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Here is an idiotic decision by a judge. He states that if the government does something illegal to everybody, then there can be no judicial relief because the complaint is no longer a "particularized injury" but instead is a "generalized grievance". Under that judicial theory, if the government decides to demand that citizens quarter troops in their homes (great way to reduce the cost of the military!), then there would be no judicial redress. This would be a "generalized grievance" despite the fact that the American revolution was fought over arbitrary state authority that demanded citizen open up their homes and allow British troops to be quartered for free. According to this judicial idiot, if the government simply made a blanket rule that everybody had a "duty" to quarter troops, then nobody could take the case to court because it wouldn't be a "particularized injury". Similarly, I guess, if the government decreed that citizen gather into groups of ten and then each group had to pick out one from the group and then proceed to burn that person at the stake, that outrageous law couldn't be fought by a court case because the government had created a "generalized grievance" and there was no "particularized injury" because everybody ran the same risk of being killed. Nutty!
A federal judge has dismissed Jewel v. NSA, a case from the Electronic Frontier Foundation (EFF) on behalf of AT&T customers challenging the National Security Agency's mass surveillance of millions of ordinary Americans' phone calls and emails.
"We're deeply disappointed in the judge's ruling," said EFF Legal Director Cindy Cohn. "This ruling robs innocent telecom customers of their privacy rights without due process of law. Setting limits on Executive power is one of the most important elements of America's system of government, and judicial oversight is a critical part of that."
In the ruling, issued late Thursday, U.S. District Court Chief Judge Vaughn Walker held that the privacy harm to millions of Americans from the illegal spying dragnet was not a "particularized injury" but instead a "generalized grievance" because almost everyone in the United States has a phone and Internet service.
"The alarming upshot of the court's decision is that so long as the government spies on all Americans, the courts have no power to review or halt such mass surveillance even when it is flatly illegal and unconstitutional," said EFF Senior Staff Attorney Kevin Bankston. "With new revelations of illegal spying being reported practically every other week -- just this week, we learned that the FBI has been unlawfully obtaining Americans' phone records using Post-It notes rather than proper legal process -- the need for judicial oversight when it comes to government surveillance has never been clearer."


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The next section, themarketing plan, gets into the details of what your business offers and what market it serves. Marketing is the communication of how your products and services "ease customer pain." Show the problem and how your business solves it. Marketing is a necessity for every business because once your doors are open, you must invite customers to come in. Everything you do in your business that affects customers is marketing because it sends a message about your company.
Right now, growth may sound like an unattainable goal as businesses are grappling just to survive, but hey, "flat is the new up." If a business can keep its doors open and lights on, then it's doing better than many others. But lights and open doors don't make sales, so making changes that attract business is in a sense, striving for growth. It won't be this tough forever, but for now, putting some growth strategies into action may be what keeps your business alive, if not thriving.
Marketing Plan
An income statement shows revenues minus expenses, in order to calculate net income or net loss. Start_ups should project these expected results for the first twelve months of business, then quarterly for the next two years. A list of a company's assets (what you own), liabilities (what you owe), and net worth (assets minus liabilities) is called a balance sheet. The statement of owner's equity shows the owner's initial investment, additional investments, and retained earnings, minus owner withdrawals.
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Faith Life at LeBlond
Students at LeBlond are always encouraged to get involved in the faith life of the school. We offer several different ways for students to develop their relationship with God and the Catholic Faith.
Sacramental Life
Students have regular opportunities for Mass, Confession and Adoration, as well as opportunities throughout the liturgical year for further faith formation.
Mass Schedule:
All-School masses are held every Thursday at 10:45
Rosary/Morning Prayer is held every day at 7:00 in the Chapel
Eucharistic Adoration: First Fridays, all day
Confessions: Held before All School Mass on Thursdays from 9:45-10:30
Campus Ministry
Campus Ministry meets every other Monday at 8:00 a.m., and EVERY student is welcome to attend. This is a student-led group that is dedicated to promoting and developing the spiritual life of the school. The students brainstorm different events and activities during the year that benefit not only the school, but in many cases the greater community. Some examples of this include:
Delivering Christmas cards to the residents of Carriage Square nursing home
Sponsoring the Catholic Schools Week Trivia Contest: this is a faith-based trivia game between the northwest Missouri Catholic grade schools held during Catholic Schools Week. The categories correspond to the subjects taught in the LB theology department
Sponsoring the Lenten Charity Drive: this charity drive happens during Lent and benefits the Noyes Home for children and Grace House charities.
St. Joseph devotions: during the month of March, a traveling statue of St. Joseph visited every classroom and was accompanied by a special prayer to St. Joseph. The school was consecrated under the spiritual patronage of St. Joseph by Bishop Johnston.
Student Retreats
We offer retreats for each grade level - and they often form some of the best memories of the entire four year experience. Our retreats are at Benedictine College and are run in conjunction with the Benedictine Retreat team. Activities include:
Full day away from school
Mass and Adoration
Talks and small group discussion
Meal at Benedictine


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GET GEARED UP is the Triathlon Club of Burlington's Season Opener.
Come out to meet new members, hear about all the awesomeness the club has planned and learn a little to make this season your best.
The event will have a social of wine and appetizers. We will be offering tire change lessons, bike maintenance how to's and everything you possibly need to know about triathlons.
Awesome door prizes are available such as fitbits, TCoB tri kits, TCoB swag, bike tune ups and more.
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DNC Chair Debbie Wasserman Schultz Resigns
By Michael A. Memoli
The Hillary Clinton campaign, responding to leaked internal Democratic Party emails that threatened to revive tensions with Sen. Bernie Sanders' followers, moved quickly to squelch the problem Sunday as the party's embattled chair, Rep. Debbie Wasserman Schultz, announced she would step down at the end of the convention week.
Wasserman Schultz, who has long been under fire for the appearance of partiality toward Clinton in the Democratic presidential primaries, announced her resignation in a statement Sunday afternoon.
The move culminated a series of steps by the Clinton campaign to sideline her _ first by appointing a new party executive last month to run the party's operations, then by taking away her speaking role at the convention and removing even the simple task of gaveling the convention in and out of session.
The announcement came after internal emails newly disclosed by the website WikiLeaks revived long-running suspicions on the part of Sanders supporters that the Florida congresswoman had tilted the scales in favor of Clinton.
On Friday, WikiLeaks released nearly 20,000 emails from a 17-month span that appeared to have been obtained by someone who hacked into the accounts of seven top party officials. The site said it was the first part of a forthcoming "Hillary Leaks" series.
One email, in particular, caught the attention of Sanders loyalists. In it, the party's chief financial officer _ an ally of Wasserman Schultz _ indicated interest in raising questions about whether Sanders lacked active involvement with his Jewish heritage.
Clinton's campaign said Sunday that the hack may have been the work of Russian operatives seeking to boost Republican nominee Donald Trump's campaign. The Democratic National Committee had reported last month that its computer system had been hacked and that the cyber-security firm hired to investigate the breach had traced it to two groups tied to Russian intelligence organizations.
Much of the newly revealed correspondence was routine in nature, showing the typical internal machinations of a major political organization. The main exception was the email from Brad Marshall, the finance chief, which asserted that Sanders had "skated on saying he has a Jewish heritage" and that he may actually be an atheist. If so, that revelation could cost Sanders support among Southern Baptists, the email said.
Although there's no evidence that Wasserman Schultz or other party officials acted on Marshall's suggestion, the mere existence of the email was enough to threaten to reopen the rift with the Sanders campaign that Clinton and her allies had spent the last month mending.
After a turbulent Republican convention that highlighted that party's internal rifts, Democrats are eager to showcase a more unified front and positive vision for the country. Wasserman Schultz, who has been denounced for months by Sanders and his supporters, had clearly become an impediment to that goal.
The Vermont senator seemed to indicate that he would continue to assist in the unity effort. During an interview several hours before Wasserman Schultz announced her decision, Sanders renewed a long-standing call for her ouster, but then briskly sought to change the subject.
"There is no question but the DNC was on Secretary Clinton's side from Day One," he said on NBC's "Meet the Press."
Still, he said, the most important thing was for Democrats to unite and focus on defeating Trump, whom he called "the worst Republican candidate that I have seen in my lifetime."
Trump's campaign, meanwhile, sought to fan the flames by suggesting that it was now Clinton's turn to step down, given her own email correspondence controversy.
Sanders' ire toward Wasserman Schultz has been building for months. During the primaries, he, as well as former Maryland Gov. Martin O'Malley _ the other Democratic candidate _ repeatedly criticized Wasserman Schultz, who had been a vocal supporter of Clinton during the 2008 presidential primary, for crafting a debate schedule that offered limited opportunities for lesser-known candidates to gain visibility.
The Sanders campaign also protested her decision to temporarily limit their access to the party's digital infrastructure after staffers were discovered to have taken advantage of a brief data breach and gained access to other candidates' voter profiling.
In her statement, Wasserman Schultz did not mention the emails or any other points of tension, but said she was "proud" of her role in laying the groundwork for a united convention.
"We arrived here in Philadelphia with the most inclusive and progressive platform the party has ever proposed and a unified recommendation from the Rules Committee on our path forward as Democrats," the statement said.
Noting her other roles as a mother and a member of Congress representing parts of South Florida _ she faces a primary challenge next month from a Sanders supporter _ Wasserman Schultz said the best way to help serve her constituents and ensure Clinton carries her home state, a key battleground, was to step down from her Democratic National Committee leadership role.
"I've been proud to serve as the first woman nominated by a sitting president as chair of the Democratic National Committee and I am confident that the strong team in place will lead our party effectively through this election to elect Hillary Clinton as our 45th president," she said.
Donna Brazile, the DNC vice chairwoman who was a campaign manager for Al Gore in 2000, will serve as interim chair through the election. She will be the first African-American woman to head the party.
Wasserman Schultz's resignation will end a more than five-year tenure during which the party retained the White House in 2012 but then lost control of the Senate and saw its ranks in the House and governorships further reduced. But it was her role overseeing the 2016 primary process that ultimately led to her undoing.
In a statement, Clinton thanked Wasserman Schultz for her leadership of the party and said she would transition to a role as honorary chair of a 50-state program to elect Democrats across the country while also serving as a surrogate in Florida.
"I look forward to campaigning with Debbie in Florida and helping her in her re-election bid _ because as president, I will need fighters like Debbie in Congress who are ready on Day One to get to work for the American people," Clinton said.
Obama tapped Wasserman Schultz to lead the party in 2011 when Tim Kaine, now Clinton's designated running mate, stepped down to launch a bid for the U.S. Senate in Virginia. Wasserman Schultz spoke Saturday at the rally in Miami at which Kaine was introduced.
Wasserman Schultz, the first Jewish woman elected to Congress from Florida, had been seen as a rising star in the party and a potential candidate for House leadership at the time. But in the last couple of years, she had reportedly fallen out of favor with some Obama loyalists who saw her interests in self-promotion conflicting with her party role.
Obama praised Wasserman Schultz in a statement, saying that for eight years she has had his back.
"Her fundraising and organizing skills were matched only by her passion, her commitment and her warmth," the statement said. "And no one works harder for her constituents in Congress."
(c)2016 Tribune Co.


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Jul 05, 2019, 1:03
Then, keep an eye to the skies with scattered strong to possibly severe storms after 3 p.m. today.
Tonight partly cloudy with temperatures falling in the upper 70s-low 80s this evening as overnight lows drop in the low-mid 70s. Heat and humidity brings the chance for isolated showers and thunderstorms in the afternoon and evening hours. Again, limited impacts are expected. "But as with Tuesday and Wednesday, scattered thunderstorms will be possible in the afternoon and evening, capable of localized damaging winds". This pattern holds on through the end of the week, with slightly better chances for storms towards the weekend. Factor in the humidity and the heat index is reaching the upper 80s or low 90s.
Partly cloudy skies with some morning fog possible. Wind: S 10-20; gusty. South wind 7 to 10 miles per hour. South wind 9 to 13 miles per hour. The heat index will stay in the low 90s all week.
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Pyongyang demanded that South Korea stop attempting to mediate between the U.S. and North Korea in a surprisingly curt statement . He previously lived in China for three years and is interested in socialism. "He was very diplomatic and sensible", said Petrov.
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Next year will be GABC South's fifth annual Kidfish! We're excited to bring this great day of fun and sun to South Tyler. Kidfish is a free event for all kids where they can learn the basics of fishing by hooking some worms, catching some fish, and maybe even winning a prize along the way!
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Mallory-Neely House -- Videography Intern
Please see instructions on how to apply at the bottom of this page.
The Intern will complete a series of five short films related to the Mallory-Neely House that will be uploaded to web - exact content TBD, but will hopefully include details about restoration projects. Additionally, the Intern will research and learn about the house and the people who lived here, to be highlighted in some of the "films" they create. This project will provide real-world experience for a film student, function as significant portfolio building for a film student, and will eventually be used for marketing and educational purposes for our institution.
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- Research and compile information pertaining to the history of the Mallory-Neely House
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1) Will be enrolled as a full time undergraduate student as of the Fall 2019 semester. You must be a rising Junior or Senior.
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On Friday, May 24th join us at The Radisson Hotel for the Featured Artist Stroll, beginning at 6:30pm and ending at The ARTS Council of the Southern Finger Lakes.
The Featured Artist Stroll begins at the Radisson Hotel Corning and ends with a reception at The ARTS Council. Stops along the stroll include:
Radisson Hotel Corning
Gustin Gallery Goldsmiths
Pure Design
West End Gallery
Throughout the weekend stop by these storefronts to see work by the Glassfest Featured Artists:
Alan Bennett
Scott Griffin
Crow's Nest Artists - Gwen Quigley & Tony Moretti
Glass Makers from Corning Museum of Glass
On Saturday, The ARTS Council will host CareFirst for a program about achieving and visualizing life goals. Artist Members, Filomena Jack, Lynn Dates and Michael Czarnecki will be there to create visual and literary articulation of your bucket list to take home - for FREE! Filomena, Lynn and Michael will bring a selection of their art works for sale.
The Evelyn Peeler Peacock Gallery will have the Pride Invitational Exhibition on view. A selection of works by members of the LBGTQ Community representing a range of styles and media have been chosen for this exhibition.
Join Pat Kane and friends for an Irish Jam Session at The ARTS Council during GlassFest on Sunday May 26 from noon to 3:00pm. We'll be outside on the street in front of The ARTS Council weather permitting, inside if it is rainy. All are welcome to come listen, play, and dance! For more information about the jam session, click here.


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Table of contents
The question of sexual abuse on victims by UN workers
Issue in GA 2: Human Rights
Historically, United Nations peacekeepers have been protected from prosecution when carrying out peacekeeping duties because they have been granted 'immunity.' Immunity allows peacekeepers to carry out their duties without being prosecuted by the governments of UN member countries.
However, many peacekeepers protected by immunity have been found to abuse this privilege, by sexually harassing and assaulting those they are sent to help. Women and children are particularly vulnerable to this.
There is, however, no current system in place to prosecute the perpetrators of sexual assault, many of whom are in positions of trust. Victims have no way of coming forward and whilst many high-profile accusations have been made, there have been no prosecutions.
In the past 15 years, over 1700 allegations of sexual abuse have been made, however, due to the difficulty in reporting an incident, many more are suspected.
There are no questions for this issue.


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Ambrose Property Group, Exhibit Columbus, and Central Indiana Community Foundation have partnered to transform sections of the former General Motors Stamping Plant site, now called Waterside, into a vibrant urban art and cultural centerpiece. The organizations are combining their efforts to run a design competition to transform approximately 25,000 square feet of the 82 year old former General Motors Chevrolet Division Commercial Body Plant (the "Crane Bay"), develop its surrounding plaza, design a pedestrian bridge connection to downtown Indianapolis, and consider the public realm framework that connects these elements within the larger development site. This ambitious and aspirational public space project aims to reimagine this space into a dynamic, inclusive, and artistically and architecturally significant programmable space.
Please join us for the final presentations on October 2nd at the Indianapolis Public Library. More information and registration can be found here.
This project is Indianapolis' watershed moment and this design competition places innovation and design excellence at the center of an important development in the connection and expansion of downtown Indianapolis. The development of the former GM Stamping Plant on Indianapolis' near west side represents a rare opportunity to impact the city's identity through design at a critical point in our city's history. This ambitious and inspiring project has dramatic potential to significantly grow the city and its reputation. The Waterside Design Competition will create bold and iconic ideas for public space, art, and architecture. Waterside will be a place where residents, regardless of identity, will explore and dream. Visitors will consider Indianapolis with a fresh lens. Bringing to life this 103-acre site in downtown Indianapolis will not only raise Indianapolis' profile on a national scale, but will also bring thousands of new residents, employees, and visitors to the urban core. It will demonstrate the capacity for architecture, art, and design to play a pivotal role in catalyzing new possibilities around the city. This project will reconnect the west side of Indianapolis to downtown by creating a new center of gravity at Waterside.
To find out more about the finalists, process and public events, visit www.designwaterside.com.


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Portugal were the talk of social media as they defeated Euro 2016 hosts France in Paris and the footballing world praised Fernando Santos' side's resilience and teamwork as well as Cristiano Ronaldo's stand-in managerial skills on the touchline.
Which Tweet is your favourite? Get in touch on social media using #360fans and let us know!
É lindo ver o povo feliz!! Is beautiful to see my country happy!!!👏👏✌ pic.twitter.com/nZWRr4WYJ7— Luís Figo (@LuisFigo) July 11, 2016
My prediction from game one was spot on! #Euro2016Final #Portugal #champion2016 @Cristiano the greatest! What u y'all got to say now?!🍺 😄😄— Chris Gayle (@henrygayle) July 10, 2016
A young Portugal supporter consoles a French fan last night. Class. (via: @LMDFoot_) pic.twitter.com/JwbkjWsQLs— Footy Vines (@Footy_Vines) July 11, 2016
Te lo mereces bichooo!! Enhorabuena Pepitooo 👏🏾👏🏾👏🏾👏🏾 parabéns Portugal!!! pic.twitter.com/av2V7UvOE2— Marcelotwelve (@MarceloM12) July 10, 2016
Is this Sissoko fella the same dude from Newcastle last year! 😳#UEFA2016— Joseph Barton (@Joey7Barton) July 10, 2016
Congrats to Portugal on their #Euro2016Final win!! 🇵🇹 Party it up today, you all deserve it! pic.twitter.com/h2zWFA2rBJ— backstreetboys (@backstreetboys) July 10, 2016
Great team performance by Portugal tonight! Congratulations! #EURO2016— Basti Schweinsteiger (@BSchweinsteiger) July 10, 2016
Congratulations to Portugal and my friends @officialpepe and @cristiano who succeed us as #Euro champions— Sergio Ramos (@SergioRamos) July 10, 2016
I'm sad for France but ... Congratulations Portugal #POR ! #EURO2016 #FRAPOR pic.twitter.com/KC79xlqsbd— David Guetta (@davidguetta) July 10, 2016
Congratulations to Portugal for winning the European cup🏆. Sissoko Man Of The Match...💪🏿👊🏿✌🏿 pic.twitter.com/qwFChBfFVD— Michael Essien (@MichaelEssien) July 10, 2016
So happy for #portugal great #teamspirit— David Seaman (@thedavidseaman) July 10, 2016
Portugal win Euro 2016 a month after their U17s won their own Euros. U21s were runners-up in theirs a year ago, Euro U19s start tomorrow...— Chelsea Youth (@chelseayouth) July 10, 2016
Great footage of Sir Alex Ferguson congratulating Cristiano Ronaldo last night pic.twitter.com/ZQvu8Iie7l— Football Vines (@FootballVines) July 11, 2016
From seeing Cristiano Ronaldo lift the European Championships for Portugal to Xherdan Shaqiri's overhead kick wondergoal, Sport360 chatted with the world's media at various press rooms in France to get their view on a summer of footballing action.
What do you think of their choices? Let us know, get in touch using #360fans on social media.
Cristiano Ronaldo's special backheel goal against Hungary won your vote for Euro 2016 goal of the tournament but here you can watch the goals again in brilliant animation.
It started in Paris and ended in the French capital with Arsenal frontman Giroud opening the Euros account and Eder's match-winning strike in the final rounding it off in stunning style.
What was your favourite Euro 2016 moment? Get in touch and let us know, use #360fans on social media.


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A small fire started underneath concrete steps at the Knollwood Townhouses apartment complex Saturday morning.
Firefighters cut into the floor inside the doorway of the apartment in an attempt to find the source of the smoke. It was coming from underneath the outdoor concrete steps that lead up to the front door of apartment K105.
After breaking into the steps with sledgehammers, firefighters with the Rapid City Fire Department discovered a large amount of dry straw smoldering inside the steps.
Battalion Chief Tim Daly said the straw is used for insulation. He said he has never seen the practice, but he was told by a fellow firefighter that it is not uncommon. Daly said cracks in the concrete may have allowed something hot to reach the straw.
There were several cigarette butts on the ground beside the steps.
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The apartment manager was on scene and spoke to the firefighters. She declined to comment or identify herself to the Journal.
Residents in the two adjoining apartments were evacuated and allowed to return to their homes after firefighters determined there was no risk.
Neighbors stood outside their apartments watching the scene as the group of firemen swung sledgehammers to break the concrete steps and pull out mounds of straw, some of which was black and smoldering.
A jackhammer was eventually brought in to allow firefighters to completely remove the steps and the straw inside.
Daly said the cause of the fire is still under investigation.


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Find Top-Rated Ignacio Appliance Repair Services
There are 12 top-rated appliance repair services in your area.
Tin Lizzee Virtual Appliance Repair
Business Description: $35 for a Technician-guided appliance repair. This is a NEW kind service! This new digital solution for guided diagnoses or repairs saves our customers money and time. No waiting all day for someone to show up. You can get help immediately, online, or schedule a virtual appointment with one of our techs. Our service puts you back in control using advanced camera-sharing technology that allows the technician to draw and write on your screen in real time. It all takes place on https://tinlizzee.com/. If you have any questions, or you'd like to set up an appointment, give us a call at (833) 876 0900!
Junction Home Services, Ltd.
Business Description: Born in 2017 out of the necessity to meet an overwhelming demand, JunctionHandyman.com not only offers home services to residents of the area at the lowest possible prices while providing the best quality at the same time, but we also provide the luxury of a one-stop shop with an entirely-encompassing knowledge and experience base that can handle any and all of your needs at once! With the ability, connections, knowledge, equipment and tools to handle literally everything, we can help you to forget about your entire to-do list, restore your property to its previous glory, or tackle the smallest of projects you may need some assistance with, so you can stop worrying the moment you call! While the Jhs, Ltd. team has decades of experience and the knowledge and skills to handle just about everything, we have an expanse network of fellow highly skilled professionals, and if ever you request something of us we are unable to accommodate, we can still handle everything for you from the moment you call to the moment it's finished to the highest of standards. We are a company that prides itself most on its record of satisfied customers, its history of employee involvement in the community, and because Junction is our home and all of our customers shape the lives of everyone else in our small and wonderful valley, how could we not be want to be the best we can for you?!
Business Description: Since 1999, we've helped millions of people save money and fix their broken appliances quickly by providing quality parts and free repair help. 365 days to return any part. Amazingly fast shipping. It's better with the Pros!™ Avoid costly service calls - visit us at http://www.appliancepartspros.com
Complete Appliance Protection Inc
Business Description: CP has been providing quality home warranty products since 1998.
Business Description: Appliance Repair
RepairClinic.com Inc
Business Description: RepairClinic.com® makes fixing things easy for millions of people. Founded in 1999, it is North America's trusted online store with replacement parts for major household appliances, outdoor power equipment and heating and cooling equipment. Free repair resources including more than 2,200 how-to videos empower people to fix stuff on their own. One million parts for 160 brands are stocked at its 86,000-square-foot facility in Canton, Michigan. It offers a generous, no-hassle 365 Days. Period.® return policy. In-stock parts are guaranteed to ship the same business day. Visit RepairClinic.com for more information.
Dry Creek Electric
Business Description: Dry Creek Electric is a full service electrical contractor. We are experienced in residential, commercial and industrial- troubleshooting and repair, remodel and new construction.
Business Description: ALPINE APPLIANCE CENTER IS A FULL SERVICE RETAIL APPLIANCE DEALER. WE SPECIALIZE IN BUILDER SALES BUT WE ARE OPEN TO THE PUBLIC. WE ARE THE ONLY SERVICING DEALER IN EAGLE AND PITKIN COUNTIES. WE PRIDE OURSELVES ON PROVIDING WARRANTY SERVICE ON THE PRODUCTS WE SELL AS WELL AS PROVIDING EXCELLENT PRICING AND CUSTOMER SERVICE.
Advanced Appliance Repair
Business Description: Offering reliable honest service technician's built on the foundational values of durango life. Certified technician with more than 10 years of experience and am licensed and fully insured. Repair was created by geary parsons who set out to provide customers a service experience they can't get anywhere else. We believe in running an honest, professional service company that does not seek to profit by cutting corners & recommending unnecessary repairs.
Appliance Zone
Business Description: Appliancezone.com is a leading on-line retailer of appliance parts and accessories for do-it-yourself homeowners. Incorporated in 2008, it was started by CEO, Jim Allen and his wife Paula, who operated an appliance repair business for nearly fourteen years prior to starting the internet company Appliance Zone.


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My Journey Officially Starts Today
Today, I start a new journey / potential career as a blogger. I've never done this before so it's exciting and a little intimidating at the same time. As the saying goes - "Here goes nothing." Welcome to WorkFlowRetirement - I hope you enjoy the journey as much as me!
WorkFlowRetirement captures my journey or "flow" from working to retirement. My mission is to provide guidance for anyone nearing retirement through sharing my expertise and experience. Everyone wants to retire happily, and I want to help you achieve that! I'm glad you are here and hope you join our community. Let me tell you a little about myself and my blog.
I'm Greg Phillips - a retirement expert, business executive, technologist, and survivor of the Las Vegas shooting in 2017 which was a life changing event. After 35 years in the business world, I'm retiring which is a very exciting but stressful time. I spent the majority of my career in retirement consulting building solutions to help people plan and save for retirement. For the last twelve years, I have worked for a great company (Capital Group), one of the largest investment managers in the world and home of the American Funds.
Retirement planning is a passion of mine, and through this blog and community, I want to help others with this life-changing event. Also, creating a blog will help me transition from long days at the office and still have something to do!
Over the last year, as I have shared with friends, family and business associates my plan to retire, there are frequent questions they always ask. These questions are the inspiration behind this blog and how I plan to organize the conversation with our community. These are also the key questions I asked and answered myself.
- How much money do you need to retire (Your Number)?
- When is the right time to retire (Your Time)?
- Where will you live (Your Home)?
- How to transition from company provided to individual healthcare (Your Healthcare)?
- What is your withdrawal strategy (Your Paycheck)?
- How will you spend your time (Your Time)?
I will focus the majority of the dialogue and content on these key questions, but I would love to hear from you. Please share your questions, perspectives, and journey so our community can learn from you. Also, please invite your families and friends to join us.
Thanks for reading my story and I hope you enjoy my blog.
Take care - Greg Phillips


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Bahram Mirza, MD
The information is not available
I speak: English, Farsi, Turkish
My gender: Male
Where I practice
- Emergency service
- Urgent care services
- After-hours services
- Pharmacy services
- No emergency services
- No urgent care services
- No after-hours services
- No pharmacy services
About me
Welcome to my Web page. I am glad to have this opportunity to introduce myself and give you a brief description of our practice. I feel very fortunate to work at Kaiser Permanente. I work side by side with wonderful physicians and medical practitioners whose main focus is excellent patient care.
I am from Iran. After graduating from medical school in Turkey, I migrated to the U.S. and did my internal medicine residency in Ohio and endocrinology fellowship in Indiana. I met my wife, who is a pharmacist, in Ohio during my residency, and now we have two wonderful children.
About my practice
Our Endocrinology Department has endocrinologists, diabetes educators, dietitians, and social workers. The main focus of my practice is the treatment of diabetes, thyroid disease, bone disease, and other hormonal abnormalities. Besides treating diseases related to hormones and metabolism, my colleagues and I are committed to educating patients about their diseases, promoting preventive measurements such as lifestyle modification, and engaging them in disease management.
How I thrive
I try to maintain a peaceful life and avoid stress. I enjoy teaching, reading, music, and spending time with my family.
My office
Languages spoken by medical staff
Help is available in your language. Learn more.
My provider typePhysicians and Surgeons
Medical groups and plans
Southern California Permanente Medical GroupList Header Plans Accepted
- Exclusive Provider Organization (EPO)
- Medi-Cal Managed Care
- Point-of-Service Plan (POS)
- Senior Advantage/Medicare Cost
Contact information
- Advice/appointments
- 1-833-574-2273 (toll free)
- Information
- 951-353-3005 (TTY for the hearing/speech impaired)
- Information from Moreno Valley
- 951-247-3183
- Medical Center Operator
- 1-833-574-2273 (toll free)
My training, certifications and licensing
Medical schoolUludag Universitesi-tip Fakultesi
Board certification
Endocrinology, Diabetes, MetabolismAmerican Board of Internal Medicine
Internal MedicineAmerican Board of Internal Medicine
Medical training
Internal MedicineWestern Reserve Care System
Internal MedicineWestern Reserve Care System
Endocrinology and MetabolismIndiana University School of Medicine
Joined Permanente Medical Group2008
Active license information
License numberC53240
License typePHYSICIAN AND SURGEON
License issuing stateCALIFORNIA
National Provider Identifier (NPI)1740262674
My hospitals
All Kaiser Permanente physicians and podiatrists have admitting privileges or the ability to refer patients to our plan hospitals. Admitting privileges of other providers may vary by plan and facility. Contact your plan administrator for details.
I have admitting privileges at this hospital.
Setting the standard: Measuring care quality in our hospitals(PDF)
To find:
- a provider's office hours, search our facility directory
- providers in your plan or accepting new patients, call 1-800-464-4000 (toll free) or 711 (TTY for the hearing/speech impaired)
The information in this online directory is updated periodically. The availability of physicians, hospitals, providers, and services may change. Information about a practitioner is provided to us by the practitioner or is obtained as part of the credentialing process. If you have questions, please call us at 1-800-464-4000 (toll free). For the hearing and speech impaired: 1-800-464-4000 (toll free) or TTY 711 (toll free). You can also call the Medical Board of California at 916-263-2382, or visit their website.
We want to speak to you in the language that you're most comfortable with when you call or visit us. Qualified interpreter services, including sign language, are available at no cost, 24 hours a day, 7 days a week during all hours of operations at all points of contact. We do not encourage the use of family, friends or minors as interpreters. Only the services of interpreters and qualified staff are used to provide language assistance. These may include bilingual providers, staff, and healthcare interpreters. In-person, telephone, video, and alternative modes of communication are available. Learn more about interpreter services.
If you would like to report an error in provider or facility information, please contact us.
Kaiser Permanente enrollees have full and equal access to covered services, including enrollees with disabilities as required under the Federal Americans with Disabilities Act of 1990 and Section 504 of the Rehabilitation Act of 1973.
Kaiser Permanente uses the same quality, member experience, or cost-related measures to select practitioners and facilities in Marketplace Silver-tier plans as it does for all other Kaiser Foundation Health Plan (KFHP) products and lines of business.The measures may include, but are not limited to, HEDIS/CAHPS performance, member/patient complaints, patient safety scores, hospital quality measures, and geographic need. Members enrolled in KFHP Marketplace plans have access to all professional, institutional and ancillary health care providers who participate in KFHP plans' contracted provider network, in accordance with the terms of members' KFHP plan of coverage. All Kaiser Permanente Medical Group physicians and network physicians are subject to the same quality review processes and certifications.
Kaiser Permanente uses the same geographic distribution consideration to select hospitals in Marketplace plans as it does for all other Kaiser Foundation Health Plan (KFHP) products and lines of business. Accessibility of medical offices and medical centers in this directory: All Kaiser Permanente facilities are accessible to members.


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Crabbies Alcoholic Ginger Beer 500Ml
Product Description
- Original Alcoholic Ginger Beer
- Enjoy the curiously spicy yet deliciously refreshing blend of real steeped ginger and exotic spices.
- In the 1800s, the Scottish pioneer John Crabbie created unconventional drinks by combining exotic spices from the Far East, with the finest steeped ginger. Today, Crabbie's Alcoholic Ginger Beer comes in a wide variety of flavours but still retains the same pioneering and adventurous spirit of its founder. It's the spirit that makes Crabbie's drinks genuinely surprising and unique.
- Made to our unique steeped ginger recipe
- Gluten free
- Pack size: 500ml
Allergy Information
- Contains: Barley, Wheat & Sulphites
Alcohol Units
4.0% vol
Alcohol Type
Storage Type
Best before: See neck
Preparation and Usage
- Best served chilled
Name and address
- John Crabbie & Co.,
- Mitchell House,
- Mitchell Street,
- Leith,
- Edinburgh,
Return to
- John Crabbie & Co.,
- Mitchell House,
- Mitchell Street,
- Leith,
- Edinburgh,
- www.crabbies.co.uk
Lower age limit
18 Years
Net Contents
500ml ℮
Using Product Information
While every care has been taken to ensure product information is correct, food products are constantly being reformulated, so ingredients, nutrition content, dietary and allergens may change. You should always read the product label and not rely solely on the information provided on the website.
If you have any queries, or you'd like advice on any Tesco brand products, please contact Tesco Customer Services, or the product manufacturer if not a Tesco brand product.
Although product information is regularly updated, Tesco is unable to accept liability for any incorrect information. This does not affect your statutory rights.
This information is supplied for personal use only, and may not be reproduced in any way without the prior consent of Tesco Stores Limited nor without due acknowledgement.
Tesco © Copyright 2019


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Almost a 1/2 Acre residential building lot in Established Neighborhood at the end of a dead end street closed to schools, Stetson and Downtown
Listing information courtesy of Charles Rutenberg Realty Orlando.
All listing information is deemed reliable but not guaranteed and should be independently verified through personal inspection by appropriate professionals. Listings displayed on this website may be subject to prior sale or removal from sale; availability of any listing should always be independently verified.
Listing information is provided for consumer personal, non-commercial use, solely to identify potential properties for potential purchase; all other use is strictly prohibited and may violate relevant federal and state law.
Listing data comes from My Florida Regional MLS DBA Stellar MLS.


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ZAP-70, a Syk family cytoplasmic protein tyrosine kinase (PTK), is required to couple the activated T-cell antigen receptor (TCR) to downstream signaling pathways. It contains two tandem SH2 domains that bind to phosphorylated TCR subunits and a C-terminal catalytic domain. The region connecting the SH2 domains with the kinase domain, termed interdomain B, has previously been shown to have striking regulatory effects on ZAP-70 function, presumed to be due to the recruitment of key substrates. Paradoxically, deletion of interdomain B preserves ZAP-70 function. Recent structural studies of several receptor tyrosine kinases (RTKs) revealed that their juxtamembrane regions negatively regulate their catalytic activities. In EphB2 and several other RTKs, this autoinhibition depends upon interaction between the kinase domain and tyrosine residues within the juxtamembrane region. Autoinhibition is released when these tyrosines become phosphorylated following receptor stimulation. Sequence homology suggested analogous regulation for ZAP-70. Based on mutagenesis analysis of ZAP-70 interdomain B, we find that this region downregulates ZAP-70 catalytic activity in a similar manner as the juxtamembrane region of EphB2. Similar regulation was also noted for the related Syk kinase. These findings suggest that a general autoinhibitory mechanism employed by RTKs is also used by some cytoplasmic tyrosine kinases.
Stimulation of the T-cell antigen receptor (TCR) leads to a series of signaling events that result in changes in T-cell function and gene expression. Signaling is initiated by two families of protein tyrosine kinases (PTKs) (17). Src-family kinases first phosphorylate specific immunoreceptor tyrosine-based activation motifs (ITAMs) (4) in the cytoplasmic tails of the CD3 and ζ subunits of TCR. Each ITAM is composed of a pair of tyrosines separated by 9 to 11 amino acids which, when phosphorylated, bind the Syk family PTK ZAP-70. In turn, ZAP-70 becomes activated and subsequently phosphorylates a number of key downstream signaling molecules necessary for further signal propagation.
ZAP-70 consists of two N-terminal SH2 domains, responsible for binding to doubly phosphorylated ITAMs, and a C-terminal tyrosine kinase domain (6). The SH2 domains are separated by a linker region, termed interdomain A. The region between the SH2 domains and the kinase domain is known as interdomain B.
Upon binding to phosphorylated ITAMs of the activated TCR, ZAP-70 itself becomes phosphorylated on multiple tyrosine residues, both by Src family PTKs and via autophosphorylation. It is well established that tyrosine phosphorylation plays an important role in ZAP-70 activation, but the precise mechanism responsible is still incompletely understood. Among the best-studied phosphorylation sites are tyrosines 492 and 493 (Y492 and Y493) in the activation loop of the kinase (5, 33, 34). A model has been proposed whereby Y493 becomes phosphorylated first by the Src-family PTK Lck. This phosphorylation is then followed by autophosphorylation at Y492 (5). Phosphorylation at both sites is most likely required for displacement of the activation loop from the catalytic site and, hence, activation of the kinase. However, the recent crystal structure of the isolated nonphosphorylated ZAP-70 kinase domain complexed with staurosporine fails to show blockade of the catalytic site by the activation loop (19). This suggests that some other mechanism may be responsible for regulation of the catalytic domain in the context of the intact ZAP-70 molecule.
Another possible means of regulation could involve other phosphorylation sites, such as Y292, Y315, and Y319, in interdomain B of ZAP-70. Y292 has a negative regulatory function, possibly due to the recruitment of the E3 ubiquitin ligase c-Cbl. The interaction between phosphorylated Y292 and c-Cbl is well documented, and a crystal structure of the Y292-containing phosphopeptide bound to the phosphotyrosine binding (PTB) domain of c-Cbl has been solved (22, 25, 27). However, the importance of this interaction has been challenged by a recent study where knock-in mice expressing a c-Cbl mutant that is unable to bind ZAP-70 did not show major alterations in ZAP-70 expression or function (31).
Interdomain B tyrosines Y315 and Y319 have been proposed to be positive regulatory sites of phosphorylation. It has been demonstrated that both tyrosines become phosphorylated following TCR cross-linking, and ZAP-70 itself (8) and Lck (36) have both been suggested to be responsible for this phosphorylation. Additionally, c-Abl has recently been shown to be able to phosphorylate Y319 (41). In multiple experimental systems, mutations of Y315 or Y319 to phenylalanines substantially impaired ZAP-70-dependent signaling events, thought to be primarily due to the inability of mutated ZAP-70 to recruit downstream effector molecules (8, 12, 13, 23, 36, 37). Probably best studied is the interaction between phosphorylated Y319 and the SH2 domain of Lck, which has been proposed to play an important role in the subsequent Lck-mediated phosphorylation of Y493 of ZAP-70 as a part of a positive feedback mechanism (26, 30). There also is evidence that other molecules, including Vav1 (37), phospholipase C-γ1 (PLC-γ1) (36), and possibly Crk (11) bind interdomain B tyrosines. Therefore, it was rather surprising that an internal deletion which removed most of interdomain B, including Y292, Y315, and Y319, did not have any major effect on ZAP-70-dependent signaling events (40). This suggests that interdomain B has a regulatory function independent of any interacting molecules.
Such regulatory elements have been recently discovered in the juxtamembrane regions of several receptor tyrosine kinases (RTKs) (for review, see reference 15). In RTK EphB2, tyrosines Y604 and Y610 in the juxtamembrane region promote an interaction between the juxtamembrane region and the kinase domain. This leads to substantial alterations in the structure of the kinase domain and inhibition of kinase activity (38). Ligand-induced oligomerization of EphB2 leads to autophosphorylation of Y604 and Y610, which disrupts the autoinhibitory interaction and results in an upregulation of the kinase activity. Based on the sequence similarity between the juxtamembrane region of EphB2 and interdomain B of ZAP-70, we hypothesized that interdomain B plays an analogous role in autoinhibition of ZAP-70 kinase activity. Our data support a novel model of ZAP-70 activation in which Y315 and Y319 of interdomain B play an important structural role in autoinhibition of ZAP-70 and their phosphorylation is an essential prerequisite for putative structural changes leading to the full activation of the kinase.
Cells, cDNA constructs, and antibodies.P116, a ZAP-70-deficient Jurkat-derived T-cell line, was obtained from R. Abraham (Burnham Institute, La Jolla, CA). 293 cells, a kidney epithelial cell line, were obtained from the American Type Culture Collection (Manassas, VA). A QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and standard PCR techniques were utilized to prepare the ZAP-70 mutations (Y315/319F, Y315/319A, Y315/319E, K369A, Δ265-331) in the expression plasmid pcDNA3 (Invitrogen, Carlsbad, CA) and the Syk mutations (Y342/346F, Y342/346A) in the expression plasmid pEF6 (Invitrogen). Hemagglutinin (HA)-tagged rat wild-type (WT) Syk was provided by J. B. Bolen (DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA). C-terminal, Myc-tagged wild-type, and ΔSH3 c-Abl were constructed by cloning EcoRI fragments of wild-type and ΔSH3 c-Abl from pSRα-MSV-TKneo (obtained from Marie Anne Pendergast, Duke University Medical Center, Durham, NC), into pBS SK. A BglII-XbaI PCR product covering the last 90 coding base pairs of c-Abl with the XbaI site destroying the stop codon was cloned in frame with c-Abl or ΔSH3 c-Abl from pBS (NotI-partial BglII fragment) into NotI-XbaI-digested pEF6Myc/HisA. Lck (24), FynT (7), FLAG-tagged linker for activation in T cells (LAT) (2), non-T-cell activation linker (NTAL) (2), and CD8-TCR-ζ (16) constructs have been described previously. Monoclonal antibody (MAb) 1F6 (anti-Lck) was obtained from J. B. Bolen. Anti-phosphotyrosine MAb 4G10 and mixed MAbs against PLC-γ1 were purchased from Upstate Biotechnology (Charlottesville, VA). Anti-FLAG-tag MAb M2 was from Sigma (St. Louis, MO). Rabbit antisera to PLC-γ1-pY783, LAT-pY132, LAT-pY191, and SLP-76-pY145 were purchased from Biosource (Camarillo, CA); rabbit antiserum against LAT was from Novus Biologicals (Littleton, CO); rabbit antiserum against SLP-76 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA); and mouse monoclonal antibody 9B11 against Myc epitope and rabbit antisera against ZAP-70, pY493, and pY319 were from Cell Signaling Technology (Beverly, MA). Rabbit antisera against Fyn were from EMD Biosciences (La Jolla, CA). Monoclonal antibodies C305 (anti-TCR-β) (35), 2F3.2 (anti-ZAP-70) (17), 6B10 (anti-TCR-ζ) (32), and NAP-4 (anti-NTAL) (2) have been described previously.
Transfections and transcriptional assays.For transient transfections of P116 cells, 20 μg of NFAT luciferase reporter plasmid DNA (29) and 20 μg of ZAP-70 or Syk expression constructs were electroporated at 250 V, 960 μF, using the Bio-Rad Gene Pulser electroporator (Bio-Rad Laboratories, Hercules, CA). Sixteen hours after the transfection, cells were stimulated with anti-TCR (1:1,000 C305 ascites) or phorbol 12-myristate 13-acetate (PMA; 20 ng/ml) and ionomycin (1 μM). Approximately 8 h later, the cells were harvested, lysed, and assayed for luciferase activity. Stably transfected cell lines were generated by transfection of 20 μg WT or mutated ZAP-70 expression constructs into the parental P116 cell line. Clones were isolated by limiting dilution in the presence of G418 (2 mg/ml; Invitrogen). Transient transfections of 293 and Cos cells were carried out in 24-well plates using Lipofectamine and PLUS reagents (Invitrogen) according to the manufacturer's instructions.
Measurement of free intracellular calcium concentration.Cells were loaded with the fluorescent dye indicator Indo-1 (Molecular Probes, Eugene, OR) and stimulated with anti-TCR MAb C305 (1:1,000 ascites). The fluorescence at 400- and 500-nm wavelengths was measured with a Hitachi F-4500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan), and the concentration of free intracellular calcium was calculated from the ratio of fluorescence at the two wavelengths (14).
Cell stimulation, lysate preparation, immunoprecipitation, and Western blot analyses.P116 cells (108/ml phosphate-buffered saline) transfected with ZAP-70 constructs were stimulated with anti-TCR MAb C305 (1:100 or 1:1,000 ascites) at 37°C for the time indicated, immediately pelleted, and lysed in ice-cold lysis buffer (20 mM Tris, pH 7.6, 150 mM NaCl, 1% NP-40, 0.01% NaN3, and a cocktail of protease and phosphatase inhibitors). Postnuclear supernatant was either used for immunoprecipitation with antibody prebound to Sepharose-protein A or -protein G beads (Amersham Biosciences, Buckinghamshire, United Kingdom) or diluted with 2× concentrated sodium dodecyl sulfate (SDS) sample buffer containing 2-mercaptoethanol for whole-cell lysates. Where indicated, whole-cell lysates were prepared by resuspending the cells in 2× concentrated SDS sample buffer (200 μl per 5 × 106 P116 cells or 60 μl per well of 293 cells) followed by ultracentrifugation (30 min, 300,000 × g). Samples were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting with the indicated primary and horseradish peroxidase-conjugated secondary antibody. Proteins were then detected by chemiluminescence (Western Lightning; Perkin-Elmer, Wellesley, MA) on a Kodak Image Station (Kodak, Rochester, NY).
Mutation of tyrosines 315 and 319 of ZAP-70 to alanines does not compromise ZAP-70 function in Jurkat T cells.In a number of experimental models, mutations of Y315 or Y319 to phenylalanine had adverse effects on ZAP-70-mediated signaling events. Surprisingly, removal of amino acids 265 to 331 from interdomain B, containing these tyrosines, had only mild consequences (40). To reconcile these conflicting observations, we analyzed the amino acid sequence of interdomain B and found that Y315 and Y319 reside within motifs that have striking homology to motifs containing negative regulatory tyrosines located in the juxtamembrane region of EphB2 (Fig. 1A). Similar sequence homology was seen in the Syk PTK. Moreover, the distance of these residues from the kinase domains of ZAP-70 and Syk was similar to that of the tyrosines in EphB2. Therefore, we hypothesized that the segment of interdomain B of ZAP-70 containing Y315 and Y319 autoinhibits its catalytic function via a mechanism similar to that of the EphB2 juxtamembrane region. In EphB2, mutation of juxtamembrane tyrosines Y604 and Y610 to structurally related but unphosphorylatable phenylalanines constrained the kinase into an inactive conformation (38). We reasoned that ZAP-70 could be under similar regulatory constraint. According to this model, replacement of Y315 and Y319 in ZAP-70 with phenylalanines should lock ZAP-70 into an inactive state. We also hypothesized that mutation of these residues to structurally distant alanines could free ZAP-70 from this inhibitory constraint. To test this hypothesis, we made several expression constructs of ZAP-70. In addition to WT ZAP-70, we made a mutant form of ZAP-70 with Y315 and Y319 mutated to phenylalanine residues (YYFF) and other mutants with substitution for Y315 and Y319 with alanine residues (YYAA) or glutamic acids (YYEE). Additional control constructs included a kinase-inactive ZAP-70 (KA) where lysine 369 in the ATP binding site was mutated to alanine and a ZAP-70 construct lacking amino acids 265 to 331 in interdomain B (Δ IB) (Fig. 1B).
Stimulation of the TCR on the ZAP-70 deficient Jurkat mutant P116 fails to induce signaling events downstream of the receptor, including the activation of NFAT (nuclear factor of activated T cells) transcriptional reporter constructs, calcium flux, mitogen-activated protein (MAP) kinase activation, and phosphorylation of multiple important downstream signaling molecules. To analyze the role of Y315/Y319 in NFAT activation, we transiently transfected P116 cells with the ZAP-70 constructs described above together with an NFAT-driven luciferase reporter construct and measured TCR-induced luciferase production in these transfectants. We also established clones stably expressing the same ZAP-70 constructs and analyzed them in a similar fashion. The TCR induction of NFAT could be restored by transient or stable transfection of WT ZAP-70 (Fig. 2A and B). Whereas the YYFF mutant and a catalytically inactive (KA) mutant failed to restore NFAT responses, both the interdomain B deletion and the YYAA mutant restored TCR-mediated NFAT responses. These results are consistent with the notion that the Y315/Y319 sites are involved in a negative regulatory function and that recruitment of critical effector molecules is not necessary for downstream signaling. Consistent with this, TCR-induced calcium responses (Fig. 2C) and activation of MAP kinases Erk1 and Erk2 (Fig. 2D) were restored not only by WT ZAP-70 but also by the YYAA mutant in the stable transfectants.
YYAA ZAP-70 rescues TCR-dependent tyrosine phosphorylation of signaling proteins in P116 cells.One of the most proximal measures of ZAP-70-dependent TCR activation is tyrosine phosphorylation of a number of substrates by PTKs. To elucidate the influence of interdomain B tyrosines on TCR-mediated tyrosine phosphorylation of downstream signaling proteins, we analyzed unstimulated and TCR-stimulated P116 cells stably transfected with WT and mutant ZAP-70 constructs by antiphosphotyrosine immunoblotting. Analysis of whole-cell lysates revealed that TCR stimulation of the cells transfected with WT ZAP-70 resulted in phosphorylation of multiple proteins (Fig. 3A), including a 36- to 38-kDa phosphoprotein likely representing an important ZAP-70 substrate transmembrane adaptor protein, LAT (39). Consistent with the data in the previous figure, inducible tyrosine phosphorylation could also be reconstituted by the YYAA mutant, albeit to a modestly reduced extent compared to the wild-type protein. In contrast, the cells expressing the YYFF mutant exhibited reduced responses, while the inactive KA mutant failed to restore inducible tyrosine phosphorylation. An exception to this was the observation of a constitutively hyperphosphorylated 70-kDa phosphoprotein in the lysates of the KA-transfected cells, which most likely represented ZAP-70 itself, as discussed below. Consistent with overall tyrosine phosphorylation, a similar pattern of responsiveness was also observed for several specific substrates as detected by site-directed phospho-specific antibodies against the key downstream signaling molecules, including the ZAP-70 substrates LAT and SLP-76 as well as PLC-γ1 (Fig. 3B).
Recruitment of ZAP-70 to the TCR is altered in ZAP-70 mutant cells.One of the first events thought to occur following TCR engagement is the phosphorylation of the TCR-ζ chain by the PTK Lck, which leads to the recruitment of ZAP-70 via its SH2 domains to the newly phosphorylated ITAMs (17). Since this is one of the necessary steps in ZAP-70 activation, we next analyzed how mutations in interdomain B affected binding of ZAP-70 to TCR-ζ. We immunoprecipitated TCR-ζ from unstimulated and TCR-stimulated P116 transfectants and analyzed the proteins by immunoblotting (Fig. 4). All cell lines showed an inducible tyrosine phosphorylation of TCR-ζ. However, there was a reproducibly marked increase in basal and inducible TCR-ζ phosphorylation in the YYAA mutant. Inducible TCR-ζ phosphorylation was also reproducibly elevated above wild-type levels in both YYFF and KA mutants.
In addition to the phosphorylated TCR-ζ, tyrosine-phosphorylated ZAP-70 was also found in these TCR-ζ immunoprecipitates (Fig. 4). Surprisingly, compared to the WT ZAP-70, the KA mutant was hyperphosphorylated in both the basal and activated states, while phosphorylation of YYAA and YYFF was only barely detectable. The blot containing these TCR-ζ immunoprecipitates was stripped and reprobed for the total level of ZAP-70 associated with TCR-ζ. Unexpectedly, in all ZAP-70 mutant-expressing cell lines, there was an increase in the amount of ZAP-70 associated with TCR-ζ. Assuming that some level of tonic signaling is present via the TCR pathway in unstimulated Jurkat cells, as we have previously reported (28), it would appear that the Y315/Y319 sites not only may play a role in negatively regulating ZAP-70 catalytic activity, but also may serve to regulate its association or turnover on TCR-ζ ITAM phosphorylation sites. The intrinsic ability of ZAP-70 mutants to bind more efficiently to TCR-ζ ITAMs may also lead to the protection of TCR-ζ ITAMs from dephosphorylation by cellular phosphatases, thus accounting for the observed increase in TCR-ζ phosphorylation.
Phosphorylation of mutant ZAP-70 proteins.In an effort to understand the contributions of Y315/Y319 phosphorylation to ZAP-70 activation, we used phospho-specific antibodies to Y319 and Y493 to analyze the phosphorylation status of ZAP-70 proteins in the whole-cell lysates of P116 transfectants. TCR stimulation of cells expressing WT ZAP-70 led to inducible phosphorylation of Y319. Consistent with previous experiments, we observed constitutive hyperphosphorylation of Y319 in the inactive KA mutant that could be further enhanced by TCR stimulation. TCR-inducible phosphorylation of the activation loop Y493 could be detected for all ZAP-70 constructs (Fig. 5A). The phosphorylation of the YYAA mutant at Y493 was reduced compared to WT ZAP-70. The Y493 phosphorylation of YYFF mutant was even more reduced, especially considering the higher expression level of YYFF protein compared to the YYAA. Similar to Y319, Y493 was also hyperphosphorylated in the inactive KA mutant. The constitutive hyperphosphorylation of Y319 and Y493 in the KA-inactive mutant may reflect the failure of negative regulatory pathways to be activated in the absence of an active ZAP-70 kinase.
In order to simplify these phosphorylation events and examine the in vivo catalytic function of the ZAP-70 interdomain B double-tyrosine mutants, we overexpressed these mutants in 293 cells alone, or together with Lck, and performed similar analyses using site-directed phospho-specific antibodies. In the absence of Lck, only the YYAA mutant exhibited constitutive phosphorylation of Y493 in the activation loop (Fig. 5B). Phosphorylation of Y493 and Y319 in WT ZAP-70 required the presence of Lck. Interestingly, although Y319 was phosphorylated in the KA mutant, Y493 was not phosphorylated in this mutant or in the YYFF mutant. We also expressed these ZAP-70 constructs together with Lck and a CD8-TCR-ζ chimera (as a simplified TCR surrogate), and immunoprecipitated CD8-TCR-ζ from the lysates of the transfected cells. All constructs appeared to be bound equivalently to phosphorylated TCR-ζ sequences (Fig. 5C). Very similar site-specific tyrosine phosphorylation of ZAP-70 was observed, even in the presence of a CD8-TCR-ζ chain chimera (Fig. 5C). These findings are consistent with the notion that Y319 (and possibly Y315) are Lck phosphorylation sites and regulate accessibility of Y493 in the activation loop to phosphorylation. Results of experiments with single-tyrosine mutants (Y315F, Y319F, Y315A, Y319A) recapitulated those with double mutants; in all cases, the alanine mutants were more active than phenylalanine mutants (data not shown). To study the kinases involved in phosphorylation of Y319 and Y493 in greater detail, we performed a similar experiment using expression constructs of Fyn, c-Abl, and a constitutively active form of c-Abl (c-Abl Δ SH3). Fyn has been shown to interact with and to phosphorylate ZAP-70 (10), although specific phosphorylation sites in ZAP-70 have not been identified. c-Abl has been previously shown to phosphorylate Y319 of ZAP-70 (41). Since we could not observe any kinase activity of FynT in 293 cells, we employed Cos cells in this experiment. As shown in Fig. 5D, all of the kinases were able to induce a similar pattern of ZAP-70 phosphorylation as that induced by Lck. Relative efficiency of the kinases used, however, could not be evaluated since the relative level of their expression could not be estimated in this experiment.
Kinase activity of ZAP-70 and Syk mutants expressed in 293T cells.To examine the catalytic activities of these mutants, we immunoprecipitated them from P116 lysates and performed in vitro kinase assays using a glutathione S-transferase (GST) fusion to the cytoplasmic domain of band 3 protein as a substrate, but failed to detect substantial differences in catalytic activity (data not shown). However, when we transfected 293 cells with the lipid raft-anchored ZAP-70 kinase substrate LAT together with the ZAP-70 constructs in the presence or absence of Lck, we observed substantial differences when we examined the in vivo phosphorylation of LAT. Among the constructs used in the previous experiments, only the YYAA mutant could phosphorylate LAT in the absence of Lck (Fig. 6A). Cotransfection of Lck with the WT ZAP-70 was necessary to activate it, as evidenced by LAT phosphorylation. Lck also modestly augmented the ability of the YYAA mutant to phosphorylate LAT. As an additional control, we used a ZAP-70 construct in which Y315 and Y319 were both mutated to glutamic acids (YYEE) to better mimic the negative charge associated with tyrosine phosphorylation at this site. With this mutant, we obtained the same results as with the YYAA construct (Fig. 6A). Similar results were also obtained when another substrate of Syk-family kinases, NTAL (2) (also known as LAB) (18), was used instead of LAT as the ZAP-70 substrate (data not shown). Moreover, the YYEE mutant was able to reconstitute transcriptional activation of NFAT in P116 Jurkat cells to a similar extent as the YYAA mutant (data not shown).
Our experiments, together with previously published data suggest that inhibition of the kinase activity by tyrosines adjacent to the N terminus of the kinase domain may represent a broadly used regulatory mechanism employed by many receptors as well as cytoplasmic PTKs. Therefore, we next analyzed PTK Syk, the other member of the Syk family of PTKs. It has the same domain structure as ZAP-70, and the interdomain B phosphorylation sites are well conserved between the two members of the family (Fig. 1A). Analogous to previous experiments, we mutated corresponding tyrosines 342 and 346 (orthologs of Y315 and 319 of ZAP-70) to alanines or phenylalanines, expressed these constructs together with the Syk substrate NTAL in the presence or absence of Lck in 293 cells, and analyzed NTAL phosphorylation in the whole-cell lysates (Fig. 6B). Results similar to the previous ZAP-70 experiments were obtained, although this region did not appear to constrain this kinase to the same absolute extent. The YYAA mutant did not require Src family kinases to phosphorylate NTAL in this system, while WT Syk was substantially less active and the YYFF mutant displayed only marginal kinase activity in the absence of Lck. Cotransfection of Lck augmented the in vivo kinase activity of all the Syk constructs, quite surprisingly even that of the YYFF mutant. The previously observed increased activity of Syk (20) may reflect its somewhat reduced dependency on Src kinases to release the inhibitory constraints of interdomain B. The sequence differences in the ZAP-70 and Syk interdomain B (Fig. 1A) might also be partially responsible. On the other hand, it is also possible that the high kinase activity of Syk may be outside the range of sensitivity of the assay.
Our findings reveal an autoinhibitory switch in ZAP-70 and Syk and suggest that, in contrast to the previously held view, Y315 and Y319 in ZAP-70 do not play a requisite scaffolding function in order to recruit substrates. Rather, they are primarily responsible for an autoinhibitory switch that regulates kinase function. Our findings suggest that Lck or another kinase activates ZAP-70 by phosphorylating Y319 (and possibly Y315), which, by analogy to the juxtamembrane region of several RTKs (15), relieves a hydrophobic autoinhibitory interaction with the N-terminal lobe and the activation loop of the kinase domain. This likely induces a conformational change that enhances the kinase activity and/or accessibility of the activation loop tyrosines for phosphorylation.
This hypothesis is supported by experiments in 293 and Cos cells as well as in the P116 Jurkat T-cell line. The YYAA mutants of ZAP-70 and Syk appeared dysregulated in 293 cells, as measured by tyrosine phosphorylation of the ZAP-70 activation loop and the ability of both of these YYAA mutants to phosphorylate their substrates. In contrast to the YYFF mutant, YYAA ZAP-70 substantially reconstituted a number of signaling events in P116 cells, including tyrosine phosphorylation, calcium flux, transcriptional activation of NFAT, and activation of the Erk1/2 MAP kinase pathway. Also of note is the fact that we had difficulties generating wild-type expression levels of the interdomain B deletion or YYAA mutants in stable P116 transfectants, suggesting some toxicity of these potentially unregulatable mutants. This may be explained by the fact that activation of the TCR can induce apoptosis in Jurkat cells via Fas-dependent pathways (9).
Although our experiments with the YYAA mutant of ZAP-70 suggest that binding of signal transducing molecules to ZAP-70 tyrosines Y315 and Y319 has only partial amplifying effects on several major TCR-dependent signaling pathways, the scaffolding function of these tyrosines cannot be entirely dismissed. Our observation that some of the signaling events, most notably full tyrosine phosphorylation of LAT, SLP-76, and PLC-γ1, could not be completely rescued by YYAA ZAP-70 in P116 cells suggests that there still may be some, perhaps only quantitative, function for Y315 and Y319 in the recruitment of downstream signaling molecules.
Apart from directly affecting the downstream signaling pathways, the molecules interacting with phosphorylated Y315 and Y319 potentially can be involved in the regulation of ZAP-70 itself. Binding of SH2 domain-containing proteins to phosphorylated interdomain B tyrosines may stabilize the active conformation of ZAP-70 and protect Y315/Y319 from dephosphorylation. In addition, the effector functions of these molecules could directly influence ZAP-70 kinase activity. Indeed, Pelosi et al. suggested that binding of Lck via its SH2 domain to phosphorylated Y319 followed by Lck-mediated phosphorylation of Y493 in the activation loop of ZAP-70 is an important positive feedback mechanism in ZAP-70 activation (26).
Up until now, Lck has been viewed as the major PTK responsible for the phosphorylation of ZAP-70 Y493. This was primarily based on mass spectrometry and two-dimensional analysis of tryptic peptides generated from purified recombinant ZAP-70 protein phosphorylated by Lck (5, 34) as well as the data of Pelosi et al. (26). These studies however, did not investigate the role of other kinases, including Fyn and c-Abl, in ZAP-70 phosphorylation. The first two studies also failed to examine phosphorylation of Y315/Y319 and thus did not take into account the influence of these sites on ZAP-70 activity and the ability to autophosphorylate at Y493. Our data from the 293 cell overexpression system revealed that, unlike WT ZAP-70, mere overexpression of the ZAP-70 YYAA mutant led to its phosphorylation at Y493, suggesting that YYAA was able to autophosphorylate at Y493, perhaps via an intermolecular trans-phosphorylation mechanism (Fig. 7). Moreover, with Lck as well as with Fyn and c-Abl cotransfection, we observed high levels of Y493 phosphorylation in WT ZAP-70 but only marginal phosphorylation of Y493 in the kinase-inactive mutant, despite comparable phosphorylation at Y319. These results suggest that these kinases are not very efficient at phosphorylating Y493, even when Y319 is phosphorylated and potentially available for binding. This seems to suggest that in 293 or Cos cells phosphorylation of interdomain B Y315/Y319 is the only major event dependent on kinase(s) other than ZAP-70 itself, which in turn results in an increase of ZAP-70 kinase activity and its trans-autophosphorylation of the activation loop. These data also seem to rule out the possibility that phosphorylation of Y315/Y319 alone substantially enhances accessibility of Y493 for phosphorylation by Lck, Fyn, or c-Abl. In P116 Jurkat cells, however, the KA mutant was hyperphosphorylated at both Y319 and Y493, and its phosphorylation was further increased upon TCR stimulation. This apparent discrepancy could result from differential regulation of ZAP-70 in the P116 Jurkat T-cell line versus 293 or Cos cells, which are of epithelial origin. T cells likely have multiple regulatory mechanisms to control their key signaling molecules, including ZAP-70. Some of these mechanisms may be missing in 293 or Cos cells because they do not express endogenous ZAP-70 and therefore would have no need to specifically control its activity. The observed hyperphosphorylation of KA ZAP-70 in P116 cells indeed suggests the existence of a ZAP-70-driven negative feedback loop in T cells. By impairing ZAP-70 activity by the KA mutation, this putative regulatory mechanism is turned off and nonphysiological hyperphosphorylation of Y319 and Y493 occurs. A similar regulatory mechanism might also explain the incomplete reconstitution of several tyrosine phosphorylation events in P116 cells expressing the potentially hyperactive YYAA ZAP-70 mutant, which would enhance the activity of the same negative feedback loop. However, considering the difficulties we had in obtaining the cells stably expressing YYAA ZAP-70, it is also possible that only the clones able to compensate for increased ZAP-70 activity by reinforcing some of the negative regulatory pathways were selected.
The observation that KA ZAP-70 could be phosphorylated at Y493 also shows that ZAP-70 is not the only PTK able to phosphorylate Y493 and that other PTKs, most likely Lck (26), participate in phosphorylating this site. Additional studies are needed to dissect the contribution of different kinases to Y493 phosphorylation. Nevertheless, based on currently available data, we favor the possibility that Lck and ZAP-70 are the major kinases phosphorylating Y493 in vivo. It is possible that Lck is responsible only for the initial but rather inefficient phosphorylation of Y493 on several ZAP-70 molecules. These activated ZAP-70 molecules may in turn trans-autophosphorylate other ZAP-70 molecules with which they are dimerized by paired ITAMs in the TCR-ζ and CD3 chains, much like dimerized RTKs. Since there certainly is rapid turnover of ZAP-70 binding to ITAMs (3), it is likely that one active ZAP-70 molecule can activate many other ZAP-70 molecules leading to substantial amplification of the initial signal. Consistent with this model, it has been demonstrated that phosphorylated TCR-ζ chain dimers potentiate ZAP-70 in vitro catalytic activity substantially more than do monomers (21). Also, this mechanism may explain why ITAMs are almost always found as pairs in homo- or heterodimeric non-ligand-binding chains within the receptors of the hematopoietic lineage involved in antigen recognition: i.e., to allow for induced dimers of ZAP-70 or Syk to trans-autophosphorylate.
The importance of the activation loop phosphorylation of Syk family kinases has recently been questioned by the studies describing the structures of the isolated kinase domains of ZAP-70 and Syk (1, 19). In both kinase domains, the unphosphorylated activation loop assumes an extended conformation, typical for an activated kinase. In ZAP-70, this might have been driven by the interactions between the activation loop and the kinase domain of neighboring molecules in the crystal, but it also might suggest that phosphorylation of the activation loop is not as critical for ZAP-70/Syk activation as previously was thought. Interdomain B phosphorylation or YYAA mutation may be sufficient to increase ZAP-70/Syk activity enough to autophosphorylate and/or phosphorylate its downstream substrates. By analogy with EphB2, interdomain B in fact may regulate the activation loop conformation and the observed structure may reflect the lack of interdomain B in the construct used for structure determination. In such a circumstance, phosphorylation of the activation loop probably would have mainly stabilizing effects.
It appears that ZAP-70 activation is a complicated multistep process requiring proper spatial as well as temporal coordination of several key events (Fig. 7). Upon TCR stimulation and phosphorylation of TCR ITAMs by Src family kinases, ZAP-70 is recruited to phosphorylated ITAM sequences. Subsequently, Y315 and Y319 in interdomain B of ZAP-70 become phosphorylated by Src family or Abl/Arg family PTKs. This results in the release of ZAP-70 from an autoinhibited state, the upregulation of kinase activity, and the stabilization of the interaction between ZAP-70 and phosphorylated TCR chains. Subsequent steps are not clear yet, but they likely involve phosphorylation of the ZAP-70 activation loop, possibly via the cooperative action of Lck and ZAP-70 itself resulting in the fully active enzyme.
Collectively, our findings suggest that the cytoplasmic PTKs ZAP-70 and Syk share with RTKs a regulatory mechanism involved in inhibiting catalytic activity that involves two tyrosine residues N terminal to their kinase domains. The paired autoinhibitory tyrosines are localized within the juxtamembrane regions of RTKs, whereas they are within interdomain B of ZAP-70 and Syk. Thus, one could view ZAP-70 and Syk essentially as RTKs in which membrane localization is just another means of regulation. However, once localized to the membrane, these kinases are activated through mechanisms similar to those involved in RTK activation.
We thank R. Abraham, J. B. Bolen, C. L. Chan, M. Cooper, V. Horejsi, G. A. Koretzky, M. A. Pendergast, and R. M. Perlmutter for cell lines, constructs, and antibodies. We thank Weiss lab members for advice and discussion.
This work was supported, in part, by A135297 from the National Institutes of Health.
- Received 5 January 2005.
- Returned for modification 25 January 2005.
- Accepted 27 March 2005.
- American Society for Microbiology