text:
2000 Ford Explorer • 100,000 miles
What could be wrong with Ford Eplorer 2000 4x4 when it don't kick in?
December 31, 2012.
December 31, 2012.
Check to see if the actuator on the transfer case to see if it is working and getting power. If it is getting power, make sure it is operating properly.
Dec 31, 2012.
Dec 31, 2012.
My Transfer Case has a bad oil pump, in it, and the truck itself won't shift when driving in 2 wheel drive, but when I put it in 4 Low it will shift up to 3rd gear. It will not shift ...
1 answer • 1993 Ford F-150 • 150,000 miles
My 97 ford explorer 5 sped manual trans 4x4 part time has a bad transfer case. The one I that it currently has on it ends in 195ga. I was able to get a case ending in CA. it has a 4 ...
1 answer • 1997 Ford Explorer • 174 miles
Enter your question...I have a 2003 Dodge Ram 1500 4x4. I have a few concerns about it. I think either the transfer case or something is going out. I was driving it in 4Low and every ...
2 answers • 2003 Dodge Ram • 147,000 miles
2 questions; Both on a cadillac escalade transfer case with full time all wheel drive; I drained & refilled it with the blue ''auto track II'' gm stuff that my gmc truck takes, then ...
3 answers • 2002 Cadillac Escalade • 150,000 miles


text:
B and W Type 65 Black Outdoor CaseItem # HN-BWN017
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subset: commoncrawl


text:
More New and Used
from Private Sellers
Note: Supplemental materials are not guaranteed with Rental or Used book purchases.
Kalaam Gamiil : An Intensive Course in Egyptian Colloquial Arabic. Volume 2by Al-Tonsi, Abbas; Al-Sawi, Laila; Massoud, Suzanne
The American University in Cairo Press
Kalaam Gamiil Volume 2 further develops learners' skills in Egyptian Colloquial Arabic, along parallel tracks of vocabulary and grammar. It is designed to enable students to communicate effectively with native speakers in a wider range of social situations, with a higher degree of accuracy andfluency. It aims to do so by expanding learners' vocabulary and idiomatic expressions, increasing their command of grammar (both syntax and morphology), as well as instilling a measure of cross-cultural understanding.Structured around more sophisticated topics that go beyond those of daily life conversations to cover social and cultural issues and concepts, each lesson includes two situations (often dialogues), a vocabulary list, preparatory sentences using the new vocabulary items and grammatical structures,explanations of the grammar in English, relevant cultural information, in addition to a variety of mechanical drills and communicative exercises.The book, volume two of a two-part series, focuses on the speaking and listening skills that will enable high intermediate to advanced students to handle a variety of more complicated communicative tasks successfully. One CD is included.
Author Biography
Abbas Al-Tonsi is a senior lecturer in Georgetown University's School of Foreign Service in Qatar. He is the author or co-author of a number of Arabic coursebooks, including the Al-Kitaab series.
Laila Al-Sawi and Suzanne Massoud are both senior instructors at the Arabic Language Institute of the American University in Cairo.


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soProject (S.T.A.L.K.E.R. Online Project) - a modification for the game S.T.A.L.K.E.R. Call of Pripyat. Mod adds a multiplayer game scripting support, including ALife simulator. Returns artificial intelligence, monsters and stalkers will take part in online battles killing both players and enemy creatures.
Beta testing of soProject 1.0.0 is completed and version 1.0.0 is now available for download, with source code. Repeat the major changes during the beta test:
- Recovered binder objects
- Fixed bug with moving specator's camera outside the map
- Fixed player's configuration so that the monsters can attack at a distance closer
- Fixed crash when loading a singleplayer location on a dedicated server
- Fixed a crash when the server is turned off
- Fixed crash when changing modes of play through a vote
- Fixed a crash in the 'Capture The Artifact'
- Players are loaded profile and specificity mp_actor
- Fixed a crash after a reconnect client
- Fixed crash at contact to stalkers
- Fixed crash at contact to the zombie
- Are returned mp skins of players
- Fixed crash at spawn the character of the server on team modes
- Disappearance of corpses
- Stalkers attack player
- Fixed not constant crash at connection to the server
- Fixed crash of the server after recreation
- Fixed critical bug
- Crash the server when get damage from players
current soNetwork module development progress and future plans.
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S.T.A.L.K.E.R. Online Project 1.0.1 release version
S.T.A.L.K.E.R. Online Project 1.0.0 release version
S.T.A.L.K.E.R. Online Project 1.0.0 five beta version
S.T.A.L.K.E.R. Online Project 1.0.0 fourth beta version
S.T.A.L.K.E.R. Online Project 1.0.0 third beta version
S.T.A.L.K.E.R. Online Project 1.0.0 second beta version
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is the 2nd beta the 1.6.02 version?
second beta support 126.96.36.199 and 220.127.116.11 versions of game
Youtube.com Gameplay :)
Some news about v. 1.6.02 and i want to ask you: can you send me some spawn menu for multiplayer :?
We can do network with tunggle and play it online :)
Keep up the good work!
this mod looks amazing. stalker multiplayer with ai has always been a dream of mine. keep up the great work
Wow... Its about time someone had the balls.. and tech skillz... to finally try a coop MP/SP mash up!
Few Questions though for you concerning this mod:
1) The map itself...Is it a exact clone of the Pripyat singleplayer map.. minus the NPCs/towns, labs, and faction/monster spawns?....As in is it physically the same.. or is it a smaller, simplified version of the map (to cut down on maximum map size / polygons loaded into the MP engine? When you look at the vanilla MP maps, they are small chunks of single player locations.. so wondered if there was a limitation they had to follow.
2) Once you get the monsters/npcs to spawn and work correctly, will they have normal behavior as they would in a single player map.. or just randomly spawn and wander about.. As in will you see stalkers around campfires or going around in groups.
3) How will Weapons and equipment be handled... will it be like normal MP...or are you going for a true coop experiance and try to mimic the towns and vendors in singleplayer? (not sure if this is simply a map mod, or a full MP game mode overhaul?
4) do you have any test servers setup that say someone can log into and help out with testing? ^_^
you can download mod and find answers on your questions:)
1. its actually the multiplayer map titled "prypyat". its a very big map, but not the same one used in single player.
2. no towns, npcs spawn randomly like anomalies I think
3. just like mp as far as i know
Get out of here stalker, FOR REAL ?


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Tag: ohio state university
Weinland Park Story Book
The Weinland Park Story Book project is the result of a long collaboration between the Wexner Center for the Arts and the residents and children of the Weinland Park community. The limited-edition graphic anthology offers a glimpse into the rich and colorful history of a neighborhood that has faced dramatic changes over the past 100 years.
Shakespeare and Autism
Researchers at The Ohio State University are working with the Royal Shakespeare Company to determine if using Shakespeare's plays as a teaching and therapy tool can have lasting benefits for people with autism.
Weinland Park Story Book, Kevin Farrell aka Dee W. Ieye, Shakespeare and Autism
On this episode of Broad & High, The Weinland Park Story Book project is the result of a long collaboration between the Wexner Center for the Arts and the residents and children of the Weinland Park community. It also features The Dublin Arts Council, The Franklin County Animal Shelter, and Artist Profile: Kevin Farrell.
Arnold Sports Festival, Phase Shift, Kingmakers, Christine Weigand
This time on Broad & High, we'll bring you figure painting from the Arnold Sports Festival. Take a look at Phase Shift at the Urban Arts Space, a thesis exhibition for 14 Ohio State graduating MFA students. We'll visit Kingmakers, a Short North business, working to redefine game night.
The Chimes of Orton Hall
This February marks the 100th anniversary of an Ohio State tradition. Since 1915, the chimes have been part of University life, housed in one of the oldest and most unique buildings on campus. WOSU's Tom Rieland has this profile on the Chimes of Orton Hall…
October 15, 2014 Preview
On the next Broad & High, we'll share the story of Ohio State's Sullivant Hall told through dance and an art exhibit at the Riffe Gallery that will compel you to look to the sky.
Archaeology Field School
Ohio State University Students uncover Ohio's buried past in an archeology field school located in Dublin, Ohio. This is a story we gathered while working on the Adena Mound segment as part of the Columbus Neighborhoods: Clintonville documentary. Students learn how to be archaeologists in the field and learn the history of the land.
April 9 Preview
On the next Broad & High, fowl smelling Corpse Flowers are in bloom at Ohio State University and a 97-year artist reminds us that we are never to old to do what we love.
The Best of Broad & High
On this episode of Broad & High, some of our favorite segments from the first season. Take a look at the Ohio State University's Billy Ireland Cartoon Library & Museum, global mobile photographer Amy Leibrand, the My Very Own Blanket non-profit group in Westerville and a music video by Columbus band Dave Buker & the Historians.
Web Exclusive: Eastern Europe Meets 1920s Fashion
Gayle Strege, curator of OSU's Historic Costume and Textiles Collection, shows us one dress that fits this description.


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Golly. something even naffer looking than Google Glass. They said it couldn't be done. Mind you, they also said that a VR headset couldn't be done, and they were right. Two head mounted screens and a PC to drive them isn't quite the same thing.
Posts by Ian Johnston
598 posts • joined 28 Sep 2007
Oculus Rift noggin-bucket ... heyyy, errr ... have we all got them on already?
Elon Musk: How the Billionaire CEO of SpaceX and Tesla is Shaping our Future
Did we ever find out what happened to the Tesla customer service representative who advised a journalist to give hid battery a quick charge to warm it up after the car spent a freezing night outside? He duly ran out of charge, reported this and Musk went batshit.
Facebook farewells flaky SHA-1
Re: Win/win? for who?
Wouldn't that require a processor running at some significant multiple of 90GHz? Not bad for $80.
IN YOUR FACE, Linux and Apple fans! Oculus is Windows-only for now
Is the underlying message here that they have given up trying to make a proper VR headset, and instead have cobbled together a couple of displays and few accelerometers, with a Windows programme to do all the actual work?
Oculus Rift VR bucket will be seen on noggins near you in 2016
Sounds like a desperate attempt to keep a bit of momentum as far bigger and better funded competitors move rapidly towards launching products.
Tesla Powerwall: not much cheaper and also a bit wimpier than existing batteries
There are other suppliers of li-on batteries. Mastervolt, for example, do 2.5kWh (12V) and 5kWh (12V & 24V) batteries at broadly similar prices per kWh.
Microsoft HoloLens or Hollow Lens? El Reg stares down cyber-specs' code
Is this another blow to the twitching near-corpse of the Oculus Rift?
Who was downloading smut in the office while eating ice cream?
He fixed the ethernet cable?
The Internet of things is great until it blows up your house
The solution to that problem seems obvious. Design an iron equipped with Bluetooth LE, linked to a smartphone, running an app that uses its camera to scan a QR code printed on a fabric care tags. This QR code contains all of the care information for that article of clothing, so every time that dress or dress shirt goes under the iron, the app adjusts the iron to the ideal temperature.
Neither "obvious" nor "solution" seems quite the right word here. At the moment you look inside the garment (the labels are not outside, obviously), see whether it's one, two or three dots and set the iron accordingly. A slight improvement might be to have a finer scale, with degrees rather than dots.
Finding the tag, finding the smartphone, starting the app, scanning the QR code and getting Bluetooth working is not an improvement in any conceivable way. It simply adds stages for no benefit at all.
This is why people laugh and point at those who promote the internet of things.
Apple Watch: We ROUNDUP the ROUNDUPS. Yes, Roundup-squared
Re: What about the following options?
Spot on, but you forgot the prototype "left" in a bar to be "found" by a friendly tech blogger and retrieved by Apple after bloodcurdling "threats" of legal action which curiously don't seem to come to anything.
Saturn's rings, radio waves ... poetry? At home with Scotland's Mr Physics
Maxwell's real breakthrough in electromagnetic theory was positing the displacement current, which is the rate of change of electric flux density , dD/dt. There was no empirical reason to expect this, but it made the equations nicely balanced by adding a term analogous to dB/dt. Everything else in the equations predated Maxwell, but he unified them and, by adding displacement current, git EM radiation to pop neatly out. It has - rightly, I think- be described as one of the greatest intellectual leaps ever made.
Incidentally, Maxwell's own house, Glenlair in Galloway, is regularly open to visitors. The main part of the house was destroyed by fire years ago, but the surviving bit is under restoration. While in the area you can also visit Maxwell's grave in Parton kirk yard and see a memorial window in Corsock kirk, for which (the building) he paid. It's claimed to have the only stained glass representation of Maxwell's equations in the world.
Stress me, test me, vex me ... boffins seek Hall Effect in frustrated magnets
I strongly suspect that article to have been written by the editors of Social Theory, getting their own back on Alan Sokal.
Streaming tears of laughter as Jay-Z (Tidal) waves goodbye to $56m
Re: HiFi vs Premium
Excellent find, and directional too:
IMPORTANT: Please observe the correct cable orientation during installation. The cable is marked with an arrow "→" which indicates signal direction. e.g. Bluray/DVD → AV Receiver→ TV/Projector
Force your hand: Apple 13-inch MacBook Pro with Retina Display
To force-click on an item, you simply click on it once as normal, but then maintain the pressure with your finger until you feel a second click.
It has only taken Apple five years to catch up with Android, then? How nice.
By Odin, Parallels thinks cloud service providers should buy Odin
Odin is, or perhaps now was, also the port of wine to OS/2.
AUTOPILOT: Musk promises Tesla owners a HANDS-OFF hands-on
Re: Navigation Needs Update
Or when he goes of on a screaming rant about "big oil shills" at the slightest criticism.
Adobe lifts sheet on Dropbox-style doc sharing cloud
Re: Subscription dystopia
LibreOffice Draw edits pdfs surprisingly well.
Apple Watch: Wait a minute! This puny wrist-puter costs 17 GRAND?!
Five hundred quid for a tiny secondary display for your iPhone? What a bargain.
Inside GOV.UK: 'CHAOS' and 'NIGHTMARE' as trendy Cabinet Office wrecked govt websites
Re: Presumably....
...the same people are behind the Graun's recent web disaster, which converted a reasonably useable news web site into a ghastly disorganised mashup of blogs spouting cack, reposting of "news" from fourth rate journalistic sources, confusion over what counted as opinion and what as news, confusion over when material was originally published, etc etc.
Trying to recreate the spirit of the printed paper online, then?
Vodafone didn't have a £6bn tax bill. Sort yourselves out, Lefties
Very entertaining. I don't know which bit I like best, the claim that every single HSBC customer who owed taxes has paid or the claim that call-me-Dave's meeting with Vodafone didn't happen, and that everything was settled through a curiously unreported court case.
M0n0wall comes tumbling down as dev throws in the trowel
If it's good, and since it's open source, why doesn't someone else take it over?
$10,000 Ethernet cable promises BONKERS MP3 audio experience
Re: One born every minute
I think the phrase you're looking for is "It opens up the sound stage".
They've finally solved it: Schrödinger's cat is both ALIVE AND DEAD
If a single scientist enters the room with the box in it, opens up, looks inside and then dies of a heart attack before telling anyone how the cat was, what happens? Does the cat's state uncollapse itself until someone else has a shufti?
Brain-train kid game settles with FTC over 'unsupported' claims
Re: Research
It really is depressing how bad most educational research is. The Clackmannanshire farce is a prime example: all it proved was that when you give a fairly small number of children intensive coaching their scores improve. Well whoop-de-flipping-doo.
Meanwhile in the real world, as synthetic phonics becomes mandatory and the publishers laugh all the way to their banks, reading ability at the end of primary school is deteriorating measurably. So much for the "all children will be three or four years ahead" guff the Clackmannanshire enthusiasts sell us.
Brain Gym is discredited. Can someone please do Mindfulness next?
'How a censorious and moralistic blogger ruined my evening'
Re: What this world needs is more organizations like The Guardian and The Register
The Guardian? You mean that bunch of trust-funded, nepotistic private school alumni who have form for revealing their sources without a qualm if the alternative is any sort of personal inconvenience?
Ericsson boss sticks a pin in Google's loony Loon bubble
What precisely is the Google plan for dealing with the wind where their balloons are? Blimps are fun, by I doubt many of them can do the 100kt+ which would be required to maintain station in many places.
REVEALED: Apple fanbois are 'MENTALLY UNSTABLE' - iShop staff
Feeling a bit hormonal today, are we?
Re: Be nice to shop workers
Be nice to people, full stop.
It's a long time since I did customer support, but I do remember going out of the way to look after the people who treated me decently.
Did you spot the inconsistency there?
BYOD: don't let the dream turn into a nightmare
Re: BULLSHIT
That is the professional advice and best practices of REAL network admins and directors. Not snake oil salesmen.
Nah, it's appropriate advice for a very small number of companies and paranoid, will-nobody-think-of-the-child job-preserving bullshit for most. The average university has upwards of ten thousand privately owned devices connected daily, and nobody dies as a result.
Antares apocalypse: Orbital points finger at turbopump FAIL
Before getting too sneerily superior about 1960s Russian rocket technology, it might be worth remembering where and when the only spacecraft currently capable of taking people to the ISS was designed.
Gov.UK doubles IT outsourcing to £20bn
As little as that?
ROGUE SAIL BOAT blocks SPACE STATION PODULE blastoff
There is no excuse for not listening on channel 16
Marine VHF is very limited in range, both by power and by curvature of the earth. There is no reason at all to have a VHF set switched on forty miles offshore - it's for inshore and intership use only.
Trips to Mars may be OFF: The SUN has changed in a way we've NEVER SEEN
Re: Nuclear
Not terribly effective for temperatures under several thousands degrees. You need to make the bloody thing glow to radiate heat.
Care to guess why the surface of the moon, and indeed the Sahara, get so cold at night?
Does this float your boat? Dead Steve Jobs to hijack yachts from BEYOND THE GRAVE
Re: Avast, there
SHIP OF FAIL: How do we right capsized institutions we thought would NEVER go under?
Railway risk
Various inland lines that could have been put into service had been closed and taken up by people who were unable to assess risk
No, they were closed and taken up by people who realised that there was no sense in keeping a railway line open in order to provide a diversionary route which has been closed once, for a few weeks in fifty years.
Don't bother with Apple's 9 Sept hype-day: Someone's GONE AND BLABBED IT ALL
According to some of the paper's sources, Apple has tweaked the iOS user interface to include a "one-handed mode" that can be switched on and off, for those with short fingers but who still like the idea of a phone with a larger screen.
Wow. What a stunning, ground breaking idea. Only Apple could ever come up with such a stunning breakthrough. Well, not counting Samsung, who had a one-handed mode on the original Galaxy Note three years ago.
Re: Biting on the Apple
My original Galaxy S does everything I want it to do, as fast as I want it to do it. Much as I would like some new shiny, I haven't been able to find any semi-rational reason for splashing out a few hundred quid on upgrading.
Firefox 32 moves to kill MITM attacks
New features are all very well
But I do wish they'd get round to dealing with that whole "being a bloated cycle-sucking RAM vampire" thing as well.
Raspberry Pi B+: PHWOAR, get a load of those pins
Any sign of the promised education version?
It's only two years late now.
Giving your old Tesco Hudl to Auntie June? READ THIS FIRST
I was beyond fuming
He points out that cheap tablets are often bought for children and by selling on a tablet which has the child's social network data, the parent might be unwittingly aiding a stalker who could use the identity of the child to stalk other children
He appears to be channelling the collective mind (using the term loosely) of Mumsnet.
Apple wins patent on charging iThings THROUGH THIN AIR
Re: Inefficiency is irrelevant
People aren't prepared to spent money on pointless techno bling? Haven't met many Apple fanboiz, have you?
British cops cuff 660 suspected paedophiles
Re: Good work
At which time the Boots instore advert for photo processing was a life size free standing cardboard cut-out of a child, seen from behind. A naked child.
Somerville was shopped by a presumably hypocritical Boots employee, and her arrest details were flogged to the press by a Metropolitan police officer.
Anti-snoop Android 'Blackphone' sees the light of day
Am I the only person assuming that the NSA is behind this?
Quick Q: How many FLOPPIES do I need for 16 MILLION image files?
Re: 16-page document I was working on last night won't fit onto a floppy
I have a hard-sectored 8" floppy for a Philips WP system, ca 1984. It has some useful stuff on it, and I dream of one day reading it. I'd be mildly surprised if there is a working system of that sort left, and astonished if it could transfer data to anything else.
Obscure note: the disk was used on Pip, Squeak and Wilfred.
Tim Cook: Apple's 'closer than it's ever been' to releasing new product range
Here we would be going again
If Apple really were about to release something, wouldn't we be getting the usual "Apple engineer accidentally leaves prototype in bar, prototype mysteriously finds its way to Apple-friendly blogger/magazine, Apple claims to sue Apple-friendly blogger/magazine and retrieves prototype after just long enough for plenty of details to be leaked" routine?
Look out, bankers! It's Lily Cole and her (Brit taxpayer-funded) WISH-PRINTING ATM
History of Art at Cambridge, eh? The traditional degree for well-connected but dim girls.
Batten down the hatches, Ubuntu 14.04 LTS due in TWO DAYS
Re: @asdf The desktop deadend.
I've been running Ubuntu since 6.06 and current have it on about ten machines. I have never, ever managed a successful version upgrade.
Elon Musk's LEAKY THRUSTER gas stalls Space Station supply run
Re: Say what?
The usual suspects? Is this Big Oil Shill paranoia? Perhaps Mr Musk should have charged his rocket for longer, instead of relying on what the customer support person (suesequently sacked) told him to do.
NSA's TURBINE robot can pump 'malware into MILLIONS of PCs'
Oh yeah?
"All of GCHQ's work is carried out in accordance with a strict legal and policy framework which ensures that our activities are authorized, necessary and proportionate, and that there is rigorous oversight," the British intelligence agency said in a statement.
A statement which they make so often that I expect it's a keyboard macro in the press office there. Does anyone seriously believe that they would tell the truth about this?
Anatomy of a 22-year-old X Window bug: Get root with newly uncovered flaw
Re: I have looked
Code should not cause compilers to issue dozens of warnings.
Meh. If you're not getting compiler warnings you're not trying hard enough.


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Current Issues in Healthcare (BIOS3066)
This unit introduces students to selected developments that are impacting, or are likely to impact, on the practice and management of the health care in Australia. Because health care is driven by a multitude of forces, the scope of the developments studied is broad. Topics to be covered will be drawn from the basic sciences applicable to health care, and health management. Examples of the topics under consideration include a critical analysis of complementary and alternative medicine, advances in assisted reproductive technologies and emerging diseases and infections. Material will be presented in lectures, with use of self-directed learning and individual or group projects. The unit integrates units of study completed earlier in the program, thus enabling students to apply their knowledge while developing the skills needed to analyse, understand and anticipate future directions in health care.
Our courses that offer this unit of study
- Bachelor of Applied Science (Diagnostic Radiography)
- Bachelor of Applied Science (Occupational Therapy)
- Bachelor of Applied Science (Occupational Therapy) Honours
- Bachelor of Computer Science and Technology
- Bachelor of Computer Science and Technology (Advanced)
- Bachelor of Health Sciences
- Bachelor of Health Sciences and Master of Nursing
- Bachelor of Information Technology
Further unit of study information
1x2-hr lecture fortnightly, commencing week 2, plus independent on-line learning modules with email support
Online quiz (20%), written assignment (25%), end semester exam (55%)
Faculty/department permission required?
Study this unit outside a degree
Non-award/non-degree study
If you wish to undertake one or more units of study (subjects) for your own interest but not towards a degree, you may enrol in single units as a non-award student.
Cross-institutional study
If you are from another Australian tertiary institution you may be permitted to underake cross-institutional study in one or more units of study at the University of Sydney.


text:
Sta-Rite Handle for Sta-Rite Slide Valve - 14930-0014
No. 14 Image Shown Above 14930-0014 Handle for Sta-Rite Slide Valve Used on Sta-Rite (Pentair) Valve Series Replacement PartsSta-Rite is manufactured by Pentair Pool products, Pentair Water pool and spa and Sta-Rite a common name in poolValve parts. The schematic diagram of Valve parts, Pentair pool Valve parts, Sta-Rite Products, PentairValve parts and spares (spare Valve parts) are listed. Always low prices and fast shipping. Discount Sta-Rite(Pentair) swimming pool Valve parts and other swimming pool Valve parts are always in stock and available.Back To Sta-Rite Duetta Plastic Slide Valve Parts model 14936 parts


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UCSF Benioff Children's Hospitals and Community Medical Centers have signed a long-term agreement to significantly enhance and expand specialty medical care for children in Fresno. The agreement builds upon a 40-year collaboration on medical education in a region that has one of the lowest doctor-to-patient ratios in the state.
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Today Community Medical Centers' CEO Tim Joslin testified at the House Ways and Means Committee's rural healthcare hearing in Washington, D.C., where he urged Congress to revise its process for funding graduate medical education.
The 10th annual May Day Safety Fair sponsored by the Kids Safe Coalition of Central California was an education day at Chukchansi Park baseball stadium where first-graders from local schools had the opportunity to receive valuable information on how to be safe. Community's Leon S. Peters Burn Center partnered with PG&E to present how to be safe around power lines.
More than 3,000 first-graders from local schools crowded the sidewalks of a scaled down city scene to practice looking both ways and yelling at imaginary, distracted drivers during safety lessons sponsored by Community Regional Medical Center.
A special kind of teamwork and visits by clinicians to patients' homes is helping to keep those with chronic lung problems out of the hospital.
The Central San Joaquin Valley has one of the lowest physician-per-capita ratios in California, and changes in healthcare are making it harder for people to find a doctor here. Community Medical Centers' 40-year partnership with the University of California, San Francisco – rated among the top five best medical schools in the nation – educates more than 300 physicians a year in Community's hospitals.
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Phosphorylation-Dependent 14-3-3 Binding to LRRK2 Is
Impaired by Common Mutations of Familial Parkinson's
Xianting Li1, Qing Jun Wang2¤, Nina Pan1, Sangkyu Lee3, Yingming Zhao3, Brian T. Chait2, Zhenyu Yue1*
1Department of Neurology and Neuroscience, Mount Sinai School of Medicine, New York, New York, United States of America, 2Laboratory of Mass Spectrometry and
Gaseous Ion Chemistry, The Rockefeller University, New York, New York, United States of America, 3Ben May Department for Cancer Research, The University of Chicago,
Chicago, Illinois, United States of America
Background: Recent studies show that mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are the cause of the most
common inherited and some sporadic forms of Parkinson's disease (PD). The molecular mechanism underlying the
pathogenic role of LRRK2 mutations in PD remains unknown.
Methodology/Principal Findings: Using affinity purification and mass spectrometric analysis, we investigated
phosphorylation sites and binding proteins of LRRK2 purified from mouse brain. We identified multiple phosphorylation
sites at N-terminus of LRRK2 including S910, S912, S935 and S973. Focusing on the high stoichiometry S935 phosphorylation
site, we developed an anti-pS935 specific antibody and showed that LRRK2 is constitutively phosphorylated at S935 in
various tissues (including brain) and at different ages in mice. We find that 14-3-3 proteins (especially isoforms c and g) bind
LRRK2 and this binding depends on phosphorylation of S935. The binding of 14-3-3, with little effect on dimer formation of
LRRK2, confers protection of the phosphorylation status of S935. Furthermore, we show that protein kinase A (PKA), but not
LRRK2 kinase itself, can cause the phosphorylation of LRRK2 at S935 in vitro and in cell culture, suggesting that PKA is a
potential upstream kinase that regulates LRRK2 function. Finally, our study indicates that the common PD-related mutations
of LRRK2, R1441G, Y1699C and G2019S, decrease homeostatic phosphorylation levels of S935 and impair 14-3-3 binding of
Conclusions/Significance: LRRK2 is extensively phosphorylated in vivo, and the phosphorylation of specific sites (e.g. S935)
determines 14-3-3 binding of LRRK2. We propose that 14-3-3 is an important regulator of LRRK2-mediated cellular functions.
Our study suggests that PKA, a cAMP-dependent kinase involved in regulating dopamine physiology, is a potential
upstream kinase that phosphorylates LRRK2 at S935. Furthermore, the reduction of phosphorylation/14-3-3 binding of
LRRK2 due to the common familial PD-related mutations provides novel insight into the pathogenic mechanism of LRRK2-
linked PD.
Citation: Li X, Wang QJ, Pan N, Lee S, Zhao Y, et al. (2011) Phosphorylation-Dependent 14-3-3 Binding to LRRK2 Is Impaired by Common Mutations of Familial
Parkinson's Disease. PLoS ONE 6(3): e17153. doi:10.1371/journal.pone.0017153
Editor: Tsuneya Ikezu, Boston University School of Medicine, United States of America
Received December 10, 2010; Accepted January 20, 2011; Published March 1, 2011
Copyright: ? 2011 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the US National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)
NS061152 (Z.Y.), NS060809 (Z.Y.), RNS055683A (Z.Y.), Michael J. Fox Foundation (Z.Y.). The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
¤ Current address: Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, Kentucky, United States of America
Parkinson's disease (PD) is a major human neurodegenerative
disease clinically characterized by the motor function deficits as
well as non-motor impairment. The most prominent neuropath-
ological features are the loss of midbrain dopaminergic neurons
and deposit of intracellular Lewy Body consisting mainly of a-
synuclein. The pathogenic mechanism of PD remains largely
undefined. However, the recent identification of genetic mutations
that are associated with familial PD provides an entry to uncover
cellular and molecular pathways that lead to the disease.
Mutations in Leucine-Rich Repeat Kinase 2 (LRRK2) have been
linked to the most common familial form and some sporadic forms
of PD [1,2]. Because many LRRK2 mutation carriers exhibit
typical PD symptoms indistinguishable from idiopathic PD cases,
it is hypothesized that an understanding of the biology and
pathophysiology of LRRK2 will provide new opportunities to
develop effective treatments of PD.
LRRK2 is a large complex protein of 280 kD containing two
important enzymatic domains, serine/threonine kinase and ROC
GTPase; it also carries several conserved protein motifs including
leucine-rich repeats (LRR), the C-terminal of ROC (COR)
domain and a WD40 repeat. Previous biochemical analysis
demonstrates the kinase and GTPase activity of LRRK2, and
these activities are apparently altered by several PD-linked
mutations [3,4,5,6], leading to the hypothesis of ''gain-of-
PLoS ONE | www.plosone.org1March 2011 | Volume 6 | Issue 3 | e17153
Page 2
function'' in LRRK2 mutants with enhanced kinase activity
[5,7,8]. LRRK2 protein can be modified posttranslationally (e.g.
by phosphorylation) and forms a dimer in vitro and in vivo [9,10,11].
These alterations may play a critical role in regulating LRRK2
biochemical activities and cellular functions. However, until now
little has been known about the normal function of LRRK2 or the
pathogenic pathways mediated by PD-linked mutant LRRK2.
Several studies show that the mutants of LRRK2 impair the
outgrowth or maintenance of neurites in primary neuronal
cultures, whereas reduced or abolished expression of LRRK2
has the opposite effect. In addition, inhibition of kinase activity
seems to alleviate the toxic phenotype caused by LRRK2 mutants
To elucidate the cellular pathway and pathogenic role of
LRRK2 in PD, we investigated LRRK2 protein modifications and
interactors in the brain. We show that LRRK2 is phosphorylated
at multiple sites. Our study reveals that 14-3-3s bind LRRK2 and
the binding depends on the phosphorylation of S935. Further-
more, we show that protein kinase A (PKA) causes phosphoryla-
tion of LRRK2 at S935 in vitro and in cell culture, implicating PKA
pathway in regulating LRRK2 function. Finally, our study
suggests that common PD mutations of LRRK2 impair phos-
phorylation levels of S935 as well as14-3-3 binding. Our data,
therefore, provide molecular insight into the regulation of LRRK2
and suggests a potential mechanism for LRRK2-mediated PD
Identification of phosphorylation sites in LRRK2 from
mouse brain
We previously reported the purification of FLAG-tagged LRRK2
protein from BAC transgenic mice [3]. For phosphorylation site
identification, the purified LRRK2 protein was digested in-gel using
various proteasesand the resultingproteolyticpeptideswere analyzed
by multiple mass spectrometer methods including MALDI-QqTOF,
MALDI-ion trap (LCQ DECA XP), and nano-HPLC/ velos LTQ
Orbitrap. The resulting MS/MS data were used to identify proteins
and protein modifications. The results reveal 3 serine phosphoryla-
tion sites (S910, 935 and 973) from tryptic peptides and 1 serine
phosphorylation site (S912) in chymotryptic peptides of LRRK2,
respectively (Figure 1A) (Figures S1, S2, S3 and S4). Interestingly,
stoichiometry of all 4 serine phosphorylation appears high, as the
ratios of MS/MS spectra for modified peptides versus unmodified
peptides areall more than 30%. This result indicates the relative high
probability of LRRK2 phosphorylation at these sites in the brain
(Figures S1, S2, S3 and S4).
In this initial study, we focused on the analysis of the high
stoichiometry S935 phosphorylation. We developed an antibody
raised against phosphorylated S935 (pS935) peptide. The anti-
pS935 antibody detects a strong signal in purified FLAG-LRRK2
protein from BAC transgenic brains, while the signal is completely
abolished upon the treatment with calf-intestinal alkaline phos-
phatase (CIAP) (Figure 1B). The loss of phosphorylation at S935
with alkaline phosphatase treatment was also confirmed by mass
spectrometric analysis (Figure S5). The antibody also detected
pS935 signal in FLAG-LRRK2 protein isolated from transfected
HEK-293T cells. In contrast, FLAG-LRRK2 mutant S935A,
where serine 935 was replaced with alanine, was not recognized by
this antibody, even though much more mutant protein (compared
to wild type) was loaded in the gel (Figure 1C). We also examined
the pS935 levels in purified FLAG-LRRK2 from different tissues
and at different ages in the brain. The results show that FLAG-
LRRK2 is phosphorylated at S935 in lung, spleen and kidney in
addition to brain, and the pS935 levels relative to the total FLAG-
LRRK2 protein amount are comparable among these tissues
(Figure 1D). Moreover, the relative levels of pS935 do not change
significantly at different ages in the brain (Figure 1E). The above
results suggest that pS935 are maintained at a constant level under
normal condition.
Identification of 14-3-3s in LRRK2 protein complex and
specific 14-3-3 isoforms as preferential LRRK2-binding
We sought to identify LRRK2-binding proteins in the brain by
analyzing proteins that were affinity-isolated with FLAG-LRRK2
from BAC transgenic brain. We isolated only the proteins unique
to the transgenic (compared to non-transgenic control) and
performed mass spectrometry analysis. We identified several
isoforms of 14-3-3 proteins, such as c, g, f and e (Figure 2A,
arrow) (Figures S6, S7 and S8) that are specifically isolated by
FLAG-LRRK2. Using commercial 14-3-3 isoform-specific anti-
bodies, we found 14-3-3c, g, f as well as b, h in the
immunoprecipitated products (Figure 2B). To further evaluate
various 14-3-3 isoform binding to LRRK2, we co-expressed
FLAG-LRRK2 with individual myc-tagged 14-3-3 isoforms in
HEK-293T cells and tested their binding by co-immunoprecipi-
tation (co-IP) analysis. The results indicated that, although all six
isoforms co-IP with LRRK2, the c and g forms pulled down much
more LRRK2 protein than did the other isoforms (Figure 2C). In a
parallel experiment, we incubated each purified GST-14-3-3
isoform protein with BAC-FLAG-LRRK2 transgenic mouse brain
lysate and, followed by a GST pull-down assay. The result again
showed enhanced binding of 14-3-3c and g with LRRK2,
compared to 14-3-3f, e and h (Figure 2D). The above data
collectively suggests that LRRK2 prefers 14-3-3c and g as binding
partners and that the binding is likely mediated through direct
protein-protein interactions.
Binding of 14-3-3 to LRRK2 depends on phosphorylation
of S935
14-3-3 proteins are a family of conserved regulatory molecules
that control various cellular functions by directly binding target
signaling proteins, including kinases, phosphatases and receptors
[15]. 14-3-3 binding was found frequently in a phospho-serine
dependent manner and occurs in a consensus motif (RSXpSXP
and RXY/FXpSXP) [16]. We next investigated whether pS935 of
LRRK2 is relevant to the binding of 14-3-3 (although S935
surrounding sequence does not fit well with the known consensus
motif). First, we transfected HEK-293T cells with plasmids
containing Myc-14-3-3c and FLAG-LRRK2 or FLAG-LRRK2-
S935A. We found that anti-Myc antibody co-immunoprecipitated
14-3-3c with FLAG-LRRK2 but not FLAG-LRRK2-S935A
(Figure 3A). Reciprocal Co-IP assay using anti-FLAG antibody
showed a similar result that 14-3-3 was pulled down only with
LRRK2 wild type but not S935A mutant (Figure 3B). To further
test the role of phophorylated S935 in binding, we incubated
purified GST-14-3-3c and BAC-FLAG-LRRK2 transgenic mouse
brain lysate in the presence of phospho-peptide QRHSNpSLGP
(containing pS935) and non-phospho-peptide QRHSNSLGP
(control) at various concentrations. GST-14-3-3c pulled down
FLAG-LRRK2 in the absence of the peptides; however, adding
the pS935-containing peptide effectively inhibited the co-precip-
itation of GST-14-3-3c with FLAG-LRRK2 in a dosage-
dependent manner. By contrast, the control peptide has no
inhibitory effect toward the pull-down of GST-14-3-3c and
FLAG-LRRK2 under the same conditions (Figure 3C). These
Phosphorylation and 14-3-3 Binding of LRRK2 and PD
PLoS ONE | www.plosone.org2 March 2011 | Volume 6 | Issue 3 | e17153
Page 3
results indicate that the phospho-peptide (but not the control
peptide) competed for 14-3-3 binding, therefore demonstrating the
critical role of pS935 in 14-3-3 binding with LRRK2.
Furthermore, we co-transfected FLAG-LRRK2 with Myc-14-3-
3c wild type, mutant K50E or V181D, which was reportedly
impaired in binding phospho-serine of target proteins [17,18]. In
Figure 1. Identification of LRRK2 phosphorylation sites in BAC transgenic brain. (A) Table of the identified phosphorylation sites within
tryptic peptides from purified brain FLAG-LRRK2 protein. The estimate of the stoichiometry of phosphorylation is based on the ratio of number of MS/
MS spectra observed. The exact location of each proteolytic peptide is shown in the position column. (B) Purified brain FLAG-LRRK2 protein was
treated with or without Calf-intestinal alkaline phosphatase (CIAP), followed by Western blot analysis with anti-pS935 antibody or anti-FLAG antibody.
(C) FLAG-LRRK2 WT or FLAG-LRRK2 S935A mutant plasmid was transfected into HEK-293T cells; FLAG-tagged proteins were then immunoprecipitated
and analyzed by anti-pS935 antibody or anti-FLAG antibody via Western blot analysis. (D) FLAG-LRRK2 was purified from different BAC-transgenic
tissues and western blot was performed to detect pS935 and total FLAG-LRRK2 levels. No significant difference in the ratio of pS935/total LRRK2 was
observed between different tissues. Data are presented as mean value (6 SEM) from three mice. (E) FLAG-LRRK2 was purified from 3 months and 18
months transgenic mouse brain and western blot was performed to detect pS935 and total FLAG-LRRK2 levels. No significant difference in the ratio of
pS935/total LRRK2 was observed between 3 months and 18 months. The Western blot signals were quantified by using LI-COR and Odyssey software.
Data are presented as mean value (6 SEM) from three mice.
Phosphorylation and 14-3-3 Binding of LRRK2 and PD
PLoS ONE | www.plosone.org3 March 2011 | Volume 6 | Issue 3 | e17153
Page 4
contrast to wild type 14-3-3, mutant K50E or V181D completely
lost the binding to LRRK2 as shown in experiments that either use
anti-FLAG or anti-Myc antibody for the co-IP (Figure 3 D, E),
consistent with the notion that 14-3-3 binding depends on
phospho-serine in LRRK2.
To study the relationship of 14-3-3 binding and S935
phosphorylation, we transfected FLAG-LRRK2 in the absence or
presence of 14-3-3 wild type or mutant K50E/V181D, which was
previously shown as a dominant negative mutant (DN) that
abolishes wild type14-3-3 binding to the target proteins [19]. The
result showed that the relative pS935 levels, as measured with anti-
pS935 staining over anti-FLAG staining, were significantly higher
with co-transfection of Myc-14-3-3c and FLAG-LRRK2 than
FLAG-LRRK2 transfection alone. In contrast, co-transfection of
FLAG-LRRK2withMyc-14-3-3DN markedly reduced therelative
pS935 levels of LRRK2 (Figure 3F). These results suggest that 14-3-
3 binding prevents the dephosphorylation of LRRK2 at S935.
S935A mutation does not affect LRRK2 dimerization
One of the important roles of 14-3-3 binding is to provide
structural support for the target protein dimer formation via the
self-dimerization of 14-3-3 proteins [20]. Since LRRK2 is known
to form dimers in vitro and in vivo [9,10,11], we next investigated
whether 14-3-3 interaction of LRRK2 is important for the
dimerization of LRRK2. HEK-293T cells were co-transfected
with HA-tagged LRRK2 and FLAG-LRRK2 or FLAG-LRRK2
S935A mutant. The reciprocal co-IP experiments show that
FLAG-LRRK2 wild type and FLAG-LRRK2 S935A mutant did
Figure 2. Identification of 14-3-3s as LRRK2 interaction proteins. (A) Coomassie blue staining showing the affinity-purified FLAG-LRRK2 and
its binding proteins from FLAG-LRRK2 transgenic brain. The positions of the FLAG-LRRK2 and 14-3-3 bands are labeled with arrows. Wild-type mouse
brain was used as the control for background proteins during purification. (B) Western blot analysis was performed to detect the presence of various
14-3-3 isoforms in association with the affinity purified FLAG-LRRK2 protein. Antibodies against specific isoforms of 14-3-3 were used. Wild type
mouse tissue was used as the control. (C) FLAG-LRRK2 and different Myc-14-3-3 isoform plasmids were co-transfected into HEK-293T cells and
Immunoprecipitation was performed using anti-Myc antibody, followed by the analysis of protein levels using indicated antibodies. (D) FLAG-LRRK2
plasmid was transfected into HEK-293T cells and transfected cell lysate was incubated with different purified GST-14-3-3 isoforms as indicated. The
GST-pull down assay was assayed by Western blot analysis of the FLAG-LRRK2.
Phosphorylation and 14-3-3 Binding of LRRK2 and PD
PLoS ONE | www.plosone.org4 March 2011 | Volume 6 | Issue 3 | e17153
Page 5
Figure 3. 14-3-3 binding to LRRK2 S935 is phosphorylation-dependent. (A, B) FLAG-LRRK2 Wt or S935A mutant plasmid was transfected
with or without Myc-14-3-3c into HEK-293T cells. Co-immunoprecipitation experiments were performed using either anti-myc (A) or anti-FLAG (B)
antibody, followed by Western blot analysis using the indicated antibodies. (C) Sepharose bead-conjugated GST-14-3-3c was incubated with FLAG-
LRRK2 transgenic brain lysate in the presence of various concentrations of phosphorylated peptide (pS935) or non-phosphorylated peptide. The
binding FLAG-LRRK2 protein was pulled down and analyzed by Western blot analysis using anti-FLAG antibody. (D, E) FLAG-LRRK2 was transfected
alone, with Myc-14-3-3c Wt, with Myc-14-3-3 g K50E mutant, or with Myc-14-3-3c V181D mutant into HEK-293T cells. The co-IP assay was performed
using either anti-Myc (D) or anti-FLAG (E) antibody, followed by Western blot analysis using indicated antibodies. (F) FLAG-LRRK2 was transfected
alone, with Myc-14-3-3c wt or Myc-14-3-3c dominant negative (DN) mutant (K50E/V181D) into HEK-293T cells. The total FLAG-LRRK2 was
immunoprecipitated and analyzed by Western blot using anti-pS935 and anti-FLAG antibodies. The Western blot shows the results from three
independent transfections. The quantification of the signals was done by using LI-COR imaging and Odyssey software. The data was analyzed by One-
way ANOVA (** P,0.01). Data are presented as mean value (6 SEM) from three independent experiments.
Phosphorylation and 14-3-3 Binding of LRRK2 and PD
PLoS ONE | www.plosone.org5 March 2011 | Volume 6 | Issue 3 | e17153
Page 6
not differ in pulling down together with HA-LRRK2 (Figure 4A,
B). Considering S935A mutant of LRRK2 is defective in
phosphorylation at 935 and 14-3-3 binding, this result suggest
that 14-3-3 binding to LRRK2 does not play a critical role in
LRRK2 dimer formation. Rather, the dimerization of LRRK2 is
perhaps determined by the sequence outside 14-3-3 binding region
Common familial PD mutations of LRRK2 cause a
reduction in pS935 levels and 14-3-3 binding
Binding of 14-3-3 with various kinases plays an important role in
regulating downstream signaling [15,19]. Although the LRRK2-
mediated signaling pathway has not been clearly elucidated, we
sought to investigate whether common PD mutations of LRRK2,
including G2019S, R1441G and Y1699C, affect S935 phosphor-
ylation and/or 14-3-3 binding of LRRK2. First, we examined the
pS935 levels in the PD-linked mutants of LRRK2 expressed in
HEK-293T cells. Compared to wild type LRRK2, mutants
R1441G and Y1699C isolated from cells have markedly reduced
did not change significantly in pS935 levels (Figure 5A). However,
when we assayed pS935 levels in LRRK2 wild type or mutant
proteins isolated from transgenic brains [21], G2019S mutant
showed a modest but significant decrease in pS935 levels as
compared to LRRK2 wild type (Figure 5B). R1441G mutant from
transgenic brain again exhibited a marked reduction of pS935
levels, whereas K1906M had little change in pS935 levels in the
brain (Figure 5B). Next, we examined 14-3-3 binding with LRRK2
variants in transfected cells. We transfected HEK-293T cells with
Myc-14-3-3c and FLAG-LRRK2 wild type or various mutant.
Comparedtowild typeLRRK2,all threemutants(S935A,R1441G
and G2019S)exhibited reduced co-immunoprecipitationwithMyc-
14-3-3c, suggesting that these mutants are impaired in 14-3-3
binding. But the degree of impairment in these mutants differs, with
S935A affected the most (,90% reduction) and G2019S the least
(,20%) (Figure 5C).
We further evaluated the alteration of PD mutants in 14-3-3
binding in vitro. We incubated purified GST-14-3-3c proteins with
extracts from HEK-293T cells transfected with FLAG-LRRK2
wild type or various mutants, followed by analysis of FLAG-
LRRK2 protein levels pulled down by GST-14-3-3c. The results
showed that binding of 14-3-3 to mutant R1441G or Y1699C is
markedly reduced as compared to wild type LRRK2, whereas 14-
3-3 binding is affected little in G2019S or K1906M mutant
(Figure 5D). The above results indicate a general effect of the
above PD-linked mutations on the phosphorylation of S935 and
14-3-3 binding: the reduction of pS935 levels and 14-3-3 binding
caused by R1441G and Y1699C is consistent in various assays;
while the inhibitory effect of G2019S is modest and assay-
dependent. The disparity in G2019S results may reflect the
different sensitivity of the various assay systems. It is noteworthy
that the K1906M mutation did not alter the levels of pS935 or 14-
3-3 binding of LRRK2 in all assays, suggesting that the LRRK2
kinase activity is not primarily responsible for the phosphorylation
of S935 (Figure 5A, B, D).
Protein kinase A (but not LRRK2 kinase) causes
phosphorylation of S935 in vitro and in cell culture
As LRRK2 kinase activity is unlikely the cause for the
phosphorylation of S935 on its own (Figure 5), we sought to
identify other possible kinases that may be responsible for
phosphorylation of S935. A previous study showed that cAMP-
dependent protein kinase (PKA) could phosphorylate LRRK2 in
vitro but without any knowledge about the phosphorylation sites
[22]. We therefore performed kinase assay with purified PKA
catalytic subunit and GST fusion protein containing LRRK2
fragment (800–1000aa). The result revealed that PKA phosphor-
ylates S935 as detected with anti-pS935 antibody and that the
phosphorylation is enhanced with increased input of PKA amount
(Figure 6A). In addition, the phosphorylation of S935 is inhibited
by PKA inhibitor H89 [23] (Figure 6B). Next, we tested whether
or not PKA mediates phosphorylation of S935 in cells. Co-
expression of FLAG-LRRK2 and PKA catalytic domain resulted
in a significant increase in pS935 levels as compared to that with
the transfection of FLAG-LRRK2 alone. Again, the phosphory-
lation levels are enhanced with increasing transfected PKA
Figure 4. S935A mutation does not affect LRRK2 dimerization. (A, B) FLAG-LRRK2 WT or S935A mutant construct was co-transfected with HA-
LRRK2 constructs. The Co-IP assay was performed using either anti-HA (A) or anti-FLAG (B) antibody, followed by Western blot analysis using the
indicated antibodies.
Phosphorylation and 14-3-3 Binding of LRRK2 and PD
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Page 7
Figure 5. PD-linked mutants affect S935 phosphorylation and 14-3-3 binding of LRRK2. (A) FLAG-LRRK2 WT, S935A, R1441G, Y1699C,
G2019S or K1906M mutant plasmid was transfected into HEK-293T cells. The LRRK2 variants were immunoprecipitated and analyzed by Western blot
using anti-pS935 and anti-FLAG antibodies. Quantification of the signals was performed using LI-COR and Odyssey software and the data was
analyzed by One-way ANOVA (** P,0.01). Data are presented as mean value (6 SEM) from three independent experiments. (B) FLAG-LRRK2-WT,
G2019S, R1441G and K1906M was immunoprecipitated from the brain lysates of the corresponding BAC-transgenic mice. The quantification of the
signals was performed as described above. The ratio of pS935 signal over total LRRK2 signal is shown. The data was analyzed with One-way ANOVA
(* P,0.05, ** P,0.01). Data are presented as mean value (6 SEM) from three mice. (C) Myc-14-3-3c (or control Myc vector) was co-transfected with
FLAG-LRRK2 Wt, S935A, R1441G or G2019S mutant plasmid into HEK-293T cells. Co-IP was performed by using anti-Myc antibody. The pulled down
Phosphorylation and 14-3-3 Binding of LRRK2 and PD
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Page 8
plasmid amount (Figure 6C). Phosphorylation of GSK, a known
substrate of PKA, is used for the positive control of PKA activity in
this assay. Furthermore, we used PKA activator forskolin (FSK) to
treat cells expressing FLAG-LRRK2. The result showed that
administration of FSK at 10 mM caused a significant increase in
pS935 levels of LRRK2 likely as a result of enhanced PKA activity
as indicated by the increase in pGSK levels (Figure 6D). Finally, in
an in vitro kinase assay as described above (Figure 6A), we found
that, while purified PKA directly phosphorylates S935 in GST-
LRRK2 fragment, purified FLAG-LRRK2 kinase does not cause
any change in pS935 levels in GST-LRRK2 fragment (Figure 6E),
despite the active kinase activity of FLAG-LRRK2 towards
generic substrate MBP (data not shown) [3]. This result further
demonstrates that LRRK2 kinase is not responsible for pS935,
whereas PKA is a potential upstream kinase that can cause the
phosphorylation of LRRK2 at S935.
Our study identifies serial molecular events related to the
regulation of LRRK2: multiple novel phosphorylation sites (S910,
S912, S935 and S973, all in the N-terminal region), potential
phosphorylation of S935 by PKA, and pS935-dependent 14-3-3
binding of LRRK2. These events may represent an important part
of a signaling pathway in regulating LRRK2 cellular function
under physiological conditions. Furthermore, we show that the
phosphorylation status of S935 and 14-3-3 binding of LRRK2 is
impaired by the most common familial mutations including
G2019S, R1441G and Y1699C. This result provides a new
avenue for the study of pathogenic mutations of LRRK2 in PD.
Interestingly, while our study was in final stage of submission,
Nichols and Alessi's group reported the phosphorylation of
LRRK2 at S910, S935 and S973, and pS910/pS935-dependent
14-3-3 binding of LRRK2. Their study indicated a nearly identical
role of S910 and S935 in 14-3-3 binding [24,25]. Furthermore,
they examined 41 known LRRK2 mutations and found that five
out of six pathogenic mutations (except G2019S) have markedly
reduced phosphorylation of S910/S935 and thereby disrupted 14-
3-3 binding of LRRK2. Therefore, our study and theirs
independently demonstrate the phosphorylation-dependent bind-
ing of 14-3-3 to LRRK2, which may play a critical role in
regulating LRRK2 function in vivo; PD-linked mutations impair
14-3-3 interaction with LRRK2 and thus disturb the regulation of
LRRK2 by 14-3-3.
Our study also provides additional insight into the functional
relevance of the LRRK2-14-3-3 interaction. In particular, our
results indicate that the most common mutation G2019S also
causes a reduction of pS935 levels in LRRK2 from transgenic
brain, despite little effect of G2019S seen in LRRK2 protein
isolated from cell culture. Our study has further characterized the
specific isoforms of 14-3-3 binding of LRRK2 and shown the
relevance of 14-3-3 binding to the dimer formation of LRRK2.
Thus, there are seven isoforms of 14-3-3 proteins and many 14-3-
3 target proteins only bind to selective isoforms of 14-3-3
[26,27,28]. Although several isoforms 14-3-3 were identified
through our mass spectrometric analysis, our data suggest that
LRRK2 binds preferentially to c and g form (both are abundant
in the brain). This information is important for future
biochemical and structural analyses of LRRK2-14-3-3 interac-
tions. We have also sought to investigate whether 14-3-3 is a
potential substrate of LRRK2, because previous studies had
shown that 14-3-3 is a phosphoprotein [29,30]. Our data
indicated that purified LRRK2 efficiently phosphorylates generic
substrate MBP but not 14-3-3 (data not shown). In addition,
because a body of evidence has indicated an important role of 14-
3-3 in assisting target protein dimerization [19], we tested
whether 14-3-3 regulates LRRK2 dimer formation. Our data
reveal that the LRRK2 mutant defective in phosphorylation of
S935/14-3-3 binding is not affected in dimer formation. The
result suggests that 14-3-3 does not play a critical role in
dimerization of LRRK2; rather, dimerization of LRRK2 is
mediated primarily by the sequence outside of the S910/S935
sites [9,10,11]. This result, along with the previous report, is
consistent with a model that homo or hetero-dimeric 14-3-3 bind
to two separate, neighboring phosphorylation sites (e.g. pS910
and S935) on the same LRRK2 molecule, and disruption of
either phosphorylation site would destabilize dimeric 14-3-3
binding of LRRK2 (Figure 7) [25].
We have also examined the potential role of 14-3-3 binding in
directing LRRK2 subcellular localization. However, overexpres-
sion of wild type LRRK2 or S935A mutant results in largely
diffuse localization of each protein in COS-7 or HeLa cells, thus
no obvious difference was observed between the wild type and
S935A mutant of LRRK2 (our unpublished results). This result,
however, is in contrast to the recent report that S935A mutant was
associated with punta localization in COS-7 cells [24,25]. It is
unclear what causes the discrepancy of the subcellular localization
of LRRK2 mutants in the two studies, despite that 14-3-3 binding
of S935A mutant LRRK2 was impaired in both studies. An
important question that remains to be answered is whether 14-3-3
binding represents a mechanism for LRRK2 trafficking or
distribution at different subcompartments of neurons.
Finally, our study reveals a potential link of PKA kinase to the
phophorylation of LRRK2 at S935, therefore adding a new
member to the list of known substrates of PKA that are also 14-3-3
targets [31,32,33,34]. One such an example is the PKA-mediated
phosphorylation of S413 and 14-3-3 binding of pS413 in
RIM1alpha, which is an active zone protein important for
synaptic transmission [35,36]. Future study should investigate in
detail whether PKA as well as other kinase activities are
responsible for phosphorylation of S910, S912, S935 or S973.
Furthermore, previous evidence indicated that the effect of
dopamine is largely mediated through cAMP/PKA signaling
cascade [37,38]. Given the observation of dopamine transmission
deficits in several LRRK2 mutant rodent models [21,39,40,41],
the potential connection of PKA-LRRK2 signaling indicates an
attractive cellular pathway for the future dissection of the
molecular mechanism underlying LRRK2 mutations in the
pathogenesis of PD.
LRRK2 variant levels were analyzed by Western blot analysis with the indicated antibodies and quantified using LI-COR and Odyssey system. The data
was analyzed by One-way ANOVA (** P,0.01). Data are presented as mean value (6 SEM) from three independent experiments. (D) FLAG-LRRK2-WT,
S935A, R1441G, Y1699C, G2019S or K1906M mutant plasmid was transfected individually into HEK-293T cells. Sepharose 4B beads-conjugated GST-
14-3-3c protein was incubated with different transfected cell lysates containing LRRK2-Wt or various mutants. Western blot analysis was performed to
determine the LRRK2 variant amount after the GST pull-down assay. The results were quantified as described above. The ratio of pulled down LRRK2
and input LRRK2 was measured and analyzed by One-Way ANOVA (** P,0.01). Data are presented as mean value (6 SEM) from three independent
Phosphorylation and 14-3-3 Binding of LRRK2 and PD
PLoS ONE | www.plosone.org8March 2011 | Volume 6 | Issue 3 | e17153
Page 9
Figure 6. PKA, not LRRK2 itself, can phosphorylate S935 in vitro and within cells. (A) 0.4 mg of purified GST-LRRK2 fragments (800–1000aa)
was incubated with different amount of the PKA catalytic subunit (lane 1: without PKA; lane 2: 2500 units PKA; lane 3: 10,000 units PKA). The reaction
was stopped by adding 36SDS-PAGE sample buffer and Western blot was performed by using anti-pS935 and anti-LRRK2 antibodies. (B) Different
concentrations of H-89 (0, 2.5, 5 and 10 mM) were added to the PKA and GST-LRRK2 fragment reaction mixture, and Western blot analyses was
performed by using anti-pS935 and anti-LRRK2 antibodies. (C) FLAG-LRRK2 was co-transfected with different amounts of PKA plasmid into HEK-293T
cells (The ratio of FLAG-LRRK2 verse PKA plasmid DNA are: 0 in lane 1, 1:1 in lane 2 and 1:10 in lane 3). The transfected cell lysate was harvested and
analyzed by Western blot using anti-pS935, anti-FLAG, anti-PKA, anti-pGSK and anti-b-actin antibodies. (D) FLAG-LRRK2 was transfected into HEK-
293T cells and after 40 hours transfection, 10 mM FSK was added to the cell culture medium and incubated for 30 min. Then the cells were harvested
and western blot analysis was performed to study various protein levels by using anti-pS935, anti-FLAG, anti-pGSK and anti-b-actin antibodies. The
pS935 LRRK2 and total LRRK2 signals were quantified by using the LI-COR Odyssey software system and the ratio of pS935 and total LRRK2 was
calculated and analyzed by One-way ANOVA (** P,0.01). Western blot results of three independent experiments are shown, and data are presented
as mean value (6 SEM) from three independent experiments. (E) GST-LRRK2 fragment (800–1000aa) protein was incubated with purified FLAG-LRRK2
(from mouse brains) or with PKA catalytic enzyme subunit (as positive control) (lane 1: 2500 units PKA; lane 2: 12.5 nM FLAG-LRRK2; lane 3: 25 nM
FLAG-LRRK2; lane 4: 125 nM FLAG-LRRK2; lane 5: 250 nM FLAG-LRRK2) in Kinase assay buffer and western blot was performed by using anti-pS935
and anti-LRRK2 antibodies. The arrows indicated the phosphorylated form of full length LRRK2 and GST-LRRK2 fragment; the asterisks indicated the
total full length LRRK2 and total GST-LRRK2 fragment, respectively.
Phosphorylation and 14-3-3 Binding of LRRK2 and PD
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Page 10
Materials and Methods
Modified trypsin and EDTA-free protease inhibitor cocktail
tablets were purchased from Roche Diagnostics (Indianapolis, IN).
Modified porcine trypsin and chymotrypsin were purchased from
Promega Inc. (Madison, WI). LC/MS grade water and acetonitrile
(ACN) were purchased from Honeywell Burdick & Jackson
(Muekegon, MI). Protein inhibitor tablets and CIAP were
purchased from Roche (Branchbury, NJ); FLAG-M2 beads, H89
and Forskolin (FSK) were purchased from Sigma (St. Louis, MO);
Site-mutagenesis kit was purchased from Stratagene (Santa Clara,
CA); NuPAGE Bis-Tris gels, MOP SDS running buffer,
antioxidant, and lipofectamine 2000 kit were purchased from
Invitrogen (Carlsbad, CA). GelCode Blue Stain Reagent, Tri-
fluoroacetic acid and Tris (2-carboxyethyl)-phosphine hydrochlo-
ride were purchased from Pierce (Rockford, IL). The following
antibodies were used in this study: FLAG (Sigma, St. Louis, MO),
Myc, pGSK-3-alph/beta (S21/9), b-actin and PKA C-alpha (Cell
Signaling, Danvers, MA), 14-3-3 isoforms and HA (Santa Cruz,
Ca), Phospho-S935 (developed with Cocalico Biological Inc.
Reamstown, PA). PKA catalytic subunit was purchased from
New England Biolabs (Ipswich, MA). Non-phospho- and Phospho-
peptides (QRHSNSLGPC and QRHSNpSLGPC) were synthe-
sized with acetylated N-termini and HPLC purified in The
Rockefeller University Proteomics Resource Center.
Transgenic mice and plasmids
Mice were housed in the Center for Comparative Medicine at
Mount Sinai School of Medicine. Handling procedures were in
accordance with NIH guidelines and approved by the Institutional
Animal Care and Use Committees of the institute (IACUC). The
institution has Animal Welfare Assurance on file with the Office
for laboratory Animal Welfare. The Assurance number is A3111-
BAC transgenic mice expressing FLAG-LRRK2-Wt and
FLAG-LRRK2-G2019S were described previously [3,21]. The
BAC mice expressing FLAG-LRRK2-R1441G
LRRK2-K1906M were generated using the similar BAC modi-
fication strategy. The following plasmid vectors were used to
express LRRK2, 14-3-3 isoforms and LRRK2 fragment (800–
1000aa): 36FLAG and 36Myc plasmid were purchased from
Sigma (St. Louis, MO). HA vector was purchased from Santa
Cruz (Santa Clara, CA). GST vector was purchased from (GE
Healthcare, Piscataway, NJ). Full-length LRRK2 cloning method
was described in previous report. The mutations (S935A, R1441G,
Y1699C, K1906M and G2019S) were generated by using the
Quick-change site-directed mutagenesis kit.
or FLAG-
Figure 7. PKA Phosphorylation, 14-3-3 binding of LRRK2, and the effect of common familial mutations of LRRK2 in 14-3-3 binding.
A schematic model showing PKA (or other kinase) phosphorylation of S910/S935, dimeric14-3-3 binding of LRRK2 at pS910/pS935 sites in wild type
LRRK2; PD-linked mutations R1441G, Y1699C or G2019S abolishes or reduces phosphorylation of S910/S935 and impairs 14-3-3 binding. In addition,
we propose that dimeric 14-3-3 bind to pS910 and pS935 in the same LRRK2 molecule and binding of 14-3-3 plays little role in LRRK2 dimer
Phosphorylation and 14-3-3 Binding of LRRK2 and PD
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Page 11
Affinity purification of transgenic mouse brain samples
Affinity purification of FLAG-LRRK2 and its interacting
proteins was carried out as described in [42], with slight
modification. In brief, brain extracts were obtained from non-
transgenic and FLAG-LRRK2 mice (P30) by homogenizing 2
brains with a motor-driven homogenizer (speed 2.5, 12 strokes) in
3 ml buffer containing 0.32 M sucrose, 1 mM NaHCO3, 20 mM
HEPEs/pH 7.4, 1 mM MgCl2, 0.25 mM CaCl2, EDTA-free
protease inhibitor cocktail, 200 mg/mL PMSF, pepstatin 4 mg/
mL and DNase I. The tissue extracts were centrifuge at 1,500 g for
5 min and the pellets were homogenized again in 1 ml buffer for 8
strokes and centrifuged again. Supernatants from the two
homogenization steps were pooled, centrifuged at 750 g for
10 min twice. The collected supernatants were diluted with equal
volumes of 26 pull-out buffer (16 pull-out buffer containing
20 mM HEPEs/pH 7.4, 1 mM MgCl2, EDTA-free protease
inhibitor cocktail, 200 mg/mL PMSF, 4 mg/mL pepstatin, 0.1%
triton X-100 and 150 mM NaCl). Sample equivalent to half brain
was incubated with FALG M2 beads (,10 ml bed volume) for
2 hours at 4uC. After washed 5 times with 16pullout buffer, beads
were eluted with 250 ml elution buffer (0.5 mM EDTA and 0.5 M
NH3?H2O) at room temperature.
In-gel digestion of purified LRRK2 protein with trypsin
and chymotrypsin
The purified LRRK2 was resolved in SDS-PAGE and
visualized by colloidal Coomassie blue staining. The protein band
of interest were excised, in-gel digested after reduction and
alkylation, and extracted using the clean protocol previously
described [43].
Nano-HPLC/mass spectrometric analysis
The proteolytic peptides derived from LRRK2 were analyzed in
Velos LTQ Orbitrap mass spectrometer (Thermo Fisher Scien-
tific, Waltham, MA). The peptides were separated in a home-
made capillary HPLC column (110 mm length675 mm internal
diameter, 5 mm particle size, 100 A˚pore diameter) with Jupiter
C12resin (Phenomenex, St. Torrance, CA) using a gradient from
2% to 30% solvent B in solvent A (mobile phase A: 0% ACN in
0.1% formic acid; and mobile phase B: 100% ACN in 0.1%
formic acid) for 50 min. The eluted peptides were directly
electrosprayed into the mass spectrometer using a nanospray
source. The spray voltage was set to 2.1 kV and the temperature of
the heated capillary was set to 300uC. The MS was operated in the
data-dependent mode to automatically switch between Orbitrap-
MS and Velos LTQ-MS/MS acquisition. Survey full scan MS
spectra (from m/z 350–1500) were acquired in the Orbitrap with
resolution R=60,000 at m/z=400. The 20 most intense ions were
sequentially isolated for fragmentation in the linear ion trap using
collision-induced dissociation (normalized collision energy=35%,
activation Q=0.250 and activation time=10 ms) in the Velos
LTQ. Maximal filling times were 30 ms for the full scans and
25 ms for the MS/MS scans. Precursor ion charge state screening
was enabled, and singly and unassigned charge states were
rejected. A lock-mass ion from ambient air (m/z 445.120024) was
used for internal calibration of all full scan measurements with the
Orbitrap detector [43,44]. The following parameters were
specified in MS/MS analysis: dynamic exclusion (36 seconds);
the repeat count (2) and the exclusion window (+3 and 21.5 Da).
Protein sequencing alignment
Mass spectra collected by MALDI-QqTOF and MALDI-ion trap
mass spectrometers were analyzed by the computer search engines
Xproteo (http://www.xproteo.com) and GPM (http://prowl.rock
efeller.edu/tandem/thegpm_tandem.html) using the NCBI non-
redundant mouse protein database. All MS/MS spectra were
searched against the IPI-mouse data base (v3.74) protein sequence
database (56860 sequences) using Mascot (v2.1). The specific
parameters for protein sequence database searching included serine,
threonine and tyrosine phosphorylation; cysteine carbamidomethy-
lation and methionine oxidation as variable modifications. Other
parameters used in data analysis were: four allowed missing
cleavages; mass error of 10 ppm for precursor ions and 0.5 Da for
fragment ions. Charge states of +2, and +3 were considered for
parent ions. If more than one spectrum was assigned to a peptide,
only the spectrum with the highest Mascot score was selected for
manual analysis. Allpeptides identified with peptide scoresofmascot
.20 were manually examined using rules described previously [45].
Immunoprecipitation was described as previously with modifi-
cation [3]. LRRK2 transgenic brain was homogenized with
homogenization buffer (50 mM Tris HCl at pH 7.5, 5% Glycerol,
1 mM NaHCO3, 0.25 mM CaCl2, 1 mM MgCl2, 1 mM PMSF,
10 mg/mL pepstatin, 20 mM beta-Glycerol phosphate, 1 mM Na
Vanadate, 50 mM NaF, 10 mM Pyrophosphate, 0.01%Triton X-
100 and mini complete protease inhibitor cocktail), and incubated
at 4uC on rotator for 30 min. Transfected HEK-293T cells were
harvested and suspended in cell lyses buffer (50 mM Tris HCl at
pH 7.5, 150 mM NaCl, 1 mM PMSF, 10 mg/mL pepstatin,
20 mM beta-Glycerol phosphate, 1 mM Na Vanadate, 50 mM
NaF, 10 mM Pyrophosphate, 1%Triton X-100 and mini complete
protease inhibitor cocktail). The cell lysate or homogenized brain
lysate were clarified at 12,000 g for 10 min at 4uC, the 36FLAG-
LRRK2 protein was purified using anti-FLAG Affinity Gel
according to the manual. For Myc-14-3-3 immunoprecipitation,
the HEK-293T cell lysate was precleared using agarose gel for
1 hour at 4uC, and then anti-Myc antibody was added and
incubated for overnight. The beads were washed with modified
RIPA buffer (50 mM Tris HCl at pH 7.5, 300 mM NaCl, 10 mM
MgCl2, 1 mM PMSF, 10 mg/mL pepstatin, 0.5% Sodium
Deoxycholate, 0.1% Sodium Dodecyl Sulfate). The immunopre-
cipitated protein was eluted by using 16sample buffer.
GST-pull down assay
GST-14-3-3 isoform protein was expressed in bacteria with
100 mM IPTG induction for 4 hours at 37uC, and purified
following the manufacture's instruction (GE Healthcare). The
protein concentration was measured on SDS-PAGE and Coo-
massie Staining comparing to Standard BSA. 5 mg of each GST-
14-3-3 isoform protein was incubated with 100 mg FLAG-LRRK2
transfected HEK-293T cell lyses supernatant for 4 hours at 4uC
and washed with cell lyses buffer. The bound protein was eluted
with 16 SDS-PAGE sample buffer. For the phosphopeptide
competition assay, 2 mg of brain lysate from FLAG-LRRK2 Wt
transgenic mice was incubated with different concentration of non-
phosphopeptide or phophopeptide (S935) for 1 hour. Then GST-
14-3-3c beads were added and incubated for 4 hours. The beads
were then washed and the bound protein was eluted with 16
sample buffer.
PKA kinase assay
GST-LRRK2 fragment (800–1000aa) was expressed in bacteria
with 50 mM IPTG induction at RT for overnight and purified
following the manufacture's instruction. The protein concentra-
tion was measured on SDS-PAGE and Coomassie Staining and
Phosphorylation and 14-3-3 Binding of LRRK2 and PD
PLoS ONE | www.plosone.org 11 March 2011 | Volume 6 | Issue 3 | e17153
Page 12
comparing to standard BSA. The kinase assay was performed
following the New England Biolab protocol. Briefly, 400 ng GST-
LRRK2 fragment protein was incubated with different concen-
tration of PKA catalytic subunit protein at 30uC for 20 min. Or
GST-LRRK2 fragment protein was incubated with PKA catalytic
subunit protein and different concentration of H89 at 30uC for
20 min. The reaction was stopped by adding 66 SDS-PAGE
sample buffer, followed by boiling the samples for 5 min.
Supporting Information
Figure S1
tion by MALDI QqTOF and ion trap mass spectrome-
ters. MALDI QqTOF mass spectrum of tryptic digested peptides
extracted from the affinity purified mouse brain LRRK2 (band
shown in Figure 5A). Two tryptic peptides containing the S935
phosphorylation site are labeled A* and B*. The corresponding
unphosphorylated peptides are labeled A and B.
Identification of LRRK2 S935 phosphoryla-
Figure S2
tion by MALDI QqTOF and ion trap mass spectrome-
ters. (A) MALDI ion trap MS/MS spectrum of the phosphor-
ylated peptide HSNpSLGPVFDHEDLLR. (B) MALDI ion trap
MS/MS spectrum of the phosphorylated peptide HSNpSLGP-
VFDHEDLLRR. Both MS/MS spectra show dominant peaks due
to characteristic loss of H3PO4 (98 Da). For both (A) and (B),
insets show zoomed-in regions of the spectra. Phosphorylated-
S935-containing fragment ions are labeled in red.
Identification of LRRK2 S935 phosphoryla-
Figure S3
by nano-HPLC/velos LTQ Orbitrap mass spectrometer.
MS/MS spectra of phosphorylated peptides at serine 910
(SNpSISVGEVYR) (A), serine 912 (LVKRKSNSIpSVGEVY)
(B) in purified LRRK2 protein, respectively. In this study, we
digested the purified LRRK2 protein in-gel using trypsin or
chymotrypsin. The MS/MS data were analyzed by the Mascot
algorithm to identify the protein and its posttranslational
modifications. The candidate peptides bearing the serine phos-
phorylation were further examined manually as previously
described [45]. The phosphorylated peptide can be identified by
a mass shift of 79.96633 Da at serine/threonine/tyrosine residues.
Analysis of phosphorylation sites in LRRK2
Figure S4
by nano-HPLC/velos LTQ Orbitrap mass spectrometer.
MS/MS spectra of phosphorylated peptides at serine 935
(HSNpSLGPVFDHEDLLR) (A), and serine 973 (QSDpSSSSLA-
SER) (B) in purified LRRK2 protein, respectively. In this study,
we digested the purified LRRK2 protein in-gel using trypsin or
chymotrypsin. The MS/MS data were analyzed by the Mascot
algorithm to identify the protein and its posttranslational
modifications. The candidate peptides bearing the serine phos-
Analysis of phosphorylation sites in LRRK2
phorylation were further examined manually as previously
described [45]. The phosphorylated peptide can be identified by
a mass shift of 79.96633 Da at serine/threonine/tyrosine residues.
Figure S5
tion by a combination of alkaline phosphatase treatment
and mass spectrometry. (A) Coomassie blue stain gel showing
affinity purified FLAG-LRRK2 and its interacting proteins from
FLAG-LRRK2 BAC transgenic mouse brain and lung, with and
without alkaline phosphatase (AP) treatment. LRRK2 and AP
bands are labeled by arrows. AP treatment was performed by
incubating anti-FLAG immunoprecipitation eluent in AP (Roche,
2 U/mL) at 37uC for 1 h. (B) MALDI QqTOF mass spectra of
LRRK2 tryptic digested peptides extracted from the gel bands
shown in (A). The monoisotopic peaks of the unphosphorylated
and phosphorylated LRRK2 tryptic peptide 932[HSNSLG-
PVFDHEDLLR]947 are highlighted in yellow. (C) MS3tandem
mass spectrum confirming phosphorylated LRRK2 tryptic peptide
932[HSNpSLGPVFDHEDLLR]947, using a MALDI ion trap
mass spectrometer.
Confirmation of LRRK2 S935 phosphoryla-
Figure S6
QqTOF and ion trap mass spectrometry. (A) MALDI
QqTOF mass spectrum of tryptic digested peptides extracted from
the affinity purified mouse brain 14-3-3 (band shown in Figure 5A).
(B) MALDI ion trap MS/MS spectra of unique tryptic peptides of
Identification of 14-3-3 isoforms by MALDI
Figure S7
QqTOF and ion trap mass spectrometry. MALDI ion trap
MS/MS spectra of unique tryptic peptides of 14-3-3e (A), 14-3-3g
Identification of 14-3-3 isoforms by MALDI
Figure S8
QqTOF and ion trap mass spectrometry. MALDI ion trap
MS/MS spectra of unique tryptic peptides of 14-3-3f (A), and 14-
3-3h/t (B).
Identification of 14-3-3 isoforms by MALDI
We thank The Yue lab members for critical reading of the manuscript and
Author Contributions
Conceived and designed the experiments: XL QJW BTC ZY. Performed
the experiments: XL QJW NP SL. Analyzed the data: XL QJW YZ ZY
BTC SL. Contributed reagents/materials/analysis tools: XL NP YZ BTC.
Wrote the paper: XL YZ ZY.
1. Paisan-Ruiz C, Jain S, Evans EW, Gilks WP, Simon J, et al. (2004) Cloning of
the gene containing mutations that cause PARK8-linked Parkinson's disease.
Neuron 44: 595–600.
2. Zimprich A, Biskup S, Leitner P, Lichtner P, Farrer M, et al. (2004) Mutations in
LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology.
Neuron 44: 601–607.
3. Li X, Tan YC, Poulose S, Olanow CW, Huang XY, et al. (2007) Leucine-rich
repeat kinase 2 (LRRK2)/PARK8 possesses GTPase activity that is altered in
familial Parkinson's disease R1441C/G mutants. J Neurochem 103: 238–247.
4. West AB, Moore DJ, Biskup S, Bugayenko A, Smith WW, et al. (2005)
Parkinson's disease-associated mutations in leucine-rich repeat kinase 2 augment
kinase activity. Proc Natl Acad Sci U S A 102: 16842–16847.
5. SmithWW,PeiZ,Jiang H,DawsonVL,Dawson TM,etal.(2006) Kinase activity
of mutant LRRK2 mediates neuronal toxicity. Nat Neurosci 9: 1231–1233.
6. Guo L, Gandhi PN, Wang W, Petersen RB, Wilson-Delfosse AL, et al. (2007)
The Parkinson's disease-associated protein, leucine-rich repeat kinase 2
(LRRK2), is an authentic GTPase that stimulates kinase activity. Exp Cell
Res 313: 3658–3670.
Phosphorylation and 14-3-3 Binding of LRRK2 and PD
PLoS ONE | www.plosone.org 12March 2011 | Volume 6 | Issue 3 | e17153
Page 13
7. Greggio E, Jain S, Kingsbury A, Bandopadhyay R, Lewis P, et al. (2006) Kinase
activity is required for the toxic effects of mutant LRRK2/dardarin. Neurobiol
Dis 23: 329–341.
8. MacLeod D, Dowman J, Hammond R, Leete T, Inoue K, et al. (2006) The
familial Parkinsonism gene LRRK2 regulates neurite process morphology.
Neuron 52: 587–593.
9. Greggio E, Zambrano I, Kaganovich A, Beilina A, Taymans JM, et al. (2008)
The Parkinson disease-associated leucine-rich repeat kinase 2 (LRRK2) is a
dimer that undergoes intramolecular autophosphorylation. J Biol Chem 283:
10. Deng J, Lewis PA, Greggio E, Sluch E, Beilina A, et al. (2008) Structure of the
ROC domain from the Parkinson's disease-associated leucine-rich repeat kinase
2 reveals a dimeric GTPase. Proc Natl Acad Sci U S A 105: 1499–1504.
11. Sen S, Webber PJ, West AB (2009) Dependence of leucine-rich repeat kinase 2
(LRRK2) kinase activity on dimerization. J Biol Chem 284: 36346–36356.
12. Lee BD, Shin JH, VanKampen J, Petrucelli L, West AB, et al. Inhibitors of
leucine-rich repeat kinase-2 protect against models of Parkinson's disease. Nat
Med 16: 998–1000.
13. Parisiadou L, Xie C, Cho HJ, Lin X, Gu XL, et al. (2009) Phosphorylation of
ezrin/radixin/moesin proteins by LRRK2 promotes the rearrangement of actin
cytoskeleton in neuronal morphogenesis. J Neurosci 29: 13971–13980.
14. Plowey ED, Cherra SJ, 3rd, Liu YJ, Chu CT (2008) Role of autophagy in
G2019S-LRRK2-associated neurite shortening in differentiated SH-SY5Y cells.
J Neurochem 105: 1048–1056.
15. Fu H, Subramanian RR, Masters SC (2000) 14-3-3 proteins: structure, function,
and regulation. Annu Rev Pharmacol Toxicol 40: 617–647.
16. Yaffe MB, Rittinger K, Volinia S, Caron PR, Aitken A, et al. (1997) The
structural basis for 14-3-3:phosphopeptide binding specificity. Cell 91: 961–971.
17. Wang H, Zhang L, Liddington R, Fu H (1998) Mutations in the hydrophobic
surface of an amphipathic groove of 14-3-3zeta disrupt its interaction with Raf-1
kinase. J Biol Chem 273: 16297–16304.
18. Jin Y, Dai MS, Lu SZ, Xu Y, Luo Z, et al. (2006) 14-3-3gamma binds to
MDMX that is phosphorylated by UV-activated Chk1, resulting in p53
activation. EMBO J 25: 1207–1218.
19. Darling DL, Yingling J, Wynshaw-Boris A (2005) Role of 14-3-3 proteins in
eukaryotic signaling and development. Curr Top Dev Biol 68: 281–315.
20. Braselmann S, McCormick F (1995) Bcr and Raf form a complex in vivo via 14-
3-3 proteins. EMBO J 14: 4839–4848.
21. Li X, Patel JC, Wang J, Avshalumov MV, Nicholson C, et al. (2019S) Enhanced
striatal dopamine transmission and motor performance with LRRK2 overex-
pression in mice is eliminated by familial Parkinson's disease mutation G2019S.
J Neurosci 30: 1788–1797.
22. Ito G, Okai T, Fujino G, Takeda K, Ichijo H, et al. (2007) GTP binding is
essential to the protein kinase activity of LRRK2, a causative gene product for
familial Parkinson's disease. Biochemistry 46: 1380–1388.
23. Daaka Y, Luttrell LM, Lefkowitz RJ (1997) Switching of the coupling of the
beta2-adrenergic receptor to different G proteins by protein kinase A. Nature
390: 88–91.
24. Nichols RJ, Dzamko N, Morrice NA, Campbell DG, Deak M, et al. 14-3-3
binding to LRRK2 is disrupted by multiple Parkinson's disease-associated
mutations and regulates cytoplasmic localization. Biochem J 430: 393–404.
25. Dzamko N, Deak M, Hentati F, Reith AD, Prescott AR, et al. Inhibition of
LRRK2 kinase activity leads to dephosphorylation of Ser(910)/Ser(935),
disruption of 14-3-3 binding and altered cytoplasmic localization. Biochem J
430: 405–413.
26. Wakui H, Wright AP, Gustafsson J, Zilliacus J (1997) Interaction of the ligand-
activated glucocorticoid receptor with the 14-3-3 eta protein. J Biol Chem 272:
27. Craparo A, Freund R, Gustafson TA (1997) 14-3-3 (epsilon) interacts with the
insulin-like growth factor I receptor and insulin receptor substrate I in a
phosphoserine-dependent manner. J Biol Chem 272: 11663–11669.
28. Meller N, Liu YC, Collins TL, Bonnefoy-Berard N, Baier G, et al. (1996) Direct
interaction between protein kinase C theta (PKC theta) and 14-3-3 tau in T cells:
14-3-3 overexpression results in inhibition of PKC theta translocation and
function. Mol Cell Biol 16: 5782–5791.
29. Yoshida K, Yamaguchi T, Natsume T, Kufe D, Miki Y (2005) JNK
phosphorylation of 14-3-3 proteins regulates nuclear targeting of c-Abl in the
apoptotic response to DNA damage. Nat Cell Biol 7: 278–285.
30. Dubois T, Rommel C, Howell S, Steinhussen U, Soneji Y, et al. (1997) 14-3-3 is
phosphorylated by casein kinase I on residue 233. Phosphorylation at this site in
vivo regulates Raf/14-3-3 interaction. J Biol Chem 272: 28882–28888.
31. Lizcano JM, Morrice N, Cohen P (2000) Regulation of BAD by cAMP-
dependent protein kinase is mediated via phosphorylation of a novel site, Ser155.
Biochem J 349: 547–557.
32. Feng L, Yunoue S, Tokuo H, Ozawa T, Zhang D, et al. (2004) PKA
phosphorylation and 14-3-3 interaction regulate the function of neurofibroma-
tosis type I tumor suppressor, neurofibromin. FEBS Lett 557: 275–282.
33. Palmer D, Jimmo SL, Raymond DR, Wilson LS, Carter RL, et al. (2007)
Protein kinase A phosphorylation of human phosphodiesterase 3B promotes 14-
3-3 protein binding and inhibits phosphatase-catalyzed inactivation. J Biol Chem
282: 9411–9419.
34. Thomas D, Guthridge M, Woodcock J, Lopez A (2005) 14-3-3 protein signaling
in development and growth factor responses. Curr Top Dev Biol 67: 285–303.
35. Kaeser PS, Kwon HB, Blundell J, Chevaleyre V, Morishita W, et al. (2008)
RIM1alpha phosphorylation at serine-413 by protein kinase A is not required for
presynaptic long-term plasticity or learning. Proc Natl Acad Sci U S A 105:
36. Lonart G, Schoch S, Kaeser PS, Larkin CJ, Sudhof TC, et al. (2003)
Phosphorylation of RIM1alpha by PKA triggers presynaptic long-term
potentiation at cerebellar parallel fiber synapses. Cell 115: 49–60.
37. Wolf ME, Mangiavacchi S, Sun X (2003) Mechanisms by which dopamine
receptors may influence synaptic plasticity. Ann N Y Acad Sci 1003: 241–249.
38. Svenningsson P, Nishi A, Fisone G, Girault JA, Nairn AC, et al. (2004) DARPP-
32: an integrator of neurotransmission. Annu Rev Pharmacol Toxicol 44:
39. Melrose HL, Dachsel JC, Behrouz B, Lincoln SJ, Yue M, et al. Impaired
dopaminergic neurotransmission and microtubule-associated protein tau
alterations in human LRRK2 transgenic mice. Neurobiol Dis 40: 503–517.
40. Tong Y, Pisani A, Martella G, Karouani M, Yamaguchi H, et al. (2009)
R1441C mutation in LRRK2 impairs dopaminergic neurotransmission in mice.
Proc Natl Acad Sci U S A 106: 14622–14627.
41. Li Y, Liu W, Oo TF, Wang L, Tang Y, et al. (2009) Mutant LRRK2(R1441G)
BAC transgenic mice recapitulate cardinal features of Parkinson's disease. Nat
Neurosci 12: 826–828.
42. Wang QJ, Ding Y, Kohtz DS, Mizushima N, Cristea IM, et al. (2006) Induction
of autophagy in axonal dystrophy and degeneration. J Neurosci 26: 8057–8068.
43. Cheng Z, Tang Y, Chen Y, Kim S, Liu H, et al. (2009) Molecular
characterization of propionyllysines in non-histone proteins. Mol Cell Proteo-
mics 8: 45–52.
44. Olsen JV, de Godoy LM, Li G, Macek B, Mortensen P, et al. (2005) Parts per
million mass accuracy on an Orbitrap mass spectrometer via lock mass injection
into a C-trap. Mol Cell Proteomics 4: 2010–2021.
45. Chen Y, Kwon SW, Kim SC, Zhao Y (2005) Integrated approach for manual
evaluation of peptides identified by searching protein sequence databases with
tandem mass spectra. J Proteome Res 4: 998–1005.
Phosphorylation and 14-3-3 Binding of LRRK2 and PD
PLoS ONE | www.plosone.org13 March 2011 | Volume 6 | Issue 3 | e17153


text:
Innovative online entertainment channel gives customers an up close look at MaxxForce engines
WARRENVILLE, ILL — As Navistar completes its final testing stages and prepares for the upcoming launch of its 2010 MaxxForce engines, a new online entertainment channel - MaxxForce TV - is making its debut.
Starting this month, customers will be able to learn about the benefits of Navistar's MaxxForce brand engines in a new way with the launch of MaxxForce TV, an online channel devoted to trucks and engines. The inaugural series, "Maxx IQ," is an entertaining and informative game show hosted by Joe Elmore, host of the television show "Horsepower TV" on the Spike Network.
In each fast-paced segment, Elmore tests the engine smarts of drivers at truck stops and other locations nationwide for a chance to win cash and prizes. Online viewers are encouraged to play along to increase their knowledge of MaxxForce products and hone their skills in case this traveling game show rolls into their town.
The face of Maxx IQ is Nashville's own Joe Elmore, known to car and truck enthusiasts as the co-host of America's longest-running performance car show, Horsepower TV. Every weekend on the show, he shares engine building tips and visits hot rod and automotive events around the country. Hero to millions of fellow "gearheads," he's the perfect fit for a game show that's all about trucks and engines.
"Joe already has such a great knowledge of engines," notes Mark Johnson, marketing communications manager, Navistar. "We knew he was going to bring a great presence to Maxx IQ. Viewers are definitely going to enjoy seeing him in action - whether they're watching on their computers, or lucky enough to become contestants.
"Obviously, engines are one of my passions," Elmore said. "So it was satisfying to give drivers a chance to win some money and educate them about MaxxForce engines at the same time. During the shoot, I got the opportunity to test-drive the engines myself and they have some real power. I was impressed."
The first four episodes of Maxx IQ can be viewed on www.maxxforcetv.com. The next four episodes will be released later this month.
Navistar International Corporation (NYSE: NAV) is a holding company whose subsidiaries and affiliates produce International brand commercial and military trucks, MaxxForce brand diesel engines, IC Bus brand school and commercial buses, Monaco RV brands of recreational vehicles, and Workhorse brand chassis for motor homes and step vans.
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