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There may be a new roof on the New Orleans Superdome and tourists in the French Quarter, but time is not healing all wounds in the wake of Hurricane Katrina. On the contrary, time has been a salt in the psychological wounds of hundreds of thousands of Gulf Coast residents. Even two years after the storm, mental health problems in the region are growing among the nearly 70,000 families still living in temporary housing provided by the Federal Emergency Management Agency (FEMA). The slow recovery, researchers and clinicians are finding, has bred levels of mental distress unseen in the aftermath of other disasters.
"Most of the time, distress emerges early and dissipates over the first year post-disaster," says psychologist Fran Norris of the National Center for Post Traumatic Stress Disorder at Dartmouth Medical School. Not so with Hurricane Katrina. One year after the storm a Harvard Medical School committee funded by the National Institute of Mental Health reported doubled rates of depression and anxiety in the region. A team led by David Abramson of the National Center for Disaster Preparedness (NCDP) at Columbia University, in collaboration with the Children's Health Fund, surveyed residents of FEMA-provided trailers and hotels in Louisiana and reported widespread clinically diagnosed psychiatric problems. Sixty-eight percent of female caregivers and 44 percent of children suffered new mental health issues, including depression, anxiety and sleep disorders. When the Columbia team surveyed a similar group in Mississippi six months later, it found even higher rates of distress despite the fact that Mississippi had suffered less damage and had an additional half a year to recover. Clinical care providers corroborate the studies' findings, both sets of which were scheduled to have been updated by mid-August.
"A disaster is an abnormal event, and people being affected by that is normal," allows Anthony Speier of the Office of Mental Health at the Louisiana Department of Health and Hospitals. "But Katrina falls into the realm of a catastrophic event. We are not set up to help a population recover from that," he adds.
Katrina differs from other storms not only for its sheer magnitude but also for the stymied rebuilding efforts following it. The federal disaster area spanned the size of Great Britain, at least 1,836 people perished and some 1.5 million people were displaced, creating the largest population migration in the U.S. since the dust bowl of the 1930s. Enticing people back to their neighborhoods without health, educational or criminal justice systems to support them there is difficult, so most neighborhoods have remained deserted in gray shambles with negligible visible change in the past year, according to Speier.
This open-ended holding pattern and continued displacement have perpetuated feelings of loss of control, which correlate with depression and anxiety. "Many people still live in conditions and with uncertainties that would rarely still be present nearly two years after a disaster," Norris says.
This storm was particularly cruel in that it hit people with very few resources very hard. For many it took away not only their home and friends but also their social identity, job and any sense of self-sufficiency. Abramson and NCDP director Irwin Redlener note that of those they surveyed who had annual salaries of $10,000 or less before the storm, 53 percent were still out of work a year after it. Rents have doubled, though, and the FEMA trailer parks where many now indefinitely reside have proved to be pressure cookers for despair. People feel unsafe among their neighbors and isolated from the rest of the city, and the density of depression, Abramson observes, has a community-level, spiraling effect.
These assessments bear political significance because federal disaster spending is based on the assumption that once an area's infrastructure recovers, the population will recover naturally. Direct compensation for loss is one of the lowest priorities, practically nonexistent for individuals who owned no property to begin with. And the Stafford Act, which allows for short-term mental first aid after a disaster, is not designed to support long-term therapies that help to overcome persistent distress.


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- Filed Under
I dropped mama off at her house and logged another 20 something miles to mine. The two of us had gone to Dallas to see my sister Rhonda and watch my nephew play ball. The weekend had been wonderful. The weekend had been long. It felt good to pull in my drive. I wanted to see Phil and Dixie Belle. Dixie met me at the top of the hill. Phil met me at the door.
After 30 years, I can read the man like a book. I knew immediately that something was wrong and I was right. It was Dixie. While I was gone she had somehow managed to get into some rat poison belonging to our neighbor. One emergency trip to the vet and several hundred dollars later, all signs were pointing to a full recovery. Phil reassured me she'd be fine, provided we gave her the three pills a day that would prevent her developing a serious blood disorder. If that were to happen, it could be fatal.
Phil and I said it then, and we've repeated it a few times since. We're both glad he noticed the empty packaging lying in the yard. Had he gone hunting that afternoon without seeing it, Dixie could've begun bleeding internally and - well, I don't want to think about it. Let's change the subject.
May I remind you that the culture we believers live in offers us deadly packages 24/7. It's often called entertainment but much of it would be more accurately described as poison. Often times you can consume it without any visible signs; at least none that are immediate, but that doesn't mean it's not damaging your soul. The exceedingly good news is that the Great Physician still makes house calls.
If you've been dining on what could be deadly, please call Him before it's too late.

subset: commoncrawl


text:
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Granting Your Parents Access To Your Portal Information
New Student Summer 2015 Information Series: Message Four
You should never share your AU account information with anyone else, not even your parents. However, you may want a convenient way to share your academic and financial information with them... or at least give them an easy way to add more EagleBucks to your account!
If you wish, you can grant your parents, guardians, or others the ability to access your academic and administrative information through a special "account" on the portal. You may create a separate user name for each individual and specify the level of access you authorize for him or her. To protect your privacy, the Family Educational Rights and Privacy Act requires us to obtain your permission before sharing your educational records.
To grant access, follow the steps below:
- Log in to myau.american.edu with your USER NAME and PASSWORD.
- Click on the ACADEMICS link in the personalized links section to expand the options.
- Select AUTHORIZE ACCESS TO MY DATA.
- Select the individual's name from the list provided (if you do not see him or her, click ADD SOMEONE ELSE TO MY LIST and follow the instructions).
- Create a USER NAME and PASSWORD for that individual. (Note: This type of user name always begins with "myau_" and the password must be at least six characters.)
- Check the boxes to indicate what you want this person to see.
- Click on the AUTHORIZE button. When the process is complete, you will receive an e-mail message verifying the user name.
- Share the assigned user name and password with this individual.
That's it! When the person you authorized logs in to the portal with the user name and password you created, they will see only the links that you enabled for them. You can modify the access level at any time by checking and unchecking the items available.
You can also contact the Help Desk to have the password reset or to have the account deleted. These accounts are yours to manage, and the Help Desk will only reset the passwords for you or the account holder.
If you have any problems or questions related to the parent portal, please contact the OIT Help Desk at 202-885-2550, email@example.com, or AskAmericanUHelp.
This is the fourth in a series of information messages that will introduce you to important technology services at American University. You can view previous messages and other tips for New Students by visiting www.american.edu/technology/newstudents. Click on the SUMMER 2015 INFORMATION SERIES link. Enjoy your summer.
IT Help Desk
Office of Information Technology
American University
IM: AskAmericanUHelp


text: Suspect (L) and Associate (R) - Lubbock Police [ Lubbock Police Still Searching For Suspect in 2009 Unsolved Murder of Christopher Perez By Cole Shooter August 2, 2013 This week marked the fourth anniversary of the unsolved murder of 26-year-old Christopher Perez outside of the Boss Office Bar, located at 518 East 34th Street. Read More|Comment Category: Lubbock News Tags: christopher perez, lubbock police Send to a friend! Share on Facebook Share on Twitter Pin it! Reddit This! Share on Tumblr!


text:
HOW IT ALL BEGAN...Told by Len McCulloch
I was working as a Psychotherapist in a rehabilitation facility for people recovering from Traumatic Brain Injuries when a unique patient came to our program. The man had spent the previous eighteen years in State Hospital. He was distinctive because we knew he could talk, but he wouldn't. One day, someone told me they had heard him singing! When I saw him next, I said "I know you don't like to talk but, can you sing?" He belted out a beautiful rendition of "Amazing Grace." I'm not sure why, but my response was, "We have a Choir now and you are it!" He and I met in my office and communicated by singing simple songs back and forth. After a while, he eventually began talking!
In the mean time, other patients heard our singing, asked me about the choir, and joined. Soon, we grew in numbers and gave our first public performance. It took place at a talent show held by the Brain Injury Association of Michigan at their Annual Conference. We sang 5 simple songs and wore borrowed choir robes from a local church. We didn't produce a musical masterpiece, but we were a hit with the crowd and were invited back on the spot to perform the following year.
Over the years, we have put on over 160 FREE shows (please click here to see a list of some of our performance venues). We have performed for the entire Michigan Legislative body for 14 years in a row to kick-off March as Brain Injury Awareness Month and now promoting Choir Therapy Month in Michigan. We have also sung at 12 Annual Christmas Concerts at the Historic Detroit Rescue Mission. In addition, we have produced 4 PBS Specials and 5 CDs.
In addition to providing FREE shows, we also donate clothing we collect from the community to the Historic Detroit Rescue Mission. One year we clothed 3,000 homeless!
More recently, we began to take the Therapy Choir concept to the next level by forming our own Michigan 501 (c) 3 Non-Profit Organization, which we named The Therapy Choirs of Michigan. We started our second Choir at the Macomb Oakland Regional Center (MORC) and set the predominant focus on helping people with developmental disabilities.
Although we don't know what the future has in store, we know one thing: it will be exciting!


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Julie Cox
|Julie Cox
24 April 1973
Ely, England
Julie Cox (born 24 April 1973) is an English actress, perhaps best known for her role as Princess Irulan in the Sci Fi Channel's 2000 miniseries Frank Herbert's Dune and its 2003 sequel, Frank Herbert's Children of Dune.[1][2][3]
Early life and education[edit]
Cox was born in Ely, Cambridgeshire,[4][5][dubious ] to a father who worked as an engineer in the oil business, and a mother of Irish and Scottish ancestry who died when she was a child.[6]
One of Cox's earliest roles was the Childlike Empress in the 1994 film The NeverEnding Story III. She played Diana, Princess of Wales in Princess in Love by David Greene, a film released in 1996 based upon the publication by Anna Pasternak.[7] Cox played the character Sophie Aronnax in a remake of 20,000 Leagues under the Sea in 1997, and in 1999 she appeared as Giulietta in the film adaptation of Alegría.
Cox portrayed Princess Irulan in the 2000 Sci Fi Channel miniseries Frank Herbert's Dune and its 2003 sequel, Frank Herbert's Children of Dune.[1][2][3]
Cox starred with Jean-Claude Van Damme in Second in Command (2006) and in 2007 was the female lead in The Riddle alongside Vinnie Jones, Sir Derek Jacobi, and Vanessa Redgrave. She starred in The Oxford Murders with Elijah Wood and John Hurt in 2008.
Cox portrayed a fictionalised Mary Shelley, a companion of the Eighth Doctor, in several Big Finish Productions Doctor Who audio dramas, including The Company of Friends, The Silver Turk, The Witch from the Well and Army of Death.
- ^ a b Franklin, Garth (2000). "Review: Frank Herbert's Dune". DarkHorizons.com. Retrieved December 9, 2013.
- ^ a b "Dune: Cast & Details". Movies.TVGuide.com. 2000. Retrieved December 9, 2013.
- ^ a b "Children of Dune: Cast & Details". Movies.TVGuide.com. 2003. Retrieved December 9, 2013.
- ^ Julie Cox Online - Biography
- ^ Julie Cox profile - IMDb.com
- ^ http://juliecox.free.fr/biography.html
- ^ http://dianaslegacy.com/Difilms/?page_id=30
External links[edit]
- Julie Cox at the Internet Movie Database
|This article about an English actor or actress is a stub. You can help Wikipedia by expanding it.


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Many programs in Windows 7, such as Firefox, will utilize your geographic location. This location will be used in a variety of ways including letting people know where you are when you send emails and take pictures etc.
To set your default geographic location, do the following:
1. Click the Start button, type loc, and click on Default Location
2. From this screen, you may set you default location settings
3. When you are done, click Apply Now you are ready for maximum utilization as more
location-aware programs and services are released.
Windows 7 has some excellent new features.Also read other tips and features of Windows 7. And I can say that Windows 7 is Microsoft"s best operating system to date; I am in no doubt about this statement. The future is bright.
If you like bench3, please like us on Facebook also leave your comment so that it may help others who are following you to this website. Thank You For Visiting bench3."


text:
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Plumbing & Drain Cleaning
Performance Plumbing Inc. is a Class A plumbing contractor with over 25 years of plumbing and drain cleaning experience.
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Performance Plumbing Inc. can handle any type of vac job and clean any type of pipe or storm drain 24 hours a day.
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1992 Horatio Alger Award Winner
"On the edge of the ravine, you always find the most beautiful flowers. We guide our business by that principle—taking well-placed risks is essential."
Born in 1932 in Austin, Minnesota, Richard Knowlton was third in a family of seven children. His father was a life-long employee of the nearby Hormel Company. Knowlton also worked at the plant from the age of 16. He told his father he was thinking of quitting school because he felt good about the money he was making at Hormel, but his father encouraged him to finish his education.
An excellent student in high school, Knowlton was a member of the National Honor Society. His skills in football won him a scholarship to the University of Colorado. Upon graduation, he received an offer to play professional football, but instead chose to return to Hormel, where he had worked every summer.
Following a steady line of promotions, Knowlton was elected president of Hormel in 1979, followed by appointments as chief executive officer and chairman. In January 1996, he completed his 48-year career with Hormel Foods Corporation, and has stayed on as chairman of the Hormel Foundation. Knowlton is the only food company CEO to ever receive all three of the highest awards in his industry. Recently, the Knowlton Award was established in his name to annually recognize the person who has made the best and most innovative contributions to the food industry.
Proud of his Horatio Alger Award, Knowlton says, "My passion for Horatio Alger is one that continues to grow." Knowlton has served on the Horatio Alger Board of Directors for a number of years, most recently as chairman. "My interest and commitment to Horatio Alger has intensified," he says. "Education needs have reached new dimensions. One of the most satisfying accomplishments with the Association has been the dramatic increase in the number and amount of scholarships we award. This is one of the most gratifying aspects of my involvement with the Horatio Alger Association."
When advising youth, Knowlton says, "Establish work and social habits that form a strong value system within. Later, that foundation will provide a sound basis for the thousands of decisions and choices you have to make along the way. Always remember, it's the little things you do that make up who you are as a person. Leaders should be measured by how well they serve their people, both in the workplace as well as in the community."
Knowlton believes it was his strong value system that got him through a difficult time at Hormel, when the casualty rate in the industry was nearly 100 percent. To survive, he restructured the company and guided Hormel each step of the way, using what he calls "points of difference" to help his company succeed when others were failing. "Always believe you can make a difference," he says. "Always search for a better way. Establish those qualities that are so much a part of you that it guides you in every situation."


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November 26, 2015
Six days after the violent storms that knocked out power to more than 2 million people in parts of the Midwest and the mid-Atlantic region, utility crews are still working to return lights and air conditioning to hundreds of thousands of others.
Nearly half of the 500,000 people who are still without power are in West Virginia. The state's heavily-forested and hilly terrain is slowing the thousands of workers trying to restore electricity.
The utility that covers much of the nation's capital and its Maryland suburbs, Pepco, has received some of the harshest criticism in the aftermath of the storm. As of this morning, it says it had restored power to more than 90 percent of its customers.
Meanwhile, a new round of summer storms is making its way across
Michigan and Ohio, knocking down trees and power lines. Some of the new outages are in areas where power had already been restored.
Utilities say more than 300,000 homes and businesses across Michigan had their power knocked out by the new round of thunderstorms.
Click Here to access the online Public Inspection File
Viewers with disabilities can get assistance accessing this station's FCC Public Inspection File by contacting the station with the information listed below. Questions or concerns relating to the accessibility of the FCC's online public file system should be directed to the FCC at 888-225-5322, 888-835-5322 (TTY), or firstname.lastname@example.com.


text:
A person who drinks excessively, often has sex with mudsharks and throws people in trunks of police cars.
Hey that guy got really schumered. or that guy pulled a schumer by putting those innocent people in the trunk.
by justin Igger November 21, 2007
Top Definition
An elected offical who acts solely for the benefit of publicity and cannot resist the opportunity to appear before TV cameras or radio microphones but who then does nothing at all to follow the matter they discussed up with any actual action.
We had three Schumers from the city council here two years ago for a press conference about the problem, but the situation is the same because they never did anything to follow up.
by Paula10001 August 09, 2010
1. any beverage that contains both alcohol and caffeine;
2. Sen. Charles "Chuck" Schumer, who seeks to ban the sale of such beverages in the state of New York.
2. Sen. Charles "Chuck" Schumer, who seeks to ban the sale of such beverages in the state of New York.
by NYC4Schumer November 13, 2010
An individual who so craves publicity that his/her behavior results in continual douchebaggery.
What a schumer that Senator Douchebag is.
Senator Douche is such a schumer!
Senator Douche is such a schumer!
by Sam Minardi October 27, 2011


text:
I was blessed with two great-grandchildren in the past year. This is Antwan, who was a three pound preemie! He's four months old and celebrating his cousin Taliyah's first birthday.
This is my second great-grandchild
By: katerina111Subscribe Channel: Babies Today
+ Comment!
This story is inappropriate and should be flagged for moderation. Please choose from one of the following options:


text: 2012 SASQUATCH! LINEUP ANNOUNCEMENT Feb 2, 2012 15:31:26 GMT -8 Quote Select PostDeselect PostLink to PostMemberGive GiftBack to Top Post by Lump on Feb 2, 2012 15:31:26 GMT -8 Feb 2, 2012 15:30:31 GMT -8 mazdawg said:GREAT meow I WANT PIE AND BEEF JERKYYou are a kitten inspired by other kittens.EDIT: Didn't realize Geno already posted that video. It all makes sense now. The meow is definitely a great filter touch though.


text:
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Categories: Real Weddings Posted By Real Vendors


text:
Can I buy a second lightning adapter
I have a second charger but would like a second lightning cord to go with it. That way I can have one at home and one in my office.
- Asked by Karen F from Pender Island
- Flag as inappropriate (Can I buy a second lightning adapter)
- Asked about: Apple 12W USB Power Adapter
1 Answer from the Community
Yes, of course you can buy a second one. You would have to buy it separably. But it won't come with your iPhone or any device with a lightning charging port.
- Answered by Carolyn H from Glassport
- Flag as inappropriate Answer 1
Similar Questions
Lightning Digital AV Adapter to HDMI to VGA adaptor?
- Asked by Robert P from Fishers
- 29/01/2014
- Flag as inappropriate (Lightning Digital AV Adapter to HDMI to VGA adaptor?)
- Asked about: Lightning Digital AV Adapter
Will the iPad 2 digital av adapter (with the lightning to 30 pin adapter) work on iPad 4
- Asked by Gerald C from Comstock Park
- 03/11/2013
- Flag as inappropriate (Will the iPad 2 digital av adapter (with the lightning to 30 pin adapter) work on iPad 4)
- Asked about: Apple 30-pin Digital AV Adapter


text:
For today's short product development cycles, NXP helps you to simplify your design. SimPort makes it easy to calculate efficiency, predict real-word performance, simulate a design, and track your findings.
The Active Datasheet lets you vary the test conditions and redraw many of the graphs typically provided in a conventional datasheet. The online Buck Designer prompts the user for a few basic parameters and then calculates the most efficient MOSFET combinations for a given synchronous buck regulator. The pre-designed analog simulation features let you adjust component values to reflect different devices (like inductors and capacitors), and lets you generate input, output, and switching waveforms. And, when you're done, the Design Summary tab gives you a handy summary of all the design parameters and waveforms.
It's never been easier to select a Power MOSFET, so be sure to use SimPort for your next design!
The first web based design and monitoring solution for power management ICs compliant with the current PMBus protocol specifications is now live. NXP´s PIP8000 power-supply interface lets the designer configure, sequence, and monitor power supplies using PMBus commands.


text:
Jump to content
Posted by Crisis
on 04 March 2014 - 11:22 AM
Smoke=fire. You don't need 25 legit sources to add two and two.
Smoke=fire. You don't need 25 legit sources to add two and two.
You need at least one legit source, which we're still waiting for.
Posted by Crisis
on 13 December 2013 - 03:52 PM
So it's weird for a 40yr old man to have a jersey of his favorite team with his own name on the back, but it isn't weird for a 40yr old man to wear a jersey with the name of an 18yr old boy on the back?
I am neither a 40yr old man or someone with their own name on the back of their jersey. Just an interesting question.
Community Forum Software by IP.BoardLicensed to: NJDevs.com Administration


text:
- Age / Gender:
- n/a, Female
- Location:
- Location not disclosed
- Joined:
- 9/27/10
- All Stats >
User Statshot
- Community Stats
Level 6 Blank Slate
Normal Whistle
Ranked as Civilian
Contact Info / Websites
Recent Game Medals
Giant in Armor 5 Points Kill Giant in Armor Medal Stats.
Giant 5 Points Kill Giant Medal Stats.
Brigand in Armor 5 Points Kill Brigand in Armor Medal Stats.
It's a Trap! 10 Points Make 100 mines (poisonous, energy or volcanic) of the enemies Medal Stats.
The Apotheosis of War 10 Points Defeat 2000 common enemies Medal Stats.
Look After the Pence... 10 Points Collect 10,000 gold picking up coins Medal Stats.
Drunken Master 5 Points Collect 100 mana potions Medal Stats.
V.I.P. Cemetery 10 Points Defeat 50 champions Medal Stats.
Thunderstorm 10 Points Damage 50 enemies with the thunder scroll Medal Stats.
Tastes Like Chicken 10 Points Turn 50 enemies into chickens Medal Stats.
Total Medals Earned: 671 (From 109 different games.)
Favorite Movies
- Move Your Feet - Rated 4.4404 Stars Animated to "move your feet" by Junior Senior, for the greatest super saiyan ever! Music Video Slush Invaders Rated 4.2191 Stars The stick residents of Slusher fight back hoards of invaders! Action anime munters Rated 4.4642 Stars animation for my own amusement haha! Music Video Mr. Literal Rated 4.4209 Stars You've all met this guy. But you've never seen him go over the edge... Comedy - Original


text:
On September 19 and October 3, 2001, the Department of Public Instruction received a complaint under state and federal special education law from XXXXX against the Milwaukee Public Schools. This is the departments decision regarding that complaint. The issues are whether the district failed to:
- provide the speech and language services required by the childs individualized education program (IEP) during the 2001-2002 school year;
- properly notify the parents of an IEP team meeting to be held in late September or early October 2001 and to schedule the meeting at a mutually agreed on time;
- provide the parents, prior to the IEP team meeting, with the childs recent assessment materials following their request;
- include in the childs IEP the frequency of the services to be provided; and
- consider the parents concerns for enhancing the education of their child.
The district acknowledged to the department that it failed to provide required speech and language services to the child for approximately the first three weeks of the current school year. The district convened an IEP team meeting on October 2, 2001, to determine if the child requires additional services to compensate for the delay in providing services at the beginning of the school year. The IEP team determined that the child does need additional services, and the district is now providing speech and language services in accordance with the childs October 2 IEP. Therefore, the department will not require additional corrective actions regarding this issue.
On September 21, 2001, the district called the parents and sent them an invitation for an IEP team meeting to be held on October 1, 2001. The parents declined that meeting date. On September 24, 2001, the speech pathologist sent the parents a letter requesting that they inform the district of an acceptable date for the IEP team meeting. On September 26, 2001, the district called the parents regarding rescheduling the IEP team meeting. On September 28, 2001, the speech pathologist sent the parents a letter making another attempt to reschedule the IEP team meeting, to be held on October 2, 2001, and indicating that the parents had declined that time. The letter requested that the parents let the district know as soon as possible what time would work for them. The parents did not contact the district about another time to hold the meeting. The IEP team meeting was held on October 2, 2001. Both parents arrived for the meeting, and the childs mother participated in the IEP team meeting. The department concludes that the parents were properly notified of the IEP team meeting, that the district repeatedly attempted to schedule the meeting at a mutually agreed upon time, and that a parent participated in the meeting.
On September 24, 2001, the childs father requested in writing that the district provide him with a "copy of all documentation which will be discussed at the 10/1/01" (sic). The parent informed the department that, by this request, he was asking to receive copies of his childs phonological analysis and progress report prepared by the speech pathologist. The district provided the parents with the two-page report at the beginning of the October 2 IEP team meeting. The IEP team discussed the childs progress, and the speech pathologist explained the phonological analysis to the team. The IEP indicates that the district informed the parent of the right to additional time, and the district stated that the parent did not request additional time to consider the two-page report.
A school district is required to provide parents of a child with a disability, on request, access to their childs education records without unnecessary delay and before any meeting regarding an IEP or a due process hearing and in no case more than 45 days after the request has been made. Records include any information directly related to the student and maintained by an educational agency. Here, the district provided the requested records at the beginning of the IEP team meeting. The parent, along with the entire IEP team, had time to review the two-page report, received an explanation of it from the speech pathologist, and discussed it at the IEP team meeting. In addition, the district informed the parent of the right to request additional time before proceeding with the meeting. Based upon these circumstances, the district met the requirements to provide the requested information.
The childs October 2 IEP states that she will receive specialized instruction in speech intelligibility for 90 minutes per week in 2 to 3 timed sessions. The amount and frequency of services was discussed by the IEP team, including the parent, at the meeting. The department concludes that the childs IEP includes the amount and frequency of services to be provided by the district.
The October 2 IEP also includes information provided by the parent at the meeting regarding the childs present level of educational performance. The information relates to the childs strengths and the parents concerns about the childs education. The parents of a child with a disability are expected to be equal participants along with school staff in developing, reviewing, and revising their childs IEP. This is an active role in which the parents provide, among other things, information regarding their childs strengths and express their concerns for enhancing the education of their child. The department concludes that the district considered the parents concerns for enhancing the childs education and included that information in the childs IEP.
This concludes our review of this complaint, which we are closing.
//signed 11/30/01
Carolyn Stanford Taylor
Assistant State Superintendent
Division for Learning Support: Equity and Advocacy


text:
Construction work is underway for the new 10-screen Carmike Cinema, located on the southend of South 44th Street in Manitowoc on Wednesday. The general contractor is the Twin Shores Construction Management of Bettendorf, IA. Photo taken on Wednesday, October 2, 2013. Sue Pischke/HTR Media.
- Filed Under
When the next "Star Wars" episode debuts in 2015, movie-goers in the Manitowoc area will be able to watch it on Carmike Cinema's "BIGD" screen.
The $6 million movie-restaurant development project is under construction on South 44th Street on the city's west side just off Interstate 43 and south of the Harbor Town retail center. The new Carmike 10 is set to open in May 2014.
Publicly traded on the NASDAQ (CKEC), the Columbus, Ga.-based Carmike Cinemas is one of the nation's largest motion picture exhibitors with some 250 theaters and 2,500 screens in 36 states. ...


text:
Wolverine slayed by swine flu
20th Century Fox has cancelled plans to release X-Men Origins: Wolverine in Mexico this weekend because most of the city's cinemas are closed.
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Protein phosphorylation regulates a multitude of biological processes. However, the large number of protein kinases and their substrates generates an enormously complex phosphoproteome. The cyclin-dependent kinases - the CDKs - comprise a class of enzymes that regulate cell cycle progression and play important roles in tumorigenesis. However, despite intense study, only a limited number of mammalian CDK substrates are known. A comprehensive understanding of CDK function requires the identification of their substrate network.
We describe a simple and efficient approach to identify potential cyclin A-CDK2 targets in complex cell lysates. Using a kinase engineering strategy combined with chemical enrichment and mass spectrometry, we identified 180 potential cyclin A-CDK2 substrates and more than 200 phosphorylation sites. About 10% of these candidates function within pathways related to cell division, and the vast majority are involved in other fundamental cellular processes. We have validated several candidates as direct cyclin A-CDK2 substrates that are phosphorylated on the same sites that we identified by mass spectrometry, and we also found that one novel substrate, the ribosomal protein RL12, exhibits site-specific CDK2-dependent phosphorylation in vivo.
We used methods entailing engineered kinases and thiophosphate enrichment to identify a large number of candidate CDK2 substrates in cell lysates. These results are consistent with other recent proteomic studies, and suggest that CDKs regulate cell division via large networks of cellular substrates. These methods are general and can be easily adapted to identify direct substrates of many other protein kinases.
Reversible protein phosphorylation is one of the most common posttranslational modifications and regulates virtually all cellular processes. Protein kinases are among the largest known gene families with more than 500 human kinase genes that comprise nearly 2% of the open reading frames of the human genome [1]. Moreover, approximately 30% of all cellular proteins are phosphorylated [2]. The large number of kinases and their substrates make it very difficult to determine which proteins are phosphorylated by specific kinases in vivo, but this information is critical to understanding kinase functions and the control of biological processes in general. Various strategies have been developed to identify protein kinase substrates, and several have resulted from recent technological advances in substrate detection.
Some approaches have utilized antibodies against phosphomotifs within the substrate proteins as affinity reagents to enrich for phosphorylated peptides. Examples include using antibodies that recognize conserved motifs that are highly specific to a particular kinase [3], as well as the use of phosphomotif antibodies combined with changes in physiological conditions that stimulate kinase function [4]. However, it is difficult to apply these approaches to kinases that phosphorylate broader substrate motifs, since there is less epitope conservation among substrates. Another recent method combined quantitative phosphoproteomics with kinase knock-outs and cellular perturbations to identify kinase targets in yeast [5]. However, with these cell-based approaches, it is often difficult to determine if the putative substrates are direct kinase targets. An in vitro approach employing arrays of proteins phosphorylated by isolated recombinant kinases has been successfully used in a global analysis of yeast kinase substrates, but this strategy may be difficult to apply to organisms with larger proteomes [6].
A 'chemical genetic' approach developed by Kevan Shokat's laboratory addresses many of these potential problems [7]. In this technique, the kinase to be studied is mutated by replacing a conserved bulky residue within the ATP-binding pocket with a smaller residue. This creates an enlarged ATP binding pocket that enables the mutant kinase to utilize bulky ATP analogues that cannot be used by wild-type cellular kinases, thereby isolating the activity of the mutant kinase from all other cellular kinases [8]. This technique is broadly applicable to most protein kinases and has led to important advances in substrate identification, including the description of a large number of potential cyclin-dependent kinase (CDK) substrates in yeast [9-15]. However, several technical hurdles add substantial challenges when applying this approach to mammalian kinases with broad substrate networks.
In this study, we report a method employing the Shokat strategy to identify direct cyclin A-CDK2 substrates in human cell lysates. CDK2 is activated by both the cyclin E and cyclin A subunits, and cyclin A-CDK2 plays critical roles in cell cycle control, primarily in G1 and S-phases [16,17]. We used an engineered cyclin A-CDK2 and ATP-γ-S analogue to label proteins with thiophosphates in cell lysates, and after digestion of the protein mixtures, we employed a single-step chemical enrichment procedure to selectively isolate thiophosphorylated peptides. As these studies were nearing completion, Blethrow et al. [18] independently reported a similar approach employing engineered CDK1 and thiophosphate enrichment methods that they used to identify a group of 68 putative cyclin B-CDK1 substrates within Hela cell lysates.
We identified 180 proteins and over 220 phosphopeptides that were phosphorylated in cell lysates by cyclin A-CDK2, and these proteins represented diverse cellular pathways. To validate these methods, we selected several candidate substrates and confirmed that they were phosphorylated by cyclin A-CDK2 in vitro on the same sites that we identified in the screen. Finally, we selected one novel substrate, the ribosomal protein RL12, for further study: site-directed mutagenesis and phosphopeptide mapping confirmed that CDK2 phosphorylates RL12 in vitro and in vivo on the same site determined by our methods.
Results and discussion
Utilization of ATP analogues by engineered CDKs
We generated mutant 'Shokat' CDKs containing amino acid exchanges at a conserved bulky residue in their ATP binding pockets. In the case of CDK2 and CDK3 this was a phenylalanine to alanine exchange at position 80, designated CDK2 (F80A). We also synthesized 12 ATP analogues to determine if the engineered CDKs can use these analogues to phosphorylate recombinant Retinoblastoma (Rb) protein in vitro, and found that they utilized N6-(2-phenylethyl)-ATP (PE-ATP) most efficiently (Figure 1a, and data not shown). Although both wild-type and cyclin E-CDK2 (F80A) used normal ATP to phosphorylate glutathione-S-transferase (GST)-Rb protein, only the F80A kinase could use PE-ATP. Similar results were obtained with cyclin E-CDK3, but in this case the F80A mutant could no longer use normal ATP, although the structural basis for this observation is unclear (Figure 1a). These studies confirmed that the wild-type and engineered kinases exhibit the desired ATP specificities.
Figure 1. Characterization of the engineered CDKs. (a) Cyclin E-CDK2/3 complexes, or their F80A engineered counterparts (indicated by asterisks), were immunoprecipitated from transfected U2OS cell lysates via the HA-tag on the CDK subunit and subjected to in vitro kinases assays with 10 μM of either normal ATP or PE-ATP analogue. Phosphorylation of GST-Rb was monitored by immunoblotting with a phosphospecific anti-pS780-Rb antibody (New England Biolabs). The wild-type kinases cannot use PE-ATP. (b) Thin-layer chromatography - analysis reveals hydrolysis of ATP in cell lysate and transfer to acceptor nucleotides (left). The positions of the free phosphate and ATP are indicated. In contrast, ATP-γ-S is not hydrolyzed in cell lysates (right). (c) Kinase assays were performed using wild-type and cyclin A-CDK2 (F80A) complexes purified from E. coli and GST-Rb in the presence of 200 μM of ATP, ATP-γ-S and PE-ATP-γ-S at room temperature for 2 h. Kinase reactions were analyzed by SDS gel electrophoresis and visualized by Coomassie staining. Extent of the GST-Rb phosphorylation was monitored by the electromobility shift of GST-Rb.
Whereas the mutant CDKs efficiently used PE-ATP to phosphorylate Rb in vitro, similar experiments utilizing radiolabeled PE-ATP in cell lysates failed because the labeled phosphate was cleaved from PE-ATP by an ATPase activity in the lysates (data not shown). We therefore switched to the thiophosphate form of the ATP analogue (PE-ATP-γ-S), which was not hydrolyzed by lysates (Figure 1b). Although kinases often use ATP-γ-S less efficiently than normal ATP, thiophosphorylation has several advantages in this context. First, thiophosphates are more stable and resistant to phosphatases [19]. Second, since there are no pre-existing thiophosphorylation events in the cells, thiophosphate labeling provides unique markers for proteins phosphorylated by the mutant kinase. Finally, the thiophosphate group has similar chemical properties as the sulfhydryl group and is amenable to chemical modifications. We expressed and purified soluble wild-type and cyclin A-CDK2 (F80A) complexes from bacteria and carried out a similar Rb kinase assay to test their ability to use PE-ATP-γ-S. As shown in Figure 1c, although both kinases can use ATP or ATP-γ-S to phosphorylate GST-Rb protein (as indicated by its electromobility shift) only the F80A mutant can use PE-ATP-γ-S. We thus used PE-ATP-γ-S for all of our subsequent studies.
Single-step purification of thiophosphorylated peptides
The use of engineered CDKs and ATP analogues facilitates highly specific substrate phosphorylation: the next challenge is how to identify them within a complex lysate. We sought to utilize the thiophosphate tags to covalently capture and enrich thiophosphorylated peptides after phosphorylation and digestion of the lysates. However, a key problem was to chemically distinguish thiophosphopeptides and cysteine-containing peptides. Although a chemoselective method for enriching thiophosphopeptides has been described, the overwhelming abundance of cysteine residues in a complex protein mixture makes this approach difficult [20]. We used a simple capture-and-release method to selectively isolate thiophosphorylated peptides within trypsinized cell lysates (Figure 2a). Proteins within the lysate were phosphorylated in vitro with cyclin A-CDK2 (F80A) and PE-ATP-γ-S. The protein mixture was subsequently digested and the resulting peptides were mixed with Thiopropyl Sepharose 6B [21], an activated disulfide resin that captures both cysteine-containing peptides and thiophosphopeptides through a disulfide exchange reaction. Because traditional dithiothreitol (DTT) elution releases both types of bound peptides, we utilized the qualitative differences between resin-bound thiophosphate peptides and cysteine-containing peptides at the phosphorothiolatesulfide linkage and alkyldisulfide linkage, respectively. At high pH values, the phosphorothiolatesulfide linkage is hydrolyzed and the alkyldisulfide remains intact [22]. Treatment of the resin with a strong base (such as sodium hydroxide) specifically releases thiophosphopeptides (and also converts them to normal phosphopeptides), but not the cysteine-containing peptides, by hydrolyzing the phosphorothiolatesulfide linkage (Figure 2b). Because peptides bound via cysteine are not eluted in the final step, we cannot recover cysteine-containing thiophosphopeptides. Moreover, the elution results in loss of the thiophosphate signature by converting the thiophosphopeptide to a phosphopeptide. Our thiophosphopeptide isolation method differs modestly from that of Blethrow et al. [18] in that we capture the thiophosphopeptides using disulfide exchange chemistry (disulfide resin) instead of alkylation (iodoacetamide resin), and we selectively elute them with base hydrolysis rather than oxidation.
Figure 2. Single-step purification of thiophosphopeptides. (a) General scheme for thiophosphospeptide isolation. Proteins were labeled with cyclin A-CDK2 (F80A) and PE-ATP-γ-S and subjected to tryptic digest. The resulting peptides were mixed with disulfide beads, which capture both thiophosphopeptides and cysteine-containing peptides. The beads were then treated with basic solution to selectively release only the phosphopeptides. (b) The chemistry underlying the thiophosphopeptide selectivity. Both thiophosphate and cysteine moieties contain reactive thiol groups that can be covalently captured by disulfide beads. At high pH values, the phosphorothiolatesulfide linkages (near the upper arrow) are hydrolyzed to allow the release of the bead-bound peptides while the alkyldisulfide linkages (near the lower arrow) are stable and thus peptides are retained on the beads. Note that during the hydrolysis of the phosphorothiolatesulfide linkage, thiophosphate is converted to normal phosphate.
To test the feasibility of this approach, we phosphorylated GST-Rb with cyclin A-CDK2 (F80A) and PE-ATP-γ-S, and applied our purification procedure to a trypsin digest of the reaction mixture. The isolated peptides were analyzed by electrospray tandem mass spectrometry (ESI-MS/MS) using an ion trap mass spectrometer. Peptides were identified by matching the tandem mass spectra to a human protein sequence database (with GST-Rb sequence added) using SEQUEST software [23]. The GST-Rb substrate contains five cysteines as well as seven SP/TP sites, which are sites favored for CDK phosphorylation [24]. We recovered multiple phosphopeptides containing only and all the expected phosphorylation sites (Table 1). Furthermore, we recovered no cysteine-containing peptides and very few non-specific peptides. This provided a proof-of-principle for our large-scale assays.
Table 1. Phosphopeptides identified from in vitro phosphorylated GST-Rb
Identification of human cyclin A-CDK2 substrates in cell lysates
Our goal was to identify potential cyclin A-CDK2 substrates on a proteome-wide scale. To reduce the sample complexity, we fractionated the whole cell lysate of HEK293 cells into 11 fractions using ion-exchange chromatography and ammonium sulfate precipitation (Additional data file 1). We then carried out in vitro kinase assays on each fraction. As a positive control, we also added a small amount of GST-Rb to each reaction. After digesting the reaction mixture with trypsin, we applied our purification protocol to isolate the thiophosphopeptides from the peptide mixtures. The recovered peptides were subjected to liquid chromatography-MS/MS analysis and database searching. We recovered varying numbers of peptides and at least one Rb phosphopeptide from each of the lysate fractions (Additional data file 2).
CDKs phosphorylate proteins in a proline-directed manner on either serine or threonine, and numerous studies support the idea that the motif S/T-P-X-R/K represents the CDK consensus motif. From the phosphopeptides we identified a total of 203 proteins: 180 candidates were phosphorylated within SP or TP motifs (proline-directed; Additional data file 3). These candidate substrates represent a wide range of biological processes, including cell cycle control, DNA and RNA metabolism, translation and cellular structures (Figure 3). A total of 96 out of 222 (43%) of the proline-directed sites conformed to the known CDK consensus (with a positively charged residue in the +3 position). Interestingly, about 24% of the proline-directed sites (53/222) contained a positively charged residue in either the +4 or +5 positions, suggesting that this motif may also be favored by CDK2. Indeed, Blethrow et al. [18] also noted that a substantial number of cyclin B-CDK1 substrates contain non-consensus sites. For the peptides that contained non-consensus sites, we found that about 50% of the corresponding proteins carried at least one K/RXLφ or K/RXLXφ motif (where φ is a large hydrophobic residue and X is any amino acid) distal to the phosphorylation sites, and almost all of them carried at least one minimal RXL motif (Additional data file 3). This is consistent with the well-established idea that these motifs promote cyclin A-CDK2 binding to substrates [16]. In addition to selecting for phosphorylations with CDK consensus motifs, we identified 28 proteins that have been previously implicated as CDK substrates (marked in bold in Additional data file 3) [25-52]. Thus, nearly 15% of our candidates have been previously found as CDK targets, further supporting the idea that our methods captured and enriched for CDK2 substrates. Finally, 43% of the phosphorylations we found have been previously identified in large-scale, in vivo phosphoproteome analyses, indicating that these phosphorylations are not limited to our in lysate conditions [53-58].
Figure 3. Classification of proteins by functional category. Numbers indicate identified proteins in each category.
Both our studies and those reported by Blethrow et al. [18] used similar phosphopeptide isolation schemes and related cyclin-CDKs, and we anticipated that there might be substantial overlap in the substrates revealed by both studies. Indeed, we found nearly 50% (30/68) of the cyclin B-CDK1 substrates in our list of cyclin A-CDK2 candidates; thus, these methods are robust and reproducible (Additional data file 4). However, there are also substantial differences between the two lists, and these likely resulted from many factors, including procedural differences, different cell types, incomplete peptide identification by MS, and substrate specificity conferred by the cyclin and/or kinase subunits. Some of these differences may also reflect the different biological functions of cyclin A-CDK2 and cyclin B-CDK1. For example, we found nine proteins involved in protein translation and/or ribosome function, but none of these proteins were found with cyclin B-CDK1, despite their relatively high abundance.
Although we identified a number of known CDK2 substrates, we did not identify some previously described CDK2 substrates. Some of the factors listed above may also account for the failure to find known CDK2 substrates in our analyses. In addition, substrates already phosphorylated by endogenous CDKs would not have been thiophosphorylated in vitro. It is also possible that some proteins were not solubilized during lysate preparation and/or the sonication step and excluded from our analyses. Finally, it is possible that large protein complexes may have been disrupted by the fractionation procedures prior to the kinase reaction, and that proteins that are phosphorylated by CDK2 only in the context of these complexes may not be discovered by our methods.
We also recovered four phosphopeptides corresponding to cyclin A and CDK2 and 27 additional phosphopeptides with non-proline directed sites (Additional data file 5). We suspected these phosphopeptides resulted from auto-phosphorylation of cyclin A-CDK2 and background phosphorylation by other kinases, and others have reported similar background phosphorylation [15]. To test these possibilities, we carried out a control kinase reaction using cyclin A-CDK2 (F80A) and PE-ATP-γ-S with no cell lysate added and recovered three of the four cyclin A-CDK2 peptides (Additional data file 5). Furthermore, when we performed a similar 'kinase-only' reaction in the presence of γ-32P-ATP, we observed 32P incorporation into both of these proteins in a dose-dependent manner (Additional data file 6). These experiments confirmed that there was background auto-phosphorylation of cyclin A-CDK2 in the original assays.
We also performed control kinase reactions using the lysate fractions, GST-Rb 'spike-in', and PE-ATP-γ-S without the addition of cyclin A-CDK2. These 'no-kinase' control reactions phosphorylated 7 of the 27 non-proline directed phosphopeptides on our list (Additional data file 5), suggesting that most, if not all, of these phosphopeptides resulted from background phosphorylation by kinases that are able to use the ATP analogue to a limited extent. For example, most of these peptides contain acidic residue-directed phosphorylation sites that are casein kinase 2 motifs. Casein kinase 2 is unique in that it can utilize GTP as well as ATP; thus, the active site may accommodate the bulky ATP analogues, such as PE-ATP [59]. Importantly, we did not recover any Rb phosphopeptides from these control experiments, indicating that there was no non-specific CDK activity in our assays. We also recovered 44 unmodified peptides, 12 of which contained cysteine residues. The majority of these peptides originated from several lysate fractions (Additional data file 2). We suspect these resulted from low-level non-specific binding of peptides to the resin despite stringent wash conditions, and in the case of the cysteine-containing peptides, from a small amount of hydrolysis of the alkyldisulfide linkage during the elution step. In summary, our methods were highly selective, and our studies identified a surprisingly large group of candidate cyclin A-CDK2 substrates, most of which have not been previously identified as CDK targets.
Validation of candidate substrates as cyclin A-CDK2 targets
We employed several strategies to validate some of the novel candidates in our list as cyclin A-CDK2 substrates. Because our protein identifications were based on peptide sequences, we began by confirming that cyclin A-CDK2 phosphorylated three full-length and native candidates that were immunoprecipitated via epitope tags from transfected 293 cells (EF2, TRF2, and RAP1). Because these proteins were not present on the cyclin B-CDK1 list [18], we determined if they are also phosphorylated by cyclin B-CDK1. Each CDK2 candidate was phosphorylated by both cyclin A-CDK2 and cyclin B-CDK1, although EF2 was phosphorylated to a lesser extent than either TRF2 or RAP1 (Figure 4a). We also expressed TRF2, RAP1, and the ribosomal protein RL12 as GST-fusions and purified them from Escherichia coli. When we used cyclin A-CDK2 to phosphorylate TRF2 and RAP1 in vitro, the proteins were highly phosphorylated, and MS analyses revealed that these phosphorylations occurred on the same sites we initially identified (Figure 4b). We also used cyclin A-CDK2 and cyclin B-CDK1 to phosphorylate GST-RL12, and found that both CDKs also phosphorylated RL12 in vitro (Figure 4c). These studies thus confirm that the peptides identified in our screen represent proteins that can be phosphorylated by cyclin A-CDK2, at least in vitro. Although we did find qualitative differences in the ability of cyclin A-CDK2 and cyclin B-CDK1 to phosphorylate specific proteins, in each case the candidates were phosphorylated by both CDKs. Because the enzyme preparation we used in these studies contained an excess of free CDK2 (F80A), we considered the possibility that some substrate phosphorylations might result from the association of endogenous cyclins with CDK2 (F80A) that was either monomeric, or that may have dissociated from cyclin A during the assay conditions. We found that the amount of cyclin B-CDK2 (F80A) activity in these extracts was negligible compared with cyclin A-CDK2 (F80A), and we could not detect any cyclin E-CDK2 (F80A) activity (Additional data file 7). Nonetheless, we cannot exclude the possibility that some peptides may have been phosphorylated by CDK2 (F80A) in complex with an endogenous cyclin, and the specificity of any candidate substrate for cyclin A versus other cyclins that activate CDK2 needs to be validated as described below.
Figure 4. In vitro validation of selective candidate CDK2 substrates. (a) HEK293 cells were transiently transfected with vectors expressing FLAG-tagged EF2, TRF2, and RAP1. Anti-FLAG antibody immunoprecipitates were in vitro phosphorylated with cyclin A-CDK2 or cyclin B-CDK1 in the presence of γ-32P-ATP (upper left panels). In parallel reactions, histone H1 was phosphorylated as a control to normalize the activities of cyclin A-CDK2 and cyclin B-CDK1 (right panel). 'C' denotes 'kinase only' reactions without transfected substrates (left panel) and 'no kinase' reaction (right panel). Protein samples were separated by SDS PAGE and the gels were transferred onto PVDF membranes. Phospho-signals were visualized by autoradiography. The membrane was subsequently probed with anti-FLAG antibody (Sigma-Aldrich) to confirm the identity of the phospho-signal bearing band (lower left panels). The asterisk represents a non-specific band from the commercial cyclin B-CDK2 preparation. (b) Kinase reaction was carried out using γ-32P-ATP, and GST-TRF2, GST-RAP1, GST-Rb (positive control) and GST (negative control) as substrates in the presence or absence of wild-type cyclin A-CDK2 kinase. Reactions were visualized by SDS PAGE followed by Coomassie staining and autoradiography (left panel). A similar kinase assay was carried out using wild-type cyclin A/CDK2 and ATP-γ-S, and subsequently subjected to a phosphopeptide isolation scheme. MS analysis confirmed that TRF2 and RAP1 were each phosphorylated on the exact sites we identified from the screen with one additional site for RAP1 (right panel). (c) Kinase assay was carried out using γ-32P-ATP, cyclin A-CDK2 or cyclin B-CDK1 with increasing amounts of purified GST-RL12. Samples were separated by SDS PAGE and the gel was stained with Coomassie (lower panel) followed by autoradiography (upper panel). 'C' denotes 'kinase only' reactions without transfected substrates.
Validation of RL12 as an in vivo CDK2 substrate
The above studies validated several novel candidates identified in our screen as CDK2 substrates in vitro. However, to determine if a novel substrate is also phosphorylated by CDK2 in vivo, we performed a more comprehensive analysis of the ribosomal protein RL12. We first mixed immunoprecipitates of epitope-tagged cyclin A-CDK2 and RL12 expressed in human cells in the presence of γ-32P-ATP and found that cyclin A-CDK2 phosphorylated RL12 in vitro (Figure 5a). The phosphorylation was largely abolished in a mutant RL12 where the identified phosphoserine S38 was replaced with an alanine, and it was restored when S38 was replaced with a threonine (Figure 5b). We then used phosphopeptide mapping to identify the peptide containing S38, and confirmed that it was directly phosphorylated by CDK2 in vitro (Figure 5c). To test if RL12 is also phosphorylated in vivo in a CDK2-dependent manner at the same site, we metabolically labeled cells with 32P-orthophosphate, and immunoprecipitated wild-type or RL12-S38A from cells overexpressing either cyclin E-CDK2, catalytically inactive cyclin E-CDK2, or the CDK inhibitor p21 (to inhibit endogenous CDKs) [60]. Because we cannot study endogenous RL12 phosphorylation due to the lack of a suitable anti-RL12 antibody, these studies examined the phosphorylation of ectopic RL12 in vivo (these transfection conditions led to an approximately five- to ten-fold overexpression of RL12 mRNA (Additional data file 8). We found that wild-type RL12, but not RL12-S38A, was phosphorylated in vivo (Figure 5d). This phosphorylation was enhanced in cells overexpressing cyclin E-CDK2, but not inactive cyclin E-CDK2, and was diminished in cells overexpressing the p21 CDK inhibitor (which inhibits endogenous cyclin-CDKs; Figure 5d). Finally, we used phosphopeptide mapping and phosphoamino acid analyses of RL12 protein immunoprecipitated from the labeled cells and confirmed that RL12 phosphorylation by cyclin E-CDK2 in vivo also occurred on S38 (Figure 5e). RL12 S38 phosphorylation in vivo has also been reported in phosphoproteome studies [54,55].
Figure 5. Phosphorylation of RL12 in vitro and in vivo. (a) HA-tagged RL12 or vector control ('vec') were transiently transfected into U2OS cells and immunoprecipitated using 12CA5 antibody. Cyclin A-CDK2 complexes were also transiently expressed separately in U2OS cells and immunoprecipitated using an antibody against cyclin A. Kinase assays were carried out using the RL12 (or control) immunoprecipitate with or without the cyclin A-CDK2 immunoprecipitate in the presence of γ-32P-ATP. (b) Similar assays were conducted using cyclin A-CDK2 and RL12 immunoprecipitates containing wild-type (wt) and the indicated RL12 phosphosite mutants. The asterisk denotes the light chain of the antibody. (c) Phosphopeptide mapping and phosphoamino acid analysis of the radiolabeled wild-type (left panel) and S38T mutant (right panel) of RL12. (d) Wild-type RL12, RL12-S38A, or vector control was transiently co-transfected with cyclin E-CDK2, catalytically inactive (dn) cyclin E-CDK2, or p21. All cells were subjected to 32P orthophosphate labeling. RL12 was immunoprecipitated from cell lysates and visualized by SDS PAGE followed by autoradiography. (e) Phosphopeptide mapping analysis was also carried out on the radiolabeled RL12 shown in (d). The bottom arrow shows a second and minor phosphorylation site detected in vivo.
Although we have validated each of the novel candidates that we have tested thus far by showing that the full-length proteins are phosphorylated by CDK2, some candidates on our list will likely prove not to be physiologically relevant cyclin A-CDK2 substrates. For example, the kinase reactions were performed in lysates, and in vivo subcellular compartmentalization may restrict the access of CDK2 to some candidates. Moreover, it is possible that other cellular kinases are either redundant with, or more important than, CDK2 with respect to individual substrates in vivo. It is thus critical that candidates be rigorously evaluated in as physiological a context as possible. Towards this end, in ongoing studies we are using a gene targeting approach to mutate a subset of these phosphorylation sites in the endogenous genes to study their physiological significance.
In summary, we describe a rapid and efficient method to identify candidate CDK substrates in cell lysates. We identified 180 candidate cyclin A-CDK2 substrates and found that our method is robust, sensitive, and capable of identifying novel CDK2 targets. Since most protein kinases have conserved ATP binding domains and the kinetics of thiophosphorylation can be optimized [61], these methods should be broadly applicable to the study of many kinases and their substrate networks. Moreover, thiophosphorylation-based phosphopeptide isolation should also facilitate the mapping of phosphorylation sites within individual proteins or protein complexes in vitro.
Materials and methods
Reagents, cell culture, recombinant protein expression and purification
All standard chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Triphosphate synthesis was carried out according the method of Ludwig [62]. PE-ATP was synthesized similar to as previously described [63] with N6-(2-phenylethyl)-adenosine as a precursor. PE-ATP-γ-S was custom synthesized by TriLink Biotechnologies (San Diego, CA, USA). All CDK (CDK2 (F80A) and CDK3 (F80A)) and RL12 phosphosite mutants were generated by site-directed mutagenesis with the Quick Change method (Stratagene, La Jolla, CA, USA). All cDNAs used in this study except TRF2 and RAP1 were generated from human mRNA via RT-PCR, and clones were sequenced. RAP1 cDNA was purchased from Open Biosystems (Huntsville, AL, USA). A TRF2 clone was obtained from Addgene (Addgene plasmid 16066, Cambridge, MA, USA). U2OS and HEK293 cell lines were maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum and expression of wild-type and mutant CDKs and other proteins in U2OS cells were performed by transient transfections or co-transfections via calcium phosphate precipitation using standard procedures. CDKs expressed in human cells were purified by immunoprecipitation using 12CA5 antibody for HA-tagged Cdk subunits or using antibodies against the cyclin subunit. GST-Rb (GST fused to the carboxy-terminal 156 amino acids of Rb), GST-RL12, GST-TRF2, and GST-RAP1 were expressed in bacteria and purified using standard glutathione resin. Purified cyclin A-CDK2 and cyclin B-CDK1 kinases used in Figure 4 were purchased from New England Biolabs (Beverly, MA, USA).
Preparation of cyclin A-CDK2 complexes expressed in bacteria
Active and soluble wild-type and F80A cyclin A-CDK2 complexes were produced by co-expressing Saccharomyces cerevisiae GST-CAK1, full-length untagged cyclin A and His6-tagged CDK2 in bacteria similar to the commercial version (New England Biolabs). Cyclin A and His6-CDK2 were cloned into the pRSFDuet-1 vector (Novagen, San Diego, CA, USA) and GST-CAK1 was cloned into pGEX-2T vector (GE Healthcare, Piscataway, NJ, USA). All three proteins were co-expressed by transforming an E. coli BL21 strain with both plasmids. For large scale preparation, cells expressing F80A cyclin A-CDK2 were grown in 500 ml of LB medium to OD600 of 0.8 and isopropyl β-D-thiogalactoside (IPTG) was added to 0.5 mM. Cells were then cultured overnight at 37°C before being harvested by centrifugation. The cell pellet was lysed by incubating with 20 ml lysis buffer (50 mM Tris, pH 7.5, 1 mM DTT, 1 mM MgCl2, 25 U/ml Benzonase (Novagen), 2 mg/ml lysozyme (Sigma-Aldrich) and protease inhibitors cocktail (Sigma-Aldrich)) at room temperature for 1 h. After NaCl was added to 250 mM and Triton X-100 to 0.025%, the cell slurry was sonicated followed by centrifugation. The supernatant was dialyzed against phosphate-buffered saline (PBS) before incubation with 3 ml of Ni-NTA resin (Qiagen, Valencia, CA, USA), which was washed sequentially with 15 ml PBS and low imidazole buffer (30 mM Tris, pH 7.5, 150 mM NaCl, 50 mM imidazole), and eluted with 6 ml high imidazole buffer (30 mM Tris, pH 7.5, 150 mM NaCl, 300 mM imidazole).
Immunoblotting, immunoprecipitation, orthophosphate labeling, and phosphoamino acid analysis
These procedures were described previously [64].
Preparation and fractionation of 293 native cell lysates
HEK293 cells were grown on 15 cm plates to near confluency and harvested in PBS buffer. Cell pellets were resuspended in hypotonic lysis buffer (50 mM Tris, pH 7.5, 1 mM DTT, 1 mM MgCl2, 0.1% Triton X-100, 25 U/ml Benzonase (Novagen), and protease inhibitors cocktail (Sigma-Aldrich)) and incubated at 4°C, followed by sonication in 150 mM NaCl. Cell debris was pelleted by centrifugation and the supernatant was diluted such that the salt concentration was below 25 mM. The whole cell lysate was then loaded onto a SP Sepharose (GE Healthcare) column manually by atmospheric pressure and the flowthrough was collected. After washing the column with loading buffer (30 mM Tris, pH 7.5, 25 mM NaCl, 1 mM DTT), bound proteins were eluted sequentially with load buffer containing 100 mM, 200 mM, 300 mM, 400 mM, and 600 mM NaCl. The flowthrough was loaded onto a Q Sepharose (GE Healthcare) column and similar procedures were carried out to collect flowthrough and elute the column. Proteins from Q Sepharose flowthrough were pelleted by ammonium sulfate precipitation at 60% and resuspended in load buffer. All fractions were concentrated with salt concentration adjusted between 100 and 200 mM by serial dilution and concentration. These fractions were used in the first set of experiments and subsequently dialyzed extensively against a Tris buffer (30 mM Tris, pH 7.5, 150 mM NaCl) to be used in a second set of experiments.
In vitro kinase assays and purification of thiophosphorylated peptides
Kinase assays using kinases purified from human cells have been described previously [64]. For kinase assays using cell lysate fractions (or GST-Rb) and PE-ATP-γ-S, recombinant wild-type and F80A cyclin A-CDK2 complexes purified from E. coli (as described above) were used. In each of the lysate reactions, about 100-200 μg of lysate fraction (and 100-200 ng of GST-Rb as positive control) was mixed with approximately 1-2 μg of cyclin A-CDK2 (F80A) complex, 250 mM PE-ATP-γ-S in kinase reaction buffer (40 mM Tris, pH 7.5, 10 mM MgCl2, 50 mM NaCl). The reaction was incubated at 30°C for 5 h and the protein mixture was denatured by adding acetonitrile to 15% and digested with trypsin (1/20 mass ratio) at 37°C for at least 6 h. Peptides were then incubated with 20 μl of disulfide beads Thiopropyl Sepharose 6B (GE Healthcare) with rotation at room temperature overnight. The beads were loaded onto a Micro Bio-Spin column (Bio-Rad Laboratories, Hercules, CA, USA), and washed sequentially with 3 ml water, 5 ml of 30% acetonitrile in 0.1% formic acid, 5 ml of 2 M NaCl, and 3 ml water. Beads were collected and incubated with 20 μl of 20 mM NaOH at room temperature for 2 h. The eluate was neutralized and acidified with 1% formic acid to pH 3 for direct analysis. Similar phosphopeptide capturing protocol was carried out for the GST-Rb kinase assay using 5 μg of GST-Rb and 0.5 μg of cyclin A-CDK2 (F80A) complex.
Mass spectrometry analysis and database search
Phosphopeptides samples were analyzed by microcapillary high performance liquid chromatography-electrospray ionization-MS/MS using an ion-trap mass spectrometer (LCQ, ThermoFinnigan, San Jose, CA, USA). Peptides were pressure-loaded onto a 75 μM × 12 cm self-packed C18 column and resolved by a non-linear gradient of 5-28% acetonitrile containing 0.1% formic acid at the flow rate of 200 nl/minute over the course of 2 h. Tandem spectra acquired were searched against the human NCI database (07.20.2006) using SEQUEST. Search parameters included one tryptic end and differential mass modification to serine and threonine due to phosphorylation. For listing purposes, all entries were manually updated using current Swiss-Prot nomenclature. Search results from two independent experiments on each lysate fraction were pooled and filtered using the statistical tool PeptideProphet [65]. Peptides with probabilities higher than 0.9 (error rate <1.8%) were manually validated to further exclude ones with poor MS/MS spectra before inclusion in the final list.
CDK, cyclin-dependent kinase; DTT, dithiothreitol; GST, glutathione-S-transferase; MS, mass spectrometry; MS/MS, tandem MS; PBS, phosphate-buffered saline; PE-ATP, N6-(2-phenylethyl)-ATP; Rb, Retinoblastoma.
Authors' contributions
YC designed the method, performed the method validation, in-lysate kinase assays, and substrate validation experiments, carried out MS and data analyses. MW performed the kinase mutagenesis and characterization, ATP analogue synthesis and substrate validation experiments. AAH contributed to substrate validation experiments. JJP contributed to the method design. RA provided MS and software resources. YC, RA, and BEC designed the research project. YC and BEC wrote the manuscript.
Additional data files
The following additional data are available. Additional data file 1 is a figure showing the scheme of HEK293 cell lysate fractionation. Additional data file 2 is a table listing the numbers of peptides identified in the lysate fractions. Additional data file 3 is a table listing the identified proline-directed phosphopeptide sequences, the corresponding protein names, and their functional categorizations. Additional data file 4 is a table comparing the proteins and phosphopeptides identified for cyclin A-CDK2 and cyclin B-CDK1. Additional data file 5 is a table listing the phosphopeptides with non-proline-directed sites and from cyclin A-CDK2 autophosphorylation. Additional data file 6 is a figure showing the autophosphorylation of cyclin A-CDK2 in vitro. Additional data file 7 is a figure showing that cyclin B-CDK2 (F80A) and cyclin E-CDK2 (F80A) complexes that may have formed in the lysate represent a negligible fraction of the total amount of CDK2 (F80A) activity. Additional data file 8 is a figure showing the Northern analysis of RL12 expression.
Additional data file 1. Scheme of HEK293 cell lysate fractionation.
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Additional data file 2. Numbers of peptides identified in the lysate fractions.
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Additional data file 3. Identified proline-directed phosphopeptide sequences, the corresponding protein names, and their functional categorizations.
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Additional data file 4. Proteins and phosphopeptides identified for cyclin A-CDK2 and cyclin B-CDK1.
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Additional data file 5. Phosphopeptides with non-proline-directed sites and from cyclin A-CDK2 autophosphorylation.
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Additional data file 6. Autophosphorylation of cyclin A-CDK2 in vitro.
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Additional data file 7. Cyclin B-CDK2 (F80A) and cyclin E-CDK2 (F80A) complexes that may have formed in the lysate represent a negligible fraction of the total amount of CDK2 (F80A) activity.
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Additional data file 8. Northern analysis of RL12 expression.
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We thank David Morgan, Kevan Shokat, Justin Blethrow, and Jonathan Cooper for helpful discussions and Titia de Lange for the TRF2 clone. This work was supported by grants from the NIH (to BEC), the Burroughs Welcome Foundation (to BEC), the Hearst Foundation (to YC), the Leukemia and Lymphoma Society (to MW); the National Heart, Lung, and Blood Institute, National Institutes of Health, under contract No: N01-HV-28179 (to RA) and by SystemsX.ch (to RA).
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November 27, 2015
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In April of this year, authorities say Wright stole more than $1,000 in jewelry from an Oak Street Home.
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What should I wear to my own engagement party?
Q. HELP!! My Fiance's mother is throwing us an engagement party luncheon at a country club at the end of March in Boston and I have no idea what to wear. I went looking for dresses last weekend and found a few that I liked but I think they are all a bit to fancy for a luncheon. From people I've talked to, they all say something along the lines of "Sunday Best" but I can't seem to find something that looks good and doesn't look either matronly or like a child. I have a few weeks but I'm starting to panic that I won't have anything and will have to wear my standby LBD and I don't want to wear black, it's my engagement and I think I should be in color or white or something that makes me stand out a bit…but clearly I've never done this before so I don't know. I know the LBD is always classic and I can add color with shoes, purse and jewelry, but I'm a very classic girl, at least where jewelry is concerned, so I was hoping to be able to wear my grandmother's pearls or her diamond necklace. Any tips would be really helpful!
A. Deep breath. Your engagement party is going to be wonderful, no matter what you wear, but of course you want to look great since you'll be the center of attention. Though it is a luncheon, feel free to dress a bit nicer than the guests, it's your party! A dress or pretty top and skirt is your best option. Though we are huge fans of a classic little black dress, you are correct that this occasion calls for something a bit more colorful or bridal. You're in luck – pretty spring dresses are everywhere now, you just need to find your perfect one.
Since we love the idea of you wearing your grandmother's pearls or diamond necklace, we have selected some pretty dresses with necklines to accommodate them. Here are some pretty, yet classic, ideas for you that will look great in photos 20 years from now:


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While the Tea Party is getting most of the media's attention regarding Common Core resistance, a silent majority is about to explode. Who are we? We are the parents of Mississippi's 492,847 public school students.
Some of us are, indeed, Tea Partiers. Some of us are liberal Democrats. Most of us, however, are apolitical folks who simply want what's best for our kids and our communities.
And we are fed up with top-down "reforms" like No Child Left Behind, Race To The Top, A-F labels for schools, and now, Common Core.
We have had enough.
No more testing. No more judging our students and teachers by data alone. No more scapegoating of public schools for our failure as a greater society to feed and nurture our children.
We ask the Mississippi Legislature to pass a moratorium on all forms of state and federal testing in our public schools, including any testing related to Common Core. We ask Governor Bryant to sign off on this moratorium.
Please give us our schools back.
Michael Van Veckhoven