 rabbit polyclonal antibody/ml). Slides were washed with PBS and then incubated with AlexaFluor goat anti-mouse (594 nm) and goat anti-rabbit (488 nm) antibodies (Molecular Probes, Inc., Eugene, Oreg.). Slides were washed and visualized with a Zeiss LSM510 confocal microscope.
Histone H1 kinase assay.U2OS cells were seeded into six-well plates and grown to confluence in 0.5% fetal bovine serum for 48 h prior to transfection. Cells were transfected with Lipofectamine 2000 reagent (Invitrogen Corporation), harvested after 24 h with a cell scraper, washed with PBS, and lysed on ice in 500 μl of RIPA buffer (0.5% NP-40, 10 mM Tris [pH 7.5], 2 mM EDTA, 150 mM NaCl, 1 mM EGTA, with protease and phosphatase inhibitors). Lysates were precleared and then rotated with 1 μg of cyclin A antibody overnight at 4°C. Cyclin A complexes were captured by rotating with protein A-Sepharose beads and washed with RIPA buffer. Cyclin A complexes were then washed with histone buffer (25 mM Tris [pH 7.5], 70 mM NaCl, 10 mM MgCl2, 1 mM EGTA, 1 mM DTT, with protease and phosphatase inhibitors). Complexes were incubated in 30 μl of histone wash buffer supplemented with 4 μg of histone H1 (Upstate USA, Inc., Chicago, Ill.), 10 mM cold ATP, and 0.2 μCi of [γ-32P]ATP/μl for 30 min at 37°C. The reaction was stopped by adding SDS-lysis buffer and heating to 95°C for 10 min. Labeled histone H1 was resolved by SDS-12% PAGE. Quantitation was done with ImageQuant software (Amersham Biosciences Corporation, Piscataway, N.J.).
HEK 293T ubiquitination assay.Ten million HEK 293T cells were transfected by electroporation (as described above) with 10 μg of pCDNA3-HA-Ub and 10 μg of a myc-tagged expression plasmid for EBNA3C, p27, Skp2, or Skp1. In some cases, cells were additionally transfected with 10 μg of pSG5-E3C or 10 μg of pCDNA3-HA-Roc1 as indicated. Cells were harvested at 36 h, lysed in RIPA buffer (see above), immunoprecipitated with 9E10 myc-specific ascites fluid, and resolved by SDS-PAGE. Ubiquitin-tagged proteins were detected by HA-specific Western blot analysis.
p27 degradation assay.The degradation reaction consisted of 20 μl of concentrated BJAB extract, 10 μl of in vitro-translated p27, 10 mM ATP, and SCFSkp2 complex immunoprecipitated from HEK 293T cells in a total volume of 40 μl. Samples were incubated at 30°C and mixed every 30 min. Five-microliter samples were harvested, mixed with 25 μl of SDS loading buffer, and heated at 95°C for 7 min. Concentrated BJAB extracts were prepared by washing cells in PBS and then resuspending them in 0.5 volumes (relative to pellet) of low-salt buffer (50 mM HEPES [pH 7.4], 5 mM KCl, 1.5 mM MgCl2, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, and 2 μg [each] of aprotinin, pepstatin, and leupeptin/ml). Cells were sonicated with four 10-s pulses, and cell debris was removed by centrifugation. Glycerol was added to 10%, and samples were frozen in liquid nitrogen and stored at −80°C until use. In vitro translation was with a rabbit reticulocyte-based kit from Promega. For immunoprecipitation of SCFSkp2complexes, 20 million HEK 293T cells were transfected by electroporation (as described above) with 10 μg (each) of pCDNA3-myc-Skp2, pCDNA3-HA-Cul1, pCR3.1-Skp1, pCDNA3-HA-Roc1, and pCDNA3-Cks1-FL and 15 μg of pSG5-E3C, as indicated. Samples were harvested at 36 h, lysed in RIPA buffer (see above), and immunoprecipitated with 9E10 myc-specific ascites fluid overnight, followed by a 1:1 mixture of protein A- and protein G-Sepharose beads for 2 h.
Proteasome inhibition of LCLs with MG-132.A recently immortalized LCL was passaged every 24 h for several days to establish an asynchronous population of cells. Cells were then treated with either dimethyl sulfoxide (DMSO) control or 10 μg of MG-132 (Calbiochem Biochemicals)/ml for up to 8 h. Lysates were prepared and normalized by Bradford assay. Samples were resolved by SDS-10% PAGE and transferred to a nitrocellulose membrane for Western blotting. After incubation with primary antibody, bands were visualized by incubation with conjugated secondary antibodies which fluoresce in the infrared region of the spectrum (Alexa Fluor 680). Direct detection of the fluorescence and quantification was done with the Li-Cor Odyssey (Lincoln, Nebr.).
Previously, we demonstrated that EBNA3C disrupts p27 from cyclin A complexes and that EBNA3C rescues p27-mediated suppression of cyclin A-dependent kinase activity. These data were consistent with a model whereby EBNA3C directly competes with p27 for cyclin A binding (20). However, to date, we have been unable to demonstrate competition between p27 and EBNA3C for cyclin A binding in vitro. This suggests that EBNA3C may employ a more complex mechanism to regulate cyclin A/p27 complexes.
Inhibition of kinase activity by carboxy-terminally deleted p27 is not rescued by EBNA3C.Previously, we demonstrated that EBNA3C stimulates cyclin A-dependent kinase activity in the osteosarcoma cell line U2OS and that EBNA3C rescues p27-mediated suppression of this activity in both U2OS and BJAB cell lines (20). Here, we demonstrate that deletion of the carboxy-terminal 13 amino acids of p27, including the regulatory amino acid threonine-187, eliminates the ability of EBNA3C to rescue kinase activity (Fig. 1A). U2OS cells were transfected with cyclin A, cdk2, p27, and EBNA3C expression constructs as indicated (Fig. 1B). At 24 h, cyclin A complexes were immunoprecipitated and incubated with [32P]ATP and histone H1 substrate. In repeated experiments, EBNA3C was able to rescue inhibition of kinase activity by p27 5- to 10-fold, while no reproducible rescue was seen for carboxy-terminally truncated p27 in spite of its efficient inhibition of kinase activity (Fig. 1B, compare lanes 2 and 3 with 4 and 5). This carboxy-terminal region of p27 is notable for the critical residue threonine-187, which regulates p27 stability (Fig. 1A). Phosphorylation of this residue by cyclin A/cdk2 and other kinase complexes such as cyclin E/cdk2 promotes recognition of p27 by the SCFSkp2 complex, resulting in polyubiquitination and ultimately degradation of p27 (29, 43). Thus, threonine-187 is likely critical for EBNA3C-mediated rescue of kinase activity.
EBNA3C is associated with ubiquitination activity and is itself ubiquitinated.Studies have shown that phosphorylation of threonine-187 stimulates recognition and degradation of p27 by the ubiquitin/proteasome pathway (29, 43). We therefore wanted to determine whether EBNA3C recruits ubiquitination activity in cells and, additionally, whether EBNA3C itself may be ubiquitinated. HEK 293T cells, known to prominently express the SCFSkp2 complex which regulates p27 stability (6), were transfected with expression constructs for HA-tagged ubiquitin and myc-tagged EBNA3C or EBNA3C truncation mutants, as indicated (Fig. 2A). myc-specific immunoprecipitation resulted in the coimmunoprecipitation of high-molecular-weight ubiquitin-tagged proteins from cells expressing both full-length EBNA3C and a carboxy-terminally deleted EBNA3C, amino acids 1 to 365 (Fig. 2A, bottom left panel, lanes 2 and 3). These ubiquitin-tagged species were seen predominantly at higher molecular weights than full-length EBNA3C and EBNA3C amino acids 1 to 365, respectively. And, importantly, some of these species were also reactive with the myc-specific Western blot analysis (Fig. 2A). The aforementioned observations strongly suggest that these species represent polyubiquitinated EBNA3C and that this ubiquitination can be recapitulated by the first 365 amino acids of EBNA3C (Fig. 2A, lower panels, lanes 2 and 3). In vector control, EBNA3C amino acids 366 to 620 and EBNA3C amino acids 621 to 992 samples, no ubiquitinated species were precipitated by myc-specific antibody (Fig. 2A, left panel, lanes 1, 4, and 5).
EBNA3C ubiquitination is dependent on amino acids 101 to 200.To further define the region or regions of EBNA3C that recruit this ubiquitination activity, additional truncation mutants EBNA3C amino acids 1 to 100 and EBNA3C amino acids 1 to 200 were transfected into HEK 293T cells and assayed as above. Interestingly, amino acids 1 to 100 were not ubiquitinated, while amino acids 1 to 200 were potently ubiquitinated similar to amino acids 1 to 365 and full-length EBNA3C (Fig. 2B, lanes 5 to 7). This result suggests that EBNA3C amino acids 1 to 200 are sufficient to facilitate ubiquitination, with essential residues for this phenotype residing between amino acids 101 and 200 (Fig. 2C).
Lysine-120 is dispensable for the ubiquitination of EBNA3C amino acids 1 to 200.At least two scenarios could explain the above data demonstrating that EBNA3C amino acids 101 to 200 are essential for ubiquitination. First, EBNA3C amino acids 101 to 200 may contain a critical lysine residue that accepts the polyubiquitin chain. Alternatively, EBNA3C amino acids 101 to 200 may recruit factors and ubiquitination machinery essential for formation of the polyubiquitin chain. The first possibility was easily addressed, as there is a single lysine residue within EBNA3C amino acids 101 to 200 (38). Mutation of lysine-120 to an alanine had no effect on the ubiquitination of EBNA3C amino acids 1 to 200 with two independent clones of this truncation (Fig. 3A, lanes 5 to 7). This result suggests that, instead of providing a critical ubiquitin acceptor, EBNA3C amino acids 101 to 200 likely recruit the enzymatic factors essential for ubiquitination of the EBNA3C molecule.
EBNA3C amino acids 90 to 190 recruit components of the SCFSkp2 ubiquitin ligase complex.As we had demonstrated above that EBNA3C regulation of cyclin A/p27 complexes is dependent upon the SCFSkp2-binding motif of p27, we decided to test whether EBNA3C interacts with SCFSkp2 in a manner that recruits ubiquitination activity to EBNA3C (Fig. 3B). We demonstrated above that EBNA3C amino acids 101 to 200 may contain an essential domain for the recruitment of ubiquitination machinery; to test whether this region of EBNA3C binds SCFSkp2, we prepared a GST fusion protein of EBNA3C corresponding to amino acids 90 to 190, shown previously to interact with cyclin A, and tested whether individual in vitro-translated SCF components bind to this region of EBNA3C (Fig. 3C). EBNA3C amino acids 90 to 190 strongly precipitated Skp2, Cul1, and Roc1, but not Skp1, when the proteins were expressed individually (Fig. 3C). To confirm that the precipitation of these species was not the result of their mutual association with ubiquitin, we also tested whether this region of EBNA3C may directly bind the 76-amino-acid ubiquitin protein. Importantly, EBNA3C amino acids 90 to 190 did not bind to monoubiquitin by an in vitro binding assay, nor did they nonspecifically precipitate high-molecular-weight ubiquitin chains from transfected cell lysates (data not shown).
As multiple components of SCFSkp2 bound EBNA3C in the above in vitro binding assay, we tested whether these molecules may cooperate or compete for binding to EBNA3C in cells. EBNA3C amino acids 90 to 190 precipitated myc-tagged Skp2 from singly transfected cell lysates (Fig. 3D, left panel). Interestingly, as additional components of SCFSkp2 were expressed, the interaction between EBNA3C amino acids 90 to 190 and Skp2 was reduced (Fig. 3D, right two panels). Indeed, with expression of myc-tagged Skp1 and HA-tagged Cul1, Skp2 was only weakly precipitated by EBNA3C amino acids 90 to 190, and Cul1 was also precipitated (Fig. 3D, middle panel). With the further addition of HA-tagged Roc1, Cul1 and Roc1 formed a strong complex with EBNA3C amino acids 90 to 190 and the Skp2 interaction was abolished to undetectable levels (Fig. 3D, right panel).
As it is known that F-box proteins are themselves ubiquitinated and may enter only transiently into SCF complexes (48), the above data suggest that EBNA3C may be capable of assembling active SCF ubiquitin ligase complexes. This potentially explains why Skp2 binding to EBNA3C was abolished with the recruitment of additional SCF components Cul1 and Roc1. To test whether EBNA3C recruits ubiquitination activity to Skp2 complexes, an expression plasmid for myc-tagged Skp2 was transfected into HEK 293T cells along with expression plasmids for HA-tagged ubiquitin and EBNA3C (Fig. 3E). Cells were then lysed, and Skp2 complexes were immunoprecipitated with myc-specific monoclonal antibody. Western blotting with either Skp2 or HA antibodies demonstrated enhanced ubiquitin-tagged Skp2 in the presence of EBNA3C (Fig. 3E, left panels). As a control, we also tested Skp1 ubiquitination in a similar HEK 293T transfection background (Fig. 3E, right panel). While low-molecular-weight ubiquitinated forms of Skp1 were easily detected, they were not enhanced by EBNA3C expression (Fig. 3E, right panel). We did detect some high-molecular-weight ubiquitinated species in the EBNA3C sample by HA Western blotting (Fig. 3E, right panel, rightmost lane); however, these species were not reactive with the Skp1-specific Western blot and likely represent an unidentified protein that coimmunoprecipitates with Skp1.
EBNA3C ubiquitination and SCFSkp2 recruitment are dependent upon EBNA3C amino acids 130 to 190.To more precisely define the region of EBNA3C that recruits ubiquitination activity, additional expression plasmids corresponding to EBNA3C amino acids 1 to 159 and 1 to 129 were constructed. These plasmids, along with EBNA3C amino acids 1 to 200, were transfected into HEK 293T cells and assayed for the formation of HA-ubiquitin chains. As was shown above, EBNA3C amino acids 1 to 200 were strongly ubiquitinated (Fig. 4A, lane 5). EBNA3C amino acids 1 to 159 were reproducibly ubiquitinated to a lesser extent than amino acids 1 to 200, and EBNA3C amino acids 1 to 129 were not ubiquitinated (Fig. 4A, lanes 6 and 7). This suggests that EBNA3C amino acids 130 to 159 likely comprise the predominant ubiquitination recruitment domain, while residues which lie within amino acids 160 to 200 may also be involved in regulation of this process. Importantly, neither of these regions contains a lysine residue.
To test whether the regions 160 to 200 and 130 to 159 play a role in SCFSkp2 recruitment, GST pull-down assays were performed from cell lysates as above. HEK 293T cells were transfected either with Skp2 alone or with both Cul1 and Roc1. Skp2 was precipitated with GST fusion proteins corresponding to both EBNA3C amino acids 130 to 159 and 160 to 190, although reproducibly to a greater extent with amino acids 160 to 190 (Fig. 4B, top panel). In contrast, Cul1 and Roc1 both precipitated most strongly with EBNA3C amino acids 130 to 159 and to a lesser extent with amino acids 160 to 190 (Fig. 4B, lower panel). These cumulative data suggest that the SCFSkp2 binding domain likely encompasses multiple residues in amino acids 130 to 190, which is consistent with this being the primary domain responsible for the recruitment of ubiquitination activity.
EBNA3C ubiquitination and Roc1 recruitment are dependent upon EBNA3C amino acids 140 to 149.Previous work has indicated that the RING finger protein Roc1 physically links the SCF core to the more basal E1 ubiquitin-activating and E2 ubiquitin-conjugating machinery and, consequently, is the minimal factor necessary for recruiting ubiquitination activity (15, 30). To test whether Roc1 recruitment is tightly linked to EBNA3C ubiquitination, HEK 293T cells were transfected with expression constructs encoding EBNA3C amino acids 1 to 159, 1 to 149, 1 to 139, and 1 to 129, each with a myc tag at the carboxy terminus (Fig. 4C). Cells were additionally transfected with expression constructs for HA-tagged ubiquitin and Roc1. While amino acids 1 to 159 and 1 to 149 recruited both Roc1 and ubiquitination activity, amino acids 1 to 139 and 1 to 129 recruited neither (Fig. 4C). This suggests that EBNA3C amino acids 140 to 149 represent a minimal domain for the recruitment of ubiquitination activity and that this activity is tightly linked to the recruitment of the RING finger protein Roc1 (Fig. 4C, lanes 6 and 7).
EBNA3C forms complexes with Skp2 in EBV-transformed B cells.To test the association between SCFSkp2 and EBNA3C in virally infected cells, Skp2 was immunoprecipitated from a recently transformed LCL and coimmunoprecipitating species were assessed by Western blotting. As expected, antibody specific to Skp2 coimmunoprecipitated Skp2 and known Skp2-binding partners Cul1 and cyclin A (24, 28, 45) (Fig. 5A). EBNA3C was also coimmunoprecipitated at a level similar to that of Cul1 and cyclin A, suggesting that EBNA3C and Skp2 may form a functional complex in LCLs (Fig. 5A). The reverse coimmunoprecipitation experiment was performed by cotransfecting HEK 293T cells with myc-tagged EBNA3C and untagged Skp2 expression plasmids (Fig. 5B). As expected, Skp2 coimmunoprecipitated with both full-length EBNA3C and EBNA3C amino acids 1 to 365 by using an antibody specific to the myc tag (Fig. 5B, lanes 7 and 8).
To determine whether Skp2 and EBNA3C exist in similar nuclear compartments, HeLa cells were transfected with a vector expressing myc-tagged EBNA3C and untagged Skp2. Cells were then stained with antibodies specific for myc and Skp2, respectively, and nuclei were visualized by confocal microscopy to assess colocalization. EBNA3C staining was tightly nuclear, demonstrating a finely stippled pattern with exclusion of nucleoli (Fig. 5C, middle panel); Skp2 staining was similarly concentrated in the nucleus (Fig. 5C, right panel). Importantly, EBNA3C and Skp2 colocalized at numerous points in the visualized nuclear plane, as illustrated by yellow color in the merged image (Fig. 5C, right panel). These data corroborate the in vivo association between Skp2 and EBNA3C, as these molecules localize to similar compartments in the cell nucleus.
EBNA3C functionally associates with the RING finger protein Roc1.The EBNA3C/Roc1 association was assayed as for EBNA3C/Skp2 by coimmunoprecipitation in HEK 293T cells (Fig. 6A). Similar to Skp2, Roc1 coimmunoprecipitated with both full-length EBNA3C and EBNA3C amino acids 1 to 365 when both components were transiently transfected into HEK 293T cells (Fig. 6A, lanes 6 and 7). To corroborate this experiment, GST-tagged Roc1 pulled down full-length EBNA3C and EBNA3C amino acids 1 to 365, but not EBNA3C amino acids 366 to 620 or 621 to 992 (Fig. 6B). The efficiency of pull down was similar to that seen for a known Roc1-interacting protein, Cul1.
Roc1 contains a RING finger motif which is responsible for the recruitment of E2 ubiquitin-conjugating activity to the SCFSkp2 ubiquitin ligase complex (30). The Roc1 RING finger motif conjugates three Zn2+ ions and occupies the carboxy-terminal two-thirds of this 108-amino-acid protein (47). To test whether this critical domain contributes to EBNA3C binding, we generated a Roc1 truncation mutant encompassing the entire RING finger motif, but with a deletion of the amino terminus of Roc1, which has previously been shown to mediate Cul1 binding (47). Interestingly, unlike full-length Roc1, Roc1 amino acids 36 to 108 did not coimmunoprecipitate with the amino terminus of EBNA3C, implicating Roc1 amino acids 1 to 35 in EBNA3C binding (Fig. 6C, compare lanes 3 and 4 with lanes 5 and 6). This result is consistent with EBNA3C binding Roc1 in a manner that leaves the RING finger motif available for ubiquitin recruitment.
During the course of the aforementioned studies, we made the observation that the delivery of a Roc1 expression plasmid into EBNA3C-expressing cells significantly and reproducibly enhanced EBNA3C ubiquitination (Fig. 7A, lower left panel). As Roc1 binding to EBNA3C is not dependent on the well-characterized RING finger motif (Fig. 6C), we decided to test whether mutations in this motif would disrupt EBNA3C ubiquitination without affecting binding. Several specific RING finger mutations which disrupt Zn2+ coordination and, consequently, RING finger structure have been shown to significantly impair Roc1-dependent ubiquitination (30). We chose one such mutation, C53A/C56A, and compared its properties to the wild type. While the mutant bound EBNA3C similarly to the wild type, it did not stimulate EBNA3C ubiquitination (Fig. 7A). Further, the mutant protein functioned as a dominant negative when coexpressed with the wild-type protein, eliminating the ability of wild-type Roc1 to stimulate EBNA3C ubiquitination (Fig. 7A). While our previous mapping data had already strongly linked Roc1 binding to EBNA3C ubiquitination (Fig. 4C), this functional experiment unquestionably implicates a Roc1-dependent ligase in EBNA3C ubiquitination, most likely SCFSkp2.
EBNA3C stimulates the ubiquitination of exogenously expressed p27 in human cells.Having established that EBNA3C recruits SCFSkp2-associated ubiquitination activity, we tested whether p27 ubiquitination may be regulated by EBNA3C. HEK 293T cells were transfected with expression plasmids for myc-tagged p27, HA-tagged ubiquitin, and untagged EBNA3C. myc-specific immunoprecipitation resulted in the coimmunoprecipitation of ubiquitin-tagged p27 protein, an effect significantly and reproducibly enhanced with EBNA3C expression (Fig. 8A). This result implicates EBNA3C in upregulating the polyubiquitination of p27 in the context of this plasmid-based expression system.
EBNA3C decreases p27 stability in an in vitro degradation assay.Polyubiquitination of p27 in HEK 293T cells suggested that EBNA3C may be regulating p27 stability. To more directly assess the role of EBNA3C in this process, we employed an SCFSkp2-dependent in vitro degradation assay. HEK 293T cells were transfected with expression constructs for Skp1, Cul1, Roc1, Cks1, and myc-tagged Skp2. After 48 h, cells were lysed and the SCF complex was immunoprecipitated with monoclonal antibody against the myc tag. The complex was mixed with ATP, concentrated BJAB extract, and in vitro-translated p27. p27 stability was measured by collecting time points and resolving for autoradiography by SDS-PAGE. As shown in Fig. 8B, the SCF complex specifically resulted in time-dependent destabilization of in vitro-translated p27 (Fig. 8B, lanes 3, 5, and 7).
We next tested whether EBNA3C plays a role in regulating this complex. First, EBNA3C was expressed with SCF components Skp1, Cul1, Roc1, Cks1, and myc-tagged Skp2 deleted for the F-box domain. A similar Skp2 mutant has previously been demonstrated to function as a dominant negative for wild-type Skp2, as it dissociates substrate binding from ubiquitin recruitment (27). For both control and EBNA3C-expressing cells, p27 remained stable (Fig. 8C, upper panel). In contrast, when EBNA3C was expressed with the wild-type SCFSkp2 complex, reproducible enhancement of SCFSkp2 activity by EBNA3C was observed (Fig. 8C, lower panel). This result is consistent with EBNA3C regulating p27 stability by directly targeting specific components of the SCFSkp2 complex, in particular Skp2 and Roc1.
p27 is stabilized in LCLs by treatment with the proteasome inhibitor MG-132.The aforementioned data strongly indicate that EBNA3C can stimulate p27 ubiquitination in cells and enhance p27 degradation in an SCFSkp2-dependent in vitro assay. While we did not regularly see a reduction in p27 levels with EBNA3C expression in our plasmid-driven system, a previous study detected a dramatic decrease in endogenous p27 levels with EBNA3C expression in a drug-inducible system (33). It should also be noted that, in our plasmid-driven system, we routinely detected the ubiquitination of both Skp2 and EBNA3C (Fig. 2 to 4). While our in vitro degradation assay and the work of Parker and colleagues (33) strongly support a link between p27 ubiquitination and degradation, it is less clear whether Skp2 and EBNA3C may be similarly regulated. We therefore decided to test the ability of a proteasome inhibitor, MG-132, to stabilize these proteins in asynchronously growing LCLs, an indication of whether these molecules are being significantly directed into the ubiquitin/proteasome pathway in the context of EBV latent infection. A representative experiment is shown in the upper panel of Fig. 9. Interestingly, while p27 and another molecule, cyclin A, were stabilized by 2 h of MG-132 treatment, neither Skp2 nor EBNA3C was stabilized over an 8-h treatment (Fig. 9, bottom panel). This suggests that either Skp2 and EBNA3C are not significantly ubiquitinated in LCLs (as they are in our plasmid-based system) or that there is a disconnect between their ubiquitination and degradation in LCLs. So, while the ubiquitination of EBNA3C in our plasmid-based system provided a convenient and powerful mechanism for mapping the recruitment of this activity, it is not yet clear that ubiquitination activity is an important regulator of the EBNA3C protein itself in cells.
EBNA3C recruits p27 degradation activity to cyclin A complexes.As EBNA3C apparently directly binds SCFSkp2 components and as EBNA3C regulates p27 ubiquitination and stability, an attractive hypothesis is that EBNA3C may provide a physical link between p27 and SCFSkp2, thereby facilitating and enhancing Skp2 recognition of the substrate. We and others have failed to identify a molecular association between EBNA3C and p27 either in vitro or in cells. Therefore, we hypothesized that cyclin A, which also strongly binds the amino terminus of EBNA3C (21), may serve as an intermediary molecule, in addition to EBNA3C, functionally linking p27 to the SCFSkp2 complex.
To definitely confirm that the amino terminus of EBNA3C associates not only with the SCFSkp2 complex but also with p27 degradation activity in cells, HEK 293T cells were transfected with expression constructs encoding either the amino terminus of EBNA3C, amino acids 1 to 365, or the carboxy terminus, amino acids 621 to 992 (Fig. 10A, right panel). Both proteins are myc-tagged. Cells were additionally transfected with expression constructs for components of the SCFSkp2 complex (Fig. 10A, right panel). Samples were immunoprecipitated with myc-specific antibody and incubated with in vitro-translated p27 as for the degradation assays above. While p27 was stable in the presence of the EBNA3C carboxy terminus (Fig. 10A, left panel), incubation with the amino terminus resulted in significant and reproducible degradation of p27 over a 4-h incubation period (Fig. 10A, left panel). This result confirms the association between the EBNA3C amino terminus and p27 degradation activity and is consistent with a model whereby EBNA3C facilitates the assembly of cyclin A/SCFSkp2 complexes which potentially stimulates p27 degradation in cells.
To further test this model, cyclin A complexes were precipitated from HEK 293T cells either in the presence or in the absence of EBNA3C. Complexes were then incubated with in vitro-translated p27 and other degradation reaction components as described in above experiments. Previously, we demonstrated that, without the addition of exogenous SCFSkp2, in vitro-translated p27 is stable over the course of a 2-h degradation reaction. However, if cyclin A complexes are precipitated from HEK 293T lysates with a GST-cyclin A fusion protein bound to glutathione-Sepharose beads, the degradation of p27 increases by approximately 25% after 2 h (Fig. 10B). Importantly, expression of EBNA3C in HEK 293T cells resulted in enhancement of p27 degradation with greater than 50% degraded by 2 h (Fig. 10B). This result is consistent with cyclin A complexes mediating p27-specific degradation activity in HEK 293T cells, an effect that is significantly enhanced by EBNA3C expression in this system. This result is also consistent with EBNA3C stabilizing the association between cyclin A and SCFSkp2 complexes, which may result in p27 being targeted for degradation specifically at the level of cyclin A complexes (Fig. 10C).
Previous studies have demonstrated that EBNA3C targets cell cycle regulators, resulting in deregulation of the mammalian cell cycle (3, 20, 21, 32, 33). EBNA3C manipulates the Rb-E2F axis in reporter assays and in rat fibroblast transformation experiments (32). Additionally, EBNA3C causes the accumulation of cells in S/G2 and prevents the induction of certain mitotic checkpoints, resulting in aberrant nuclear division (33). We have previously shown that EBNA3C specifically targets cyclin A complexes and stimulates cyclin A-dependent kinase activity (20, 21). Here, we propose a mechanism for this stimulation, namely the recruitment of the SCFSkp2 complex to cyclin A, where the kinase inhibitor p27 is targeted for degradation (Fig. 10C).
Our studies began with the observation that mutations at the carboxy terminus of p27, which remove the regulatory amino acid threonine-187, eliminate the ability of EBNA3C to rescue p27-dependent suppression of cyclin A kinase activity. Threonine-187 is known to be a critical regulator of p27 stability. Phosphorylation of this residue enhances the interaction of p27 with the E3 ubiquitin ligase complex SCFSkp2, directing p27 for degradation via the ubiquitin/proteasome system (29, 43). We therefore investigated whether EBNA3C plays a role in regulating p27 stability and whether this regulation contributes to the stimulation of cyclin A-dependent kinase activity by EBNA3C. We first demonstrated that EBNA3C strongly associates with ubiquitination activity in cells and is, in fact, itself ubiquitinated in our plasmid-driven system. Further, we found significant molecular links between EBNA3C and the SCFSkp2 core components Skp2 and Roc1. EBNA3C coimmunoprecipitates with Skp2 from LCL lysates and strongly colocalizes with Skp2 in HeLa cell nuclei. The interaction with Roc1 was perhaps even more fundamental to the ubiquitination phenotypes we observed. EBNA3C strongly binds Roc1 in vitro and in cells, with the amino terminus of Roc1 essential for the interaction; this is a potentially important observation, as it suggests that the carboxy-terminal RING finger motif is likely free to recruit E2 ubiquitin conjugation machinery, even when EBNA3C is bound. Indeed, point mutations in the RING finger domain, while having no effect on EBNA3C binding, eliminate the recruitment of ubiquitination activity to EBNA3C. We also, importantly, found that both EBNA3C ubiquitination and Roc1 binding map to the same 10-amino-acid region at the amino terminus of EBNA3C, strengthening our assertion that EBNA3C is not just coincidentally recruiting SCFSkp2 components but is actually assembling active ubiquitin ligase complexes.
Having demonstrated that EBNA3C associates with active SCFSkp2 complexes, we asked whether this association is important in the regulation of p27 stability. EBNA3C specifically stimulates the ubiquitination of p27 when both molecules are expressed in HEK 293T cells and destabilizes p27 in an in vitro degradation assay based on SCFSkp2 complexes immunoprecipitated from HEK 293T cells. These data are consistent with the work of others showing that endogenous p27 steady-state levels are dramatically reduced in the presence of EBNA3C (33). As SCFSkp2 and cyclin A both bind a small and conserved amino-terminal region of EBNA3C (amino acids 130 to 159), and as the regulation of p27 stability potentially plays a critical role in cyclin A-dependent kinase activity, we decided to test whether EBNA3C recruits p27 degradation activity to cyclin A kinase complexes. This question was especially interesting in the context of previous data suggesting that cyclin A and SCFSkp2form complexes in transformed but not primary cells (45). It has been hypothesized that cyclin A/cdk2 may function in these complexes to regulate the phosphorylation state of potential SCFSkp2 substrates, thereby increasing the association of these substrates with the F-box protein Skp2. However, a largely unexplored hypothesis is whether SCFSkp2 may regulate the stability of substrates like p27 that enter complex with cyclin A. In support of this second hypothesis, we show that in HEK 293T cells, p27 degradation activity is normally associated with cyclin A complexes, an association that is enhanced with EBNA3C expression (Fig. 10C).
While a recent report suggests that the papillomavirus protein E7 may be ubiquitinated by a Skp2- and Cul1-containing E3 ligase (29a), no viral molecule has previously been shown to regulate p27 stability through the SCFSkp2 complex. However, it has been demonstrated recently that adenovirus E1B-55K protein forms a complex with components of the VHL E3 ubiquitin ligase, including Cul2, elongin B, and elongin C (35). This complex is structurally and functionally homologous to SCFSkp2 and may regulate p53 stability in virally infected cells. Similarly, EBNA3C may serve as a bridge between p27 and SCFSkp2; however, in contrast to the E1B-55K association with p53, the association of p27 with EBNA3C may be tethered by another component of the complex which includes cyclin A. While EBNA3C weakly coimmunoprecipitates with p27 in cells, in vitro binding experiments with either bacterially expressed or in vitro-translated proteins have failed to demonstrate a direct interaction, although we cannot eliminate the possibility that some modification of p27 may be necessary for EBNA3C binding. Based on the results in this report, we presently favor the hypothesis that additional intermediary molecules, such as cyclin A, are necessary for EBNA3C-mediated recruitment of p27 to SCFSkp2 complexes and that perhaps only specific populations of p27 are targeted for degradation by EBNA3C. In summary, we have demonstrated that EBNA3C binds and regulates the SCFSkp2 E3 ligase complex. As SCFSkp2 is commonly deregulated in human cancers, this and future studies of the essential EBV antigen EBNA3C will potentially clarify the role of SCFSkp2 in EBV-mediated tumor development and provide new possibilities for therapeutic development.
We thank Stephen Elledge, Ed Harlow, Philip Hinds, Elliot Kieff, Maria Mudryj, Michele Pagano, Martin Rowe, George Mosialos, and Yue Xiong for generously providing reagents.
J.S.K. is supported by the Lady Tata Memorial Trust. The project is supported by NIH grants NCI CA72150-07, NCI CA91792-01, and DCR DE14136-01 to E.S.R. E.S.R. is a scholar of the Leukemia and Lymphoma Society of America.
- Received 7 October 2004.
- Returned for modification 5 November 2004.
- Accepted 1 December 2004.
- Copyright © 2005 American Society for Microbiology