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Gallium Ventures
© 2022, Gallium Ventures
Cooksy brings the world's first intelligent cooking assistant to your cooktop
Author Gallium Ventures
Using Cooksy, you'll cook like a professional chef and transform your kitchen to a culinary haven.
11 May 2021, Arizona, United States: Today, Cooksy has unveiled its intelligent cooking assistant which is set to revolutionize the kitchen. A master chef might spend years learning to understand when food is too hot or cold based on cooking sounds or visual cues, but Cooksy effortlessly brings this level of knowledge to the everyday chef. Cooksy cleverly blends video, artificial intelligence, thermal imaging technology, and an accompanying app to take the guesswork out of cooking so you can consistently enjoy delicious meals.
Indiegogo Campaign Page: https://www.indiegogo.com/project/preview/6875349a#/
Cooking delicious food is not easily accomplished using a cooktop. Most home cooks do not have the professional kitchen experience to interpret their stove's heat and the required time it takes to cook dishes properly. This often results in burnt, dry, or undercooked food. Additionally, recipes are vague and difficult to follow. Solving these common problems, Cooksy has launched a device that changes everything about how you see your cooktop, which makes cooking delicious food easy for everyone.
Cooksy easily mounts above your cooktop and intelligently guides your cooking, allowing both inexperienced cooks to take their first steps in the kitchen, as well as supporting the more experienced foodie to hone their skills and expertise. Cooksy has found a way to affordably bring the invisible world of temperature to your kitchen thanks to thermal imaging, which makes temperature something you can see and act on. Different from a simple probe or meat thermometer, Cooksy measures thousands of temperatures across the entire pan surface many times per second allowing more consistency in cooking.
Jeff Knighton, Founder and President at Cooksy explains, "Cooking is all about applying the right intensity of heat for the right amount of time. Cooksy makes this easy, giving you a 'sixth sense', guiding you to make consistently amazing food. Whether you are a home cook or a master chef, Cooksy provides the tools to elevate your cooking.
"We're excited to offer Cooksy ahead of retail during a time when home cooking is more of a family affair. We believe that everyone should feel comfortable in the kitchen, and are aiming to help everyday cooks on their journey."
Cooksy also offers a library of recipes which are easy to follow as it guides you with step-by-step video instructions, and analyzes your progress and adjusts cooking times and temperature to suit.
For those of you who prefer to create your own culinary masterpieces, Cooksy documents your cooking journey, allowing you to capture that spark of cooking genius, recreate it on demand, and even share it with the Cooksy community who can try your video recipe for themselves.
The Cooksy device is available for $389 early bird (40% off) while the Cooksy PRO is priced at $479 early bird (40% off) for a limited time.
Specs Cooksy: · 5 megapixel visual camera · Thermal Imaging Sensor – 32 x 24 array· On device storage – 8 GB (approx 15 hrs of video/thermal storage)· Choice of color: Midnight Black and Prisma SilverCooksy PRO: Best for Recipe Creators – higher thermal and visual resolution & more storage· 13 megapixel visual camera· Thermal Imaging Sensor – 80 x 62 array· On device storage – 32 GB (approx 60 hrs of video/thermal storage)· Choice of color: Midnight Black and Prisma Silver Cooksy PRO SE Signature: Special Edition Color· 13 megapixel camera· Thermal Imaging Sensor – 80 x 62 array · On device storage – 32 GB (approx 60 hrs of video/thermal storage)· Choice of color: Signature Copper
Connectivity Bluetooth, 802.11ac Wifi
Cooksy size 3.94 in x 3.94 in x 1.14 in (100 mm x 100 mm x 39 mm)
Weight 11.1 ounces (315 grams)
Mobile app Cooksy mobile app is available via Google Play Store and Apple App Store.
App capabilities CooksyCues allows step-by-step prompts while detecting ingredients. Record Cooksy recipes with auto temperature and timing, detail previously unavailable in a written recipe, share on social via SimpleShare, create recipe books and more.
Chipolo Celebrates 10 Years of Giving People Peace of Mind
A decade of Chipolo item trackers sees over 50,000 items found daily 4 January 2023, Las Vegas, Nevada – Chipolo — the award-winning item finder company — is celebrating their 10-year anniversary at CES. For over a decade, Chipolo has been providing people all over the world with peace of mind, knowing that they'll never lose their keys, wallets, luggage, and other important belongings ever again. From London to New York, from Tokyo to Paris, the Slovenian, self-funded Bluetooth item tracker brand has given items in cities all over the world the power to be reunited with their owners. They've...
WowWee Launches MINTiD Dog-E, The One In A Million Robot Dog, at CES 2023
The award-winning creator of tech toys for kids-of-all-ages is once again revolutionizing the industry with a robot dog "MINTiD" just for you! January 4, 2023, Las Vegas, Nevada: Dog-E was launched at CES (Consumer Electronics Show) by WowWee® — a global innovator of robotic toys and consumer entertainment products. Dog-E is a groundbreaking robot dog that is unique to every owner as a result of over a million possible combinations of colorful lights, sounds, and personality traits. Dog-E is a smart, app-connected robot dog with life-like movements, audio sensors to hear sounds, touch sensors on its head, nose and sides...
Deezer joins forces with LyricFind, Zebralution and 12 other partners for EU-funded project
Paris, France — December 14 2022 — Deezer has joined forces with a consortium of major music industry players, including Zebralution and LyricFind, to form BELEM. The consortium aims to build capacity for music publishers, songwriters, platforms, and labels to democratise and monetize access to lyrics. It will also strive to break down cultural barriers by making human translated lyrics accessible to all listeners. BELEM is an EU-funded four year project that has been granted €2 million in funding through the Creative Europe Programme (CREA). It will drive new revenue streams to key players in the music industry by promoting...


Smart Homes (JHS Ltd T/As Smart Homes) (Registered office: 316 Stratford Road, Shirley, Solihull, B90 3DN, Registration Number: 3591917) is committed to protecting your personal information, being transparent about what data we hold and giving you control over how we use it.
What are Smart Homes' privacy principles?
Registering for an account on our Digital Channels gives you access to a growing number of benefits, such as being able to save and compare properties that are of interest. Asking for your name means we know how to talk to you. Knowing your address and property status helps us to allocate the most appropriate member of our team to help you with your property needs and your contact details enables us to contact you regarding our services.
If requested, we will delete your personal information securely and swiftly and all the information we hold on your activity on our Digital Channels will be anonymised, so we won't know it's yours.
At Smart Homes, we make sure we stick to these data protection and e-privacy laws. As mentioned above, information about the laws, and your rights, can be found on the ICO's website - www.ico.gov.uk.
What information will Smart Homes collect about me?
Information you provide to us such as when you complete a digital service enquiry form, or register for an account, email notifications and/or newsletters (e.g. registering on smart-homes.co.uk to access the account area and/or subscribing to property alerts). On registration with smart-homes.co.uk, we currently ask for your first and surname, your email address, telephone, house number and postcode and your property status. We ask you for this information so we can offer you relevant local content, information or services. We will always be clear about how this information will be used.
This is used by us (Smart Homes referred to as 'us' and 'our') to analyse activity on all our Digital Channels. For example, this helps us to look at the number of visits to specific pages to evaluate if we have sufficient infrastructure to support and maintain a great experience for our visitors. We may also collect, store and use the following kinds of information: Information about your computer, your visits to and use of this website (including but not limiting to your IP address, browser type and version, operating system, referral source, length of visit, page views).
We collect information from social media activity, including likes, shares and tweets on our Digital Channels and about our content on social networking sites. Where you sign in to your account on Romans using social network credentials, such as a Facebook log-in, you grant permission to the social network to share your user details. Depending on the network this will include basic account information such as name, email address, property status and any other details you choose to share according to your Facebook account settings. We may from time to time, on different apps and services, request more than just basic account information from you. We will always be clear about this up front but you should also check what permissions we are asking for before you connect with these apps and services.
Your use of our Digital Channels may result in the transfer of personal information provided by you across national boundaries. By visiting our Digital Channels and communicating with Smart Homes electronically, you consent to the transfer of your Personal Information outside the European Economic Area.
To help us to monitor, analyse and improve our Digital Channels. This information helps us to understand which Smart Homes' content and services are most interesting, helpful to our users and if they are shared via social networks. The information also helps us to identify when errors occur within our Digital Channels and test different versions of a page or feature to see which one works best. All of which helps us improve our Digital Channels for our users.
To inform the way we commission future content at Smart Homes. We will use aggregated and anonymous user data to help our Digital Marketing team understand what type of content our users like so that they can make more in the future.
To market services to you. We will send you information about our Digital Channels and Services or other marketing activities but only where you have agreed to receive these from us and for the purposes stated at the collection point. We will email you or send you other marketing communications relating to Smart Homes' services which we think may be of interest to you, by post or, where you have specifically agreed to this, by email or similar technology. You can inform us at any time if you no longer require marketing communications. For instance, to provide you with password reminders or to contact you about services you have submitted to us via our Digital Channels so that we are able to deliver these services to you.
This policy only applies to our use of your information via Smart Homes Digital Channels. Therefore, it does not apply to third party websites to which Smart Homes might provide links or on which Romans content is embedded. Smart Homes is not responsible for the privacy practice or content of such websites.
Equally, it is important to note that when you access Smart Homes Digital Channels via a social networking site, such as Facebook, that site may also collect and use your information. So, we would advise you to check the privacy policies for those social networking sites and applications to see how your information is being used.
Please note that active clients may receive important information about Smart Homes' services that they have requested from us (such as changes to the Services themselves or other service administration matters). As an active user, this is not spam.
Will Smart Homes share my information with anyone else?
Employees, contractors and agents of Smart Homes may be given access to any personal information which Smart Homes collect but their use shall be limited to performance of their duties in relation to the services provided by Smart Homes. Smart Homes will take reasonable precautions to prevent the loss, misuse or alteration of your Personal Information. Those who have access to your personal information are required to keep that information confidential and are not permitted to use it for any purposed other than that to the services and products offered by the Company or the use of our Digital Channels.
How long does Smart Homes keep my personal information?
We will keep your information only for as long as it is relevant and useful for the purpose for which it was originally collected. Smart Homes will remove the personal information when requested by you.
You can update some of your personal information by contacting Smart Homes from the Contact Us page and we will be able to update your records on our data management systems - please let us know if the personal information which we hold about you needs to be corrected or updated.
If you would like to know what personal information Smart Homes holds about you, you may make a request to Smart Homes for copies of this information. You should be aware that we will need to see proof of identity before processing any such request (known as a 'subject access request') and may charge an administration fee of up to £10 (plus VAT) to cover our costs in providing you with this information. If requested, Smart Homes will use reasonable efforts to supply, correct or delete information about you on file.
All subject access requests should be made in writing and addressed to Smart Homes, 316 Stratford Road, Shirley, Solihull, B90 3DN.
How do I contact Smart Homes if have a question about privacy issues?


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Q: Client Assertion verify the signature failed I have configured app registration with client certificate .cer file in azure
Using jwt.io to create client assertion, and requested access token as in below screenshot

There were few posts about issues with key not found, but in this scenario key Is not able to generate signature. unable to understand the issue here. I used private key and public key in jwt.to to generate client assertion.
Header in JWt used base64 thumbprint value-
 {
  "alg": "RS256",
  "typ": "JWT",
  "x5t": "hfbTrE31AcSETk1BFhovA1w1SMc="
}

However, signature is verified in jwt.io.
TIA

A: I have figured this out, its issue in Private/public key. Extract Private key as PKCS #8 to use in building jwt assertion.

Verify Signature gives error with encrypted Private key, use decrypted private key

redpajama_set_name: RedPajamaStackExchange


The International Baccalaureate® (IB) Diploma Programme (DP) is one of the world's most exciting educational courses for 16-19 year olds.
It focuses on personal, professional and academic development and is globally recognized by universities for the holistic and rigorous education it provides.
The IB works hard to make sure the DP is globally recognised by universities, with its students gaining competitive offers for further study.
All DP curriculums are reviewed on a seven-year teaching cycle to ensure that each is fit for purpose in a changing world and incorporates the latest educational research and lessons learned from a thorough evaluation of the existing curriculum. The DP curriculum review is a collaborative process that aims to produce excellent, internationally minded, research-based curriculums and support material that enable students to develop the attributes of the learner profile and the IB mission, thereby providing an excellent preparation for life in the 21st century.
You can also read more about upcoming curriculum updates.
How is the IB recognized in different countries?
There are IB World Schools in nearly 150 countries throughout the world, and students send exam results to higher education institutions in nearly 90 countries annually. Given the wide diversity of local education systems it can be difficult to understand how the IB is recognized in various parts of the world.
To learn more on IB recognition information at the national level, read about the IB by country.
Higher education institutions around the world admit students based on their IB credentials, and many have specific admissions policies and guidelines for IB students. Admissions criteria can vary widely across educational systems and selectivity of the institution.
The university policy index [508 KB] includes self-reported policy information provided by universities to the IB through September 2018.
For general recognition queries, please contact us.
Where do IB students tend to go to university and how do they do once there?
There is a growing body of evidence on how universities value IB credentials, rates of college-going for IB students and types of institutions IB students tend to attend, preparedness for university level study and adjustment to university life. Read a brief introduction to research on postsecondary patterns of IB students.


Harvest Precision Components, Inc. is a pre-revenue WNY manufacturing company, formed in 2002, for production of small, tight-tolerance, precision ceramic components, offering significant, long term product advantages and superior customer economics. These features, easily understood by customers, are combined with resistance to wear, corrosion and oxidation, for sale into bio-medical and other high-value applications. This patent / trade secret protected material and manufacturing technology, has been proven in multiple trials. Mutually-advantageous relationships have been solidified with 2 world class suppliers of sophisticated capital equipment, involved in the production of these products. Sales into selected, advanced technology markets are planned through segment leaders, with proven, well-established distribution channels. FDA filings have already been accomplished and relationships have been established with the FDA, NIH, and a prestigious medical institution.
Markets for specific, individual components have been identified and developed in semiconductor capillaries (~$1 billion), precision surgical devices (>$1.0 Billion), and DNA analytical tools ($150 million) market segments, as introductory targeted products, replacing competitive products precision machined from gem-quality diamond and other expensive ceramic and metal-based materials. Component designs have been established and are in the process of being presented to selected customers for developmental purchase orders.
The company is raising $500,000 through the sale of convertible debt/equity, for financing the production of qualification samples, produced through subcontractors, to be delivered to these customers, and for staffing the initial phase of the 5-year (>$100 million Sales, >90% GM) business plan. Very attractive returns (>200% IRR) for investors are projected by virtue of the high gross margins anticipated for these very specialized, and defendable components. Traditional exit strategies are augmented by the anticipated sale of exclusive turnkey, controlled-royalty, manufacturing licenses to individual market segment leaders, while retaining the core technology and value of the company.


Anne has three kids; 2 boys, 1 girl & is married to her husband Trevor. She grew up in Appleton, MN & has lived in Fargo/Moorhead since 2005. Anne has a Bachelor's Degree in Marketing from North Dakota State University with a minor in Hospitality. While working on her degree Anne pursued her interests & obtained her Real Estate license in 2008.
Building her first home at 21 sparked a passion for Anne in the field of new construction. This lead her to obtain two different new construction designations. Anne is committed to helping buyers find the right home at the right price. She understands current market conditions and is trained to find opportunities in today's market. She is skilled in the art of negotiation and will work hard to get you the best deal on your next home.


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Q: Using pickit 3 with DSPIC30F2010 I'm just learning to use the PIC microcontrollers and am totally confused having no knowledge prior. I am using a pickit 3 and the DSPIC30F2010. I  have used to connect them via the circuit as shown below(i made it myself after lot of digging, couldn't find something that works in the internet). The thing is I am not even sure if the circuit is correct, primarily because i to had power the chip externally via 5V since the pickit seems to unable to power the chip by its own(returing errors like unable to detect VDD),but that kind of feels not normal, i mean the pickit should naturally be able to power the device right?

Despite my assumptions, the processor was detected and i was able to write programs into it, although when i click the verify option in the mplab IPE, it sometimes says 'verify failed' and other times, it is fine. Is this normal?
The program i wrote was generated using MICRO C FOR DSPIC and the generated hex file was written via the MPLAB IPE.
The program i used was obtained online. Its a program for simply blinking an LED.
//i have used crystal of 16 MHz

void main()
{
TRISE.F0=0;
  while (1)
  {
    PORTE.F0=1;
    Delay_ms(1000);
    PORTE.F0=0;
    Delay_ms(1000);

  }
}

But as i understand you can only write programs via the pickit, however you would require another circuit(using an external clock and so) to actually test the program.
please help me with this. What would be the circuit required to essentially test the program i written.

A: No, you don't need an external clock or crystal to test your circuit.
The micro has many clocking options, you can of course use an external clock, or an external oscillator circuit if you need an accurate clock accuracy is like 0.005%. Or, you can use an internal oscillator if you can work with low accuracy timing (internal RC oscillator accuracy is ±2% nominal).
If you want to use the internal oscillator, you should select the option from the configuration bits (choose the internal FRC oscillator). You may also need to specify for microC that you are running at 7.37 MHz (which is the frequency of the internal oscillator). This is necessary for the compiler so that he knows how to translate a 1000ms delay into assembly instructions.
to sum this up, just hook an LED to E0 (don't forget the series resistor), change config bits to select internal oscillator rather than external clock/oscillator, set frequency to 7.37 and you should have your LED blinking.


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British Prime Minister Theresa May facing open rebellion, and could be ousted by the end of the day
A no-confidence vote could force her out, right in the middle of Brexit negotiations.
Luke Barnes Twitter Dec 12, 2018, 10:36 am
British Prime Minister Theresa May is facing a vote of no-confidence, which could remove her from power if enough Conservative MPs support it. (Photo credit: Jack Taylor/Getty Images)
Another day another crisis in British politics.
After being forced on Monday to call off a crucial Brexit deal vote, Prime Minister Theresa May on Wednesday faced a vote of no-confidence, which could remove her from power if enough Conservative (Tory) Members of Parliament support it.
The vote was triggered when 48 Tory MPs wrote a letter demanding the vote. May will be forced to step down if 158 of the Conservative Party's 315 MPs support it. If she wins the vote, which is set to take place at 1 p.m. EST, she cannot be challenged for another year.
According to the BBC, May is confident she has the backing of enough MPs to survive the vote. However, the ballot is secret, so it's possible Tory MPs will say they support May in public but do the opposite when it comes time to cast their votes.
"I will contest that vote with everything I've got" – UK Prime Minister Theresa May says she will "stand ready to finish the job" as no-confidence vote is triggered
Follow live updates: https://t.co/gTH05gW8Yy pic.twitter.com/TeFWGMjVCj
— BBC Breaking News (@BBCBreaking) December 12, 2018
Multiple unconfirmed reports also indicated May would step down before the next election. A Downing Street spokesperson addressed those rumors Wednesday, saying May "doesn't believe the vote today is about who leads the party in the next election."
"It's about whether it's sensible to change leader at this point in the [Brexit] negotiations," they said.
Any leadership challenge would be seen as an impatient power grab — as well as strategically useless, since the new leader would have to deal with the remaining Brexit talks. If, however, May loses the vote, it could fundamentally alter the nature of those negotiations.
In November, May negotiated a deal with the European Union, which included several points that infuriated hardliners. Notably, it keeps the U.K. within the EU's customs union for the time being.
The EU has already said it won't renegotiate the deal, so if a Brexit hardliner like Boris Johnson replaced May, it would likely lead to a situation in which no deal is negotiated before the March 29 deadline. In this worse-case scenario, both British trade to the EU and supply chains of perishable items to the U.K. Regulations would disappear overnight meaning, among other things, that aircraft in the U.K. could not fly through the EU, and that materials needed for cancer treatments could not be imported into the U.K.
Here's what you should know about the latest Brexit trainwreck
Theresa May postponed a crucial Brexit vote Monday, the most recent installment in a series of chaotic developments.
As the Guardian's Jonathan Freedland noted Wednesday, even if a hardliner does succeed May, they still have to pass a deeply unpopular Brexit bill through a Parliament where the Conservative party does not have an outright majority.
"…Every second of [the next] 14 weeks should be spent striving to come up with a plan of action that MPs could agree on," he wrote. "…But instead of working day and night to avert that disaster, the Conservative party has decided to squander precious time with what several loyalist ministers are calling an act of self-indulgence."
#Brexit, #Politics, #Theresa May, #UK, #UK Politics, #World

redpajama_set_name: RedPajamaCommonCrawl


The extracellular signal-regulated kinases (ERK) 1 and 2 (ERK1/2) are members of the mitogen-activated protein kinase [MAPK] family. Upon stimulation, these kinases translocate from the cytoplasm to the nucleus, where they induce physiological processes such as proliferation and differentiation. The mechanism of translocation of this kinase involves phosphorylation of two Ser residues within a nuclear translocation signal (NTS), which allows binding to importin7 and a subsequent penetration via nuclear pores. Here we show that the phosphorylation of both Ser residues is mediated mainly by casein kinase 2 (CK2) and that active ERK may assist in the phosphorylation of the N-terminal Ser. We also demonstrate that the phosphorylation is dependent on the release of ERK from cytoplasmic anchoring proteins. Crystal structure of the phosphomimetic ERK revealed that the NTS phosphorylation creates an acidic patch in ERK. Our model is that in resting cells ERK is bound to cytoplasmic anchors, which prevent its NTS phosphorylation. Upon stimulation, phosphorylation of the ERK TEY domain releases ERK and allows phosphorylation of its NTS by CK2 and active ERK to generate a negatively charged patch in ERK, binding to importin 7 and nuclear translocation. These results provide an important role of CK2 in regulating nuclear ERK activities.
Extracellular signal-regulated kinases (ERK) 1 and 2 (ERK1/2) are central signaling proteins that mediate a variety of vital cellular processes, including proliferation, survival, and even apoptosis (1, 4, 15, 29, 50). In order to execute their functions, ERK molecules activate a large number of regulatory proteins, which are localized either in the cytoplasm or within various organelles, including mainly the nucleus (52). Indeed, the number of nuclear targets and downstream effectors of ERK, including a variety of transcription factors (17), is well over 100. These direct and indirect targets participate in the regulation of transcription as well as chromatin remodeling, and therefore they play a central role in mediating essentially all stimulated cellular processes (29). Moreover, because these ERK-induced nuclear activities are such central signaling processes, their dysregulation often leads to severe pathological processes, including oncogenic transformation, neurodegenerative diseases, and developmental diseases (15). In order to transmit their nuclear signals, ERK molecules that are localized in the cytoplasm of quiescent cells rapidly translocate into the nucleus upon stimulation. Although many details on ERK in the nucleus were already provided, the mechanisms of its translocation are not fully worked out yet.
The nucleus is separated from the cytoplasm by a double membrane envelope (25). Nuclear shuttling of proteins occurs through a specialized nuclear pore complex (NPC), which ensures high selectivity of molecules for nuclear import/export, thus supporting proper cytoplasmic/nuclear molecular balance. The majority of proteins enter the nucleus by an active transport mechanism, based on a nuclear localization signal (NLS) in the shuttling proteins that binds to importin-α (Impα) and importin-β (Impβ), which act as shuttling transport factors through the NPCs (13). However, a significant number of signaling proteins, which rapidly translocate into the nucleus upon cell stimulation, do not contain an NLS and therefore must utilize distinct mechanisms for their translocation. In a previous study, our group identified a novel NLS-independent mechanism of stimulated nuclear translocation of signaling proteins, including ERK, SMAD3, and MEK1 (6). This mechanism involves a novel nuclear translocation signal (NTS), which contains either Ser or Thr residues that are phosphorylated upon stimulation to allow binding to importin 7 (Imp7) and thereby nuclear shuttling via the NPCs. The NTS of ERK contains the sequence Ser-Pro-Ser (SPS) within its kinase insertion domain (KID), which undergoes stimulus-dependent phosphorylation by as-yet-unknown kinases.
Casein kinase 2 (CK2), formerly known as casein kinase II, is a ubiquitous protein Ser/Thr kinase that plays a central role in the regulation of a variety of cellular processes (14, 28). This kinase acts a tetramer containing two catalytic (α and/or α′) and two regulatory (β) subunits (20). It is a constitutively active kinase whose minimal consensus phosphorylation site is Ser-X-X-Glu/Asp, but additional Glu or Asp residues in some of the surrounding residues often allow the phosphorylation of more than 300 substrates identified to date (22). Upon phosphorylation, these substrates regulate a variety of processes, including proliferation, transformation, apoptosis, senescence, and also malignant cell transformation (8, 41). In the present paper, we show that CK2 is the main kinase that phosphorylates both Ser residues within the ERK NTS. The phosphorylation of one of the N-terminal Ser residues is facilitated by active ERK transautophosphorylation. We also demonstrate that the phosphorylation is dependent on ERK release from hindering cytoplasmic anchors. The crystal structure showed that CK2 phosphorylation creates an acidic patch important for the translocation. These results provide an important role of CK2 in regulating ERK activity in the nucleus as well as proliferation and oncogenic transformation.
Reagents and antibodies.Tetradecanoyl phorbol acetate (TPA), epidermal growth factor (EGF), the CK2 inhibitor TBB, and transforming growth factor (TGF)–4′,6-diamidino-2-phenylindole (DAPI) were obtained from Sigma (St. Louis, MO). Chelating Sepharose-coupled nickel-nitrilotriacetic acid agarose beads and a Resource-15Q anion-exchange column were from Amersham (England). Secondary antibody (Ab) conjugates were from Jackson ImmunoResearch (West Grove, PA). Anti-histone H1, antitubulin, antihemagglutinin (anti-HA), anti-Elk-1, anti-RSK-1, anti-CK2α, and anti-MEK1 Abs were acquired from Santa Cruz Biotechnology (CA); anti-pElk-1 (2B1, Ser383) and anti-pRSK (Ser381) Abs were from Cell Signaling Technology (Beverly, MA); and anti-green fluorescent protein (anti-GFP) Abs were from Roche Diagnostics GmbH (Mannheim, Germany). Anti-Imp7 Ab was from ABNova (Taipei, Taiwan). Abs to doubly TEY-phosphorylated ERK (pTEY-ERK) and general ERK (gERK) were from Sigma (Rehovot, Israel), who also produced and purified the polyclonal and monoclonal anti-phospho-SPS-ERK Abs.
DNA constructs and mutations.GFP-ERK2 was prepared in pEGFP-C1 vector (Clontech, Mountain View, CA). Human MEK1 was ligated into HindIII and ApaI sites downstream of the red fluorescent protein (RFP) gene of the DsRed1-N1 vector (Clontech), and glutathione S-transferase (GST)-ERK2 constructs were prepared in the pGEX vector. All mutations described were performed with a site-directed mutagenesis kit (Stratagene, La Jolla, CA) and confirmed by sequencing. C-terminally tagged wild-type HA-CK2 in pRc/CMV vector was a gift from D. W. Litchfield (University of Western Ontario, London, ON, Canada). The 6His-MEK construct was produced as described previously (3). All small interfering RNAs (siRNAs) were from Dharmacon (Lafayette, CO).
Cell culture and transfection.HeLa, MDA-MB-231, MCF-7, and HEK-293T cells were grown in Dulbecco's modified Eagle's medium (DMEM), and CHO cells were grown in F12-DMEM, all supplemented with 10% fetal bovine serum. Transfection used either polyethylenimine or Lipofectamine 2000 (Invitrogen, Carlsbad, CA). siRNAs were transfected using Oligofectamine (Dharmacon). In order to reach equal transfection efficiencies, transfected cells were distributed among the necessary number of plates 24 h after transfection.
Immunofluorescence microscopy.Cells were fixed in 3% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min and then permeabilized with 0.2% Triton X-100 in PBS-bovine serum albumin (BSA) (2%) for 20 min at room temperature. The fixed cells were sequentially incubated with appropriated Abs (diluted in PBS-BSA, 1:200) for 1 h, followed by either Cy-2- or rhodamine-conjugated secondary Abs and DAPI (diluted in PBS-BSA, 1:200) for 1 h. Slides were analyzed and photographed by a fluorescence microscope (×600 magnification; Nikon, Japan).
ImageStream analysis.Nuclear translocation of ERKs was examined by the ImageStream system (Amnis Corp., Seattle, WA) using the IDEAS image analysis program. About 1 million cells per sample were rinsed in cold PBS and gently scripted into 1.5-ml tubes. Cells were spun down by centrifugation, fixed in 3% paraformaldehyde (20 min), and then permeabilized with 0.1% Triton X-100 in PBS-BSA (2%) (20 min, 23°C). For endogenous ERK staining, fixed cells were sequentially incubated with anti-ERK Abs (1:200 in PBS-BSA; 1 h, 4°C) followed by incubation with Cy-2-conjugated secondary Abs (1:200) and propidium iodide (PI) (1 μg/ml, for nuclear staining) in PBS containing 100 μg/ml of RNase and 0.1 mM EDTA (1 h, 40°C). The cells transfected with GFP-containing plasmids were incubated with secondary Abs and PI. The green ERK1/2 image and the red nucleus image were first compensated into separate channels, and then an overlay of green and red (creating yellow) was quantified. Additional information regarding the ImageStream technique can be found at the Internet site of the company (Amnis Corp.).
Cell extraction, cell fractionation, Western blotting, and coimmunoprecepitation. (i) Preparation of cellular extracts for Western blotting.Cells were rinsed twice with ice-cold PBS and scraped into radioimmunoprecipitation assay buffer (RIPA) (137 mM NaCl, 20 mM Tris [pH 7.4], 10% glycerol, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride [PMSF], and 20 μM leupeptin). The extracts were centrifuged (20,000 × g, 15 min, 4°C), and the supernatants were further analyzed by Western blotting. The blots were developed with horseradish peroxidase-conjugated anti-mouse or anti-rabbit Abs.
(ii) Cellular fractionation.Cells were rinsed twice with ice-cold PBS, suspended in ice-cold hypotonic buffer (10 mM HEPES [pH 7.9], 1.5 mM MgCl2, 10 mM KCl, 0.2 mM PMSF, 0.5 mM dithiothreitol [DTT], and 0.5% Nonidet P-40), incubated (10 min, 4°C), disrupted by repeated aspiration through a 20-gauge needle, and centrifuged (12,000 × g, 5 min). The supernatant containing the cytosolic fraction was boiled in sample buffer. The pellet was suspended in extraction buffer (420 mM NaCl, 50 mM β-glycerophosphate, 0.5 mM Na3VO4, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 25% glycerol), incubated on ice for 30 min, sonicated (50 W, 2 times for 7 s), and centrifuged (15,000 × g, 30 min). The supernatant containing the nuclear fraction was subjected to Western blotting with the appropriate Abs.
(iii) Coimmunoprecipitation.Cells were rinsed twice with ice-cold PBS and scraped into buffer H (50 mM β-glycerophosphate [pH 7.3], 1.5 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM sodium vanadate, 1 mM benzamidine, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 2 μg/ml pepstatin). The extracts were sonicated (50 W, 2 times for 7 s), and centrifuged (15,000 × g, 15 min, 4°C). Cellular extracts were incubated overnight at 4°C with the appropriate Abs preconjugated to A/G beads (1 h, 23°C). Subsequently, the beads were washed three times with coimmunoprecipitation washing buffer (20 mM HEPES [pH 7.4], 2 mM MgCl2, 2 mM EGTA, 150 mM NaCl, and 0.1% Triton) and once with PBS and were subjected to Western blot analysis.
Luciferase reporter assay.The following plasmids (from the Forchheimer Plasmid Collection of the Weizmann Institute of Science, Rehovot, Israel) were used for the experiment: FR-Luc (reporter plasmid), pFA2-Elk1 (fusion trans-activator plasmid), pFC2-dbd (negative control), and pFC-MEK1 (positive control). The plasmids were transfected into HEK-293T cells with Lipofectamine 2000 according to the manufacturer's protocol. At 36 h after transfection, cells were starved overnight, pretreated with TBB (10 μM, 2 h) or left untreated as a control, and then stimulated with EGF (50 ng/ml, 6 h) or left untreated as a control. The assay was performed using the dual-luciferase reporter assay system (Promega) in accordance with the manufacturer's protocol. The luminescence was detected using a Viktor2 multilabel counter (Perkin Elmer).
In vitro kinase assay.The substrates used for the in vitro kinase assay were wild-type or mutated GST-ERK2, 6His-MEK1, and dephosphorylated α-casein (the last two from Sigma, St. Louis, MO). The substrates were mixed with cold ATP (0.5 mM), [γ-32P]ATP (4,000 cpm/pmol), and CK2 or active ERK2 (250 units each; New England BioLabs, MA) or with GST-ERK2 (ERK), active GST-ERK2 (Act-ERK), or kinase-dead GST-KA-ERK2 (KA-ERK) produced in our lab, in appropriate kinase buffers (supplied by the company; 1× CK2 buffer contained 20 mM Tris-HCl [pH 7.5], 50 mM KCl, and 10 mM MgCl2; 1× ERK2 buffer contained 50 mM Tris-HCl [pH 7.5], 10 mM MgCl2, 2 mM DTT, and 1 mM EGTA, 0.01% Brij 35; both were used in a total volume of 20 μl). The mixture was shaken at 900 rpm (30 min, 30°C), mixed with 4× SDS, and boiled for 5 min. Proteins were separated by 12 to 15% SDS-PAGE, followed by Western blotting. The loading of α-casein was assayed by protein staining with Reagent Blue (Pierce, IL).
Expression and purification of ERK2 mutants for crystallization purposes.The SPE-ERK2 proteins were expressed in Escherichia coli strain Rosetta as N-terminally hexahistidine-tagged proteins. The bacteria were grown, lysed, and then purified with chelating Sepharose-coupled nickel-nitrilotriacetic acid agarose beads. Protein elution was obtained with a linear gradient of imidazole from 10 mM to 250 mM, using 50 mM Tris-HCl (pH 8.0), 0.3 M NaCl, and 250 mM imidazole buffer. The fraction that contained ERK2 protein was then dialyzed against 50 mM Tris-HCl (pH 8.0), 0.1 M NaCl, and 1 mM EDTA for 4 h at 4°C and then transferred to dialysis buffer containing Tris-HCl (pH 8.0), 0.1 M NaCl, 1 mM DTT, and 5% glycerol for 16 to 18 h. The protein solution was then subjected to a second purification step on a 23-ml Resource 15Q anion-exchange column equilibrated with 50 mM Tris-HCl (pH 8.0), 0.1 M NaCl, 5% glycerol, and 1 mM DTT. The protein was eluted using a linear gradient of NaCl with the buffer as follows: 50 mM Tris-HCl (pH 8.0), 1 M NaCl, 5% glycerol, and 1 mM DTT. Purified protein was concentrated using Vivaspin (VivaScience) up to 6 to 12 mg/ml, as determined by A280. The purified protein was divided into aliquots and stored at −80°C.
Crystallization, data collection, and solution of the SPE-ERK2 mutant.The SPE-ERK2 proteins were expressed in E. coli (data not shown). Crystals of SPE-ERK2 protein were obtained by the vapor diffusion sitting drop technique by mixing equal amounts of protein solution (2.3 to 2.5 mg/ml) and reservoir solution in 61 mM ammonium sulfate, 100 mM Bis-Tris (pH 7.0), 12 to 15% (vol/vol) polyethylene glycol (PEG) 3350. Streak seeding (42) using wild-type (WT)-ERK2 crystals was conducted in order to initiate crystallization. Crystals appeared within a day of incubation at 20°C and reached their final size several days later. For data collection, crystals were transferred into cryoprotectant solution containing the crystallization solution and 15% glycerol. Crystallographic data were collected from a single crystal at 100 K using an Oxford Cryostream cryosystem cooling device on an ADSC Quantum 315R charge-coupled-device (CCD) detector with an oscillation range of 1.0° at beam line ID23-1 at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. The crystals belonged to the monoclinic space group P21 with one ERK2 molecule in the asymmetric unit (data not shown). Data were integrated, reduced, and scaled using the HKL2000 suite (27). The structures were solved via molecular replacement methods using Molrep (45) implemented in the CCP4i suite (30) with the ERK2 structure (1ERK) as the search model after removing all solvent molecules. Following molecular replacement, the models were refined using rigid-body and then restrained options in REFMAC5 (23). Solvent molecules were added utilizing ARP/WARP (19). The models were fitted into electron-density maps using the graphics program Coot (9).
CK2 is important for nuclear translocation of ERK.The two phosphorylated Ser residues of the ERK NTS lie within consensus phosphorylation sites of several protein kinases. Since the C-terminal Ser seems to fall within a CK2 site, we undertook to examine the role of the latter kinase in the nuclear translocation of ERK. For this purpose, we first pretreated CHO cells with CK2 inhibitors, stimulated the cells with TPA for 15 min, and followed the nuclear translocation of endogenous ERK by staining with an anti-ERK antibody (Ab). As expected, most ERK molecules were localized in the cytoplasm of resting cells and translocated into the nucleus after TPA stimulation (Fig. 1A). However, pretreatment with the CK2 inhibitors TBB (Fig. 1A) and DMAT (data not shown) prevented the stimulated nuclear translocation, without any significant effect on the localization of ERK in resting cells. Similar effects of CK2 inhibitors were observed with MCF7 (Fig. 1A), MBA-MB-231, and HeLa cells (data not shown). The effect of the CK2 inhibitors was not due to modulation of the activating TEY phosphorylation, as judged by Western blotting with anti-pTEY Ab (Fig. 1B). Since pharmacological inhibitors are often not fully specific, we ascertained the CK2 effect by specifically knocking down its expression. For this purpose, we used siRNA of CSNK2A1 (α subunit of CK2), which reduced the expression of CK2α (but not CK2α′) in HeLa and (unexpectedly) CHO cells (Fig. 1C). As with the pharmacological inhibitors, knockdown of CK2α resulted in prevention of TPA-induced nuclear translocation of ERK (Fig. 1C).
CK2 plays a role in the nuclear translocation of ERK. (A) CK2 inhibition prevents nuclear translocation of endogenous ERK. CHO and MCF7 cells were serum starved (16 h, 0.1%), pretreated with either TBB (10 μM, 2 h) or dimethyl sulfoxide (DMSO) control, and then either stimulated with TPA (250 nM, 15 min) or left untreated as a control (NS). The cells were stained with anti-gERK Ab and DAPI. Scale bar, 15 μm. (B) TBB does not affect TEY-ERK phosphorylation. HeLa cells were serum starved, pretreated with TBB (10 μM) or DMSO (indicated times), and then stimulated with TPA (250 nM, 15 min). Cell extracts were subjected to Western blot analysis using anti-pERK (low exposure and high exposure) and anti-gERK Abs. (C) Knockdown of CK2 prevents nuclear translocation of endogenous ERKs. CHO and HeLa cells were transfected with either siCK2α or nonrelevant siRNA (siCon). At 36 h after transfection the cells were serum starved, stimulated, and processed as above. The efficiency of CK2α knockdown is shown compared to endogenous tubulin levels. (D) Analysis of CK2 effect on the nuclear translocation of endogenous ERK by ImageStream. HeLa cells were serum starved and treated as above, and then the cells were analyzed by ImageStream. The graph represents fluorescence of nuclear ERK (two independent experiments), where the fluorescence of nonstimulated control was considered 1. N, number of cells analyzed. *, P < 0.02 (Student t test). (E) Cell fractionation confirms CK2's role. HeLa cells were treated as described for panel A. After fractionation, cytoplasmic and nuclear samples were subjected to Western blotting using the indicated Abs. Tubulin and histone H1 served as localization controls. The graph represents densitometric analysis of nuclear ERK (two independent experiments). *, P < 0.05.
In order to quantify our results, we used ImageStream Analyzer, which simultaneously detects the localization of a fluorescent dye in many individual cells, thus obtaining statistically significant data. For this purpose, we treated HeLa cells with the CK2 inhibitor TBB, stimulated the cells with TPA for 15 min, and subjected them to the machine. Pictures of the sorted cells (see a representative cell in Fig. 1D, left), as well as quantification of the nuclear fluorescence of the cells (Fig. 1D, right), revealed that in the great majority of resting cells the ERK molecules were localized in the cytoplasm. Upon stimulation, most ERK staining was shifted to the nucleus, while TBB pretreatment prevented TPA-stimulated ERK translocation. Finally, another quantitative measure of nuclear translocation of ERK was the subcellular fractionation of the treated cells. Similar to the results in Fig. 1D, this method also demonstrated that TBB significantly prevents nuclear accumulation of ERK upon stimulation (Fig. 1E). Taken together, our results strongly suggest that CK2 participates in the regulation of nuclear ERK translocation upon stimulation.
CK2 phosphorylates the ERK NTS and is involved in the activation of nuclear ERK targets.The finding that CK2 inhibitors prevent the nuclear translocation of ERK suggested that CK2 may act as an NTS kinase. In order to verify this point, we examined the ability of CK2 to phosphorylate purified GST-ERK2 in vitro. As expected, CK2 phosphorylated WT ERK2 but not ERK2 mutated in the Ser residues within the NTS (ERK-APA; Fig. 2A); this indicates that the Ser residues within the NTS are the only CK2 sites within ERK2. This phosphorylation was abolished by TBB, signifying that the phosphorylation is indeed mediated by CK2 and not caused by autophosphorylation. We then examined the in vivo effect of CK2 inhibition or knockdown on NTS phosphorylation using anti-pSPS Ab, which primarily recognizes the doubly phosphorylated NTS. We found that TBB as well as siCK2 from two different sources (Fig. 2B and C and data not shown) results in the prevention of NTS phosphorylation in HeLa and CHO cell lines. Since this effect is not dependent on changes in TEY phosphorylation, at any time after stimulation (Fig. 1B and 2C), it is likely that CK2 is not involved in the upstream activation of the ERK cascade but rather directly phosphorylates the Ser residues in the NTS. We also found that MEK inhibition had an effect similar to that of CK2 inhibition, and a possible reason for it will be explained below. In order to further verify the role of the CK2 effect, we postulated that the impaired nuclear translocation of active ERK should lead to a decrease in the phosphorylation of nuclear ERK targets, without much change in the phosphorylation of cytoplasmic substrates. Indeed, pretreatment of cells with CK2 inhibitor or knockdown of CK2α resulted in a significant decrease in the phosphorylation and activation of the transcription factor Elk1, which is a well-known nuclear target of ERK (Fig. 2A, B, and D). On the other hand, the phosphorylation of a cytoplasmic ERK target (RSK) was reduced by MEK inhibition but not significantly affected by the inhibition of CK2 activity. We also examined the effect of CK2 inhibition on cell viability, which is known to be regulated by the ERK cascade, and found that TBB indeed inhibits it in nontransfected cells as well as in cells overexpressing constitutively active MEK1. Interestingly, overexpression of the ERK2-EPE construct rescued the TBB effect (data not shown), indicating that although CK2 affects cell proliferation by inhibiting various cellular processes, its effect in our system might be mediated, at least in part, by the nuclear activity of ERK. These results clearly indicate that NTS phosphorylation is executed primarily by CK2, acting downstream of TEY phosphorylation, and therefore does not affect the activation of ERK. Thus, CK2 affects only the nuclear targets of ERK, without much influence on cytoplasmic activity.
CK2 phosphorylates the ERK NTS and is involved in the activation of nuclear ERK targets. (A) CK2 phosphorylates the ERK NTS in vitro. The indicated GST-ERK2 constructs (0.1 μg per sample) were subjected to in vitro CK2 phosphorylation, which was detected by autoradiography (upper rows). The loaded ERK and CK2 constructs were detected using anti-gERK and anti-CK2α Abs (middle and lower rows, respectively). (B) CK2 inhibition prevents ERK NTS and Elk1 but not RSK1 phosphorylation. HeLa cells were serum starved (0.1%, 16 h), pretreated with TBB (10 μM, 2 h), UO126 (5 μM; 2 h), or dimethyl sulfoxide (DMSO) control, and then stimulated with TPA (250 nM, 15 min) or left untreated as a control. Cell extracts were subjected to Western blot analysis with the indicated Abs. (C) CK2 knockdown prevents ERK NTS and Elk1 but not RSK1 phosphorylation. CHO cells were transfected with either siCK2 or nonrelevant siRNA (siCon). At 36 h after transfection, the cells were serum starved and then either stimulated with TPA (250 nM, 15 min) or left untreated as a control. Cell extracts were subjected to Western blot analysis using the same Abs as in panel B. Efficiency of CK2 knockdown is shown in Fig. 1B. (D) Inhibition of CK2 attenuates the ERK-dependent activation of Elk1. HEK-293T cells were transfected with plasmids pFC2-Elk1, pFC-MEK1 (for constitutively active MEK1), and pFC2-dbd as a control. The cells were then either pretreated with TBB (10 μM, 2 h) or left untreated as a control, followed by stimulation with EGF (50 ng/ml) for 6 h where indicated. The graph represents the ratio of the luminescence of pFR-Luc (reporter)/Renilla luminescence. The bar graph shows the means and standard errors of results of three repeats. *, P < 0.01.
The most important requirement for substrate recognition by CK2 within its consensus site is the presence of acidic amino acid 3 amino acids C-terminal to the phosphorylated site (position +3), while acidic amino acid at position +2 may contribute to the recognition as well (18, 36). In order to further verify the importance of CK2 in NTS phosphorylation, we constructed GFP-conjugated mutants of ERK2, in which the Glu and Asp residues at positions of 248 and 249 (+2 and +3 from the phosphorylated Ser246) were changed to Ala, producing the sequence SPSQAA (ERK-SQAA). The same mutation was also inserted within an NTS phosphomimetic mutant of ERK2, producing the sequence EPEQAA (ERK-EQAA). These constructs were cotransfected into CHO cells together with red fluorescent protein (RFP)-conjugated MEK1. All mutants were localized in the cytoplasm of quiescent cells, indicating that they still preserve their ability to interact with MEK1. As previously shown (6), TPA stimulation of the SPS (WT)-ERK- and EPE-ERK-expressing cells resulted in nuclear accumulation of ERK2 (Fig. 3A). TPA stimulation of the ERK-SQAA-expressing cells did not result in the nuclear translocation of the mutant, but the phosphomimetic mutant (ERK-EQAA) rescued the effect. Fractionation confirmed these results by showing that despite a lack of effect on TEY phosphorylation, ERK-EQAA is readily present in the nucleus, while SQAA is not (Fig. 3B). The abrogated nuclear translocation was not due to changes in TEY phosphorylation, as the NTS mutations had only minor effects on the activating phosphorylation (Fig. 3B, lower blot). Finally, Western blot analysis using anti-pSPS Ab showed no TPA-induced phosphorylation of the Ser residues within the NTS (Fig. 3C). These results further support the involvement of CK2 in NTS phosphorylation, and they show the importance of the acidic 248/249 residues in the process.
Mutation of E248 and D249 prevents ERK NTS phosphorylation and nuclear translocation. (A) Mutation of D248 and E249 inhibits nuclear translocation of exogenous ERK2. CHO cells were cotransfected with plasmids for GFP-tagged ERK2-WT (SPSQED), ERK2-EPEQED, ERK2-SPSQAA (SQAA), or ERK2-EPEQAA (EQAA) and for RFP-MEK1. At 36 h after transfection the cells were serum starved and either stimulated with TPA (250 nM, 15 min) or left untreated (NS). The cells were stained by DAPI and visualized by using fluorescence microscopy. Scale bar, 15 μm. (B) Cells were treated as described for panel A and then fractionated to cytosolic/nuclear fractions and subjected to Western blot analysis. Tubulin and histone H1 served as purity and loading markers. P-ERK as well as ERK and MEK expression was also detected using whole-cell extracts (lower blots). The bar graph represents densitometric analysis of nuclear ERK (three experiments). *, P < 0.05. (C) Mutation of D248 and E249 inhibits ERK NTS phosphorylation. HEK-293T cells were transfected with the indicated GFP-tagged plasmids, serum starved, and stimulated as indicated. Cell extracts were subjected to Western blot analysis with the indicated Abs.
ERK/Imp7 interaction is regulated by CK2 binding and phosphorylation of ERK.We have previously shown that the translocation of ERK to the nucleus is executed by the interaction of their phosphorylated NTS with Imp7 (6). Since Imp7 recognizes only NTS-phosphorylated ERK, we examined whether CK2 inhibition affects the endogenous ERK/Imp7 interaction in stimulated cells. As expected, stimulation of HeLa cells with TPA resulted in an enhanced interaction of ERK with Imp7 (Fig. 4A). Inhibition of CK2 phosphorylation by TBB as well as prevention of NTS phosphorylation due to SQAA mutation abolished this interaction (Fig. 4A and B). These results clearly support the involvement of CK2-mediated NTS phosphorylation in the interaction with Imp7 and thereby in the nuclear translocation of ERK. We then entertained the possibility that the phosphorylation of NTS by CK2 is dependent on its physical association with ERK. For this purpose we overexpressed GFP-ERK2 and HA-CK2 in HEK-293T cells, which were starved and then stimulated with TPA. The extracts of these cells were then used for coimmunoprecipitation with anti-GFP Ab, which revealed that the two overexpressed proteins interact with each other after cell stimulation but not in resting cells (Fig. 4C). Therefore, it was important to show that the interaction occurs between the endogenous proteins as well. To this end, coimmunoprecipitation revealed that as for the overexpressed proteins, there was very little interaction in resting cells, but TPA treatment stimulated this interaction mainly upon 5 min of stimulation (Fig. 4D). Therefore, it is likely that shortly after stimulation, ERK interacts with and is phosphorylated by CK2. The phosphorylation then allows ERK binding to Imp7, and this promotes the nuclear translocation of ERK.
ERK binding to Imp7 and CK2. (A) Imp7 interaction with ERK depends on CK2 phosphorylation of the ERK NTS. HeLa cells were serum starved and treated with TPA (250 nM) for the indicated times. ERK was immunoprecipitated using anti-gERK Abs or normal rabbit serum (NRS), which served as an irrelevant Ab control. Coimmunoprecipitated Imp7 was detected by Western blot analysis using anti-Imp7 Abs (upper panel). ERK activation and Imp7 amount were determined using the indicated Abs. (B) Mutation of E248 and D249 prevents the interaction of ERK with Imp7. HEK-293T cells were transfected with the GFP-tagged plasmids of ERK2-WT (SPS) and ERK2-SPSQAA, serum starved, and stimulated with TPA (250 nM) for the indicated times. ERK constructs were immunoprecipitated using either anti-GFP Ab or NRS control, and coimmunoprecipitated Imp7 was detected by Western blot analysis with anti-Imp Abs. ERK activation and Imp7 amount were detected using the indicated Abs. (C) Ectopically expressed CK2 interacts with overexpressed ERK2 in a stimulus-dependent manner. HEK-293T cells were transfected with plasmids for HA-tagged CK2α or GFP-ERK2 or with both plasmids. At 36 h after transfection, the cells were serum starved and then stimulated with TPA (250 nM) for the indicated times. ERK2 was immunoprecipitated using anti-GFP Ab, and the coimmunoprecipitated CK2α was detected by Western blot analysis using anti-HA Ab. The amount of expressed proteins was detected with anti-HA and antitubulin Abs. (D) Stimulus-dependent interaction of endogenous CK2 and ERK. HeLa cells were serum starved and then stimulated with TPA (250 nM) for the indicated times. Endogenous ERKs were immunoprecipitated using either anti-gERK Ab or NRS control, and coimmunoprecipitated CK2α was detected by Western blot analysis with anti-CK2α Ab. ERK activation and CK2 loading were detected using the indicated Abs.
Ser246 phosphorylation by CK2 is sufficient for full ERK translocation, while Ser244 phosphorylation accelerates it.Our previous study using APE and EPA mutants of the ERK2-NTS suggested that phosphorylation of Ser246 is more important for ERK nuclear translocation than that of Ser244 (6). In order to further study the role of the distinct residues, we mutated NTS within GFP-ERK2 to APS, SPA, and APA. These constructs were cotransfected into CHO cells together with the RFP-conjugated MEK1 for cytoplasmic anchoring (11, 34). After serum starvation, the cells were treated with TPA and the nuclear translocation of the various constructs was monitored. As expected, all overexpressed-ERK2 constructs were localized in the cytoplasm, but after stimulation the distinct constructs translocated into the nucleus with different kinetics (Fig. 5A). Thus, ERK-SPS (WT) translocation was fastest, and this construct was found in the nuclei of 65% of the cells already 6 min after stimulation. ERK-APS translocated slightly more slowly and reached this level 12 min after stimulation. The translocation of the ERK-SPA was much slower, as 15 min after stimulation only 35% of the cells had nuclear fluorescence, and ERK-APA demonstrated almost no nuclear staining after stimulation. These results indicate that Ser246 is sufficient to induce full but slower ERK translocation, while the phosphorylation of Ser244 cannot induce ERK translocation but rather facilitates the rate of translocations that occur together with Ser246 phosphorylation. The differences in nuclear translocation were not due to changes in TEY phosphorylation, as under the coexpression conditions used here, the NTS mutations had only minor effects on the activating phosphorylation (Fig. 5A, lower blot). The result that the effect of SPS mutation on TEY phosphorylation was smaller than that reported in our previous publication (6) is probably due to the overexpression of MEK1 that is probably able to overcome to some extent the compromised conformational change. Results similar to those determined by cell count were found using ImageStream as well (Fig. 5B). We then tested whether the translocation of the various SPS mutants might be regulated by CK2. For this purpose, we repeated the above experiment but stimulated the cells with TPA for 15 min in the presence or absence of TBB. As expected, most ERK-SPS (WT) and ERK-APS, a small portion of ERK-SPA, and essentially no ERK-APA were found in the nucleus at this time (Fig. 6). TBB pretreatment abolished most of the nuclear translocation of ERK-SPS and ERK-APS (>80%) but inhibited only ∼30% ERK-SPA and had no effect on ERK-APA localization. These results indicate that CK2 is the main kinase responsible for ERK translocation, but other kinases might participate in Ser244 phosphorylation and influence the kinetics of ERK translocation as well.
Kinetics of NTS-mutated ERK translocation into the nucleus. (A) CHO cells were cotransfected with RFP-MEK1 and the indicated GFP-ERK2 constructs. At 36 h after transfection the cells were serum starved and then stimulated with TPA (250 nM) for the indicated times. The cells were processed and stained as described above. Scale bar, 15 μm. About 100 cells from each group in 10 to 12 random microscopic fields were monitored. The graph represents the percentage of cells in which GFP-ERK2 was mostly localized in the nucleus. The statistical difference in the number of cells with the SPA mutant compared to those with WT ERK2 at 15 min after stimulation was assessed by the Student t test. *, P < 0.01. pERK as well as ERK and MEK expression was examined using whole-cell extract (lower Western blots). (B) Analysis of the nuclear translocation of GFP-ERK2 mutants by ImageStream. HeLa cells were cotransfected with pcDNA-MEK1 and the indicated GFP-ERK2 constructs. At 36 h after cotransfection the cells were serum starved and then stimulated with TPA (250 nM) for 6 min. The treated as well as the nontreated control cells (100 to 170 per sample) were processed as described in Materials and Methods and analyzed by ImageStream. The graph represents the geographic mean of nuclear ERK fluorescence ± standard error (SE). *, P < 0.05.
Role of distinct Ser phosphorylation within the NTS in the nuclear translocation of ERK. CHO cells were cotransfected with the following GFP-ERK2 constructs: SPS (WT), APS, SPA, APA, and RFP-MEK1. At 36 h after transfection the cells were serum starved, pretreated with either 10 μM TBB (10 μM) or dimethyl sulfoxide (DMSO) control for 2 h, and then either stimulated with TPA (250 nM; 15 min) or left untreated (NS). The cells were processed and stained as in Fig. 1A. Scale bar, 15 nm. In the parallel experiment the cells were separated into cytoplasmic and nuclear fractions, and the amount of GFP-ERK2 constructs was analyzed with anti-GFP Ab (upper panels). Tubulin and histone H1 served as loading controls for cytosolic and nuclear fractions, respectively (bottom panels).
The phosphorylation of Ser244 is mediated by both CK2 and ERK.Since Ser244 is not a classical phosphorylation site of CK2, we undertook to study the mechanism of phosphorylation of this site. As CK2 was shown above to be the kinase that was mainly responsible for the translocation, we first examined the ability of CK2 itself to phosphorylate each of the Ser residues within the NTS by in vitro phosphorylation of WT ERK2 and the various NTS mutants. As expected, CK2 efficiently phosphorylated the recombinant GST-ERK2-SPS and failed to phosphorylate the ERK2-APA construct (Fig. 7A). However, the ability of CK2 to phosphorylate the various NTS mutants was compromised. Thus, the rate of phosphorylation of the predicated CK2 site (Ser246 in ERK2-APS and ERK2-EPS) was roughly half that of ERK2-SPS phosphorylation by CK2. The reason for this reduced phosphorylation of Ser246 in APS or EPS is not clear, but it might be due to changes in ERK conformation. On the other hand, Ser244 was not phosphorylated at all when Ser246 was mutated to Ala (ERK2-SPA), but the phosphorylation was rescued when Ser246 was mutated to Glu (ERK2-SPE). These results indicate that both Ser residues within the NTS can be phosphorylated by CK2, but Ser244 can be phosphorylated only after Ser246 phosphorylation, supporting the ability of CK2 to phosphorylate Ser residues when position +2 is primed by phosphorylation (36).
CK2 phosphorylates both Ser244 and Ser246, while ERK participates in Ser244 phosphorylation. (A) CK2 phosphorylates both Ser244 and Ser246 in vitro. The indicated GST-ERK2 constructs (0.1 μg per sample) were subjected to an in vitro CK2 phosphorylation as described in Materials and Methods. The phosphorylated proteins were detected by autoradiography, and band intensities were quantified by densitometry. The substrates and kinase were detected using the appropriate Abs. (B) ERK phosphorylates the ERK NTS in vitro. The GST constructs ERK2-WT, ERK2-TEY-AAA, and ERK-APA (SPS-APA) were subjected to in vitro phosphorylation with either GST-ERK2 (ERK), active GST-ERK2 (Act-ERK), or kinase-dead GST-ERK2 (KA-ERK). The phosphorylation was detected by autoradiography. The amount of ERK was detected using anti-gERK Ab. The intensity of the phosphorylation (percentage of ERK-WT is shown on top of upper blot) was quantified by densitometry. (C) ERK phosphorylates Ser244 in vitro. The indicated GST-ERK2 constructs (0.1 μg per sample) were subjected to in vitro phosphorylation with ERK2. The amounts of phosphorylated constructs were detected by autoradiography, and those of the GST and endogenous kinase were detected by Western blot analysis using anti-gERK Ab. The bar graph represents the incorporation of 32P into GST-ERK as quantified by densitometry (*, P < 0.05). (D) ERK phosphorylates Ser244 in vivo. HEK-293T cells were transfected with GFP-ERK2-SPA, serum starved, and pretreated with dimethyl sulfoxide (DMSO) (control), TBB (10 μM), U0126 (5 μM), or both inhibitors for 2 h. Then the cells were stimulated with TPA (250 nM, 10 min) or left untreated. pSer244 was detected using polyclonal anti-pSPS Ab, and the amount of ERK was detected by anti-GFP Ab. The bar graph represents SPA phosphorylation quantified by densitometry (*, P < 0.05). (E) Phosphorylation of Ser244 in vivo is mediated mostly by CK2. HEK-293T cells were cotransfected with GFP-ERK2-SPE and RFP-MEK1, pretreated with TBB (10 μM) or DMSO control for 2 h, and stimulated with TPA (250 nM, 15 min). pSer244 was detected as described above and quantified by densitometry (bar graph; *, P < 0.05).
The ability of CK2 to phosphorylate both Ser244 and the predicted Ser246 might indicate that this kinase is sufficient to induce full phosphorylation of the NTS. However, since the results described above (Fig. 6) showed that the translocation of ERK2-SPA is only partially inhibited by TBB, we undertook to examine the possibility that this site is phosphorylated by other kinases as well. Since this site lies within an ERK consensus phosphorylation site and ERK is activated upon stimulation, we entertained the possibility that Ser244 is partially phosphorylated by this kinase as well. For this purpose, we first examined the ability of active ERK to phosphorylate the NTS in vitro, using immunoprecipitated WT GFP-ERK2, inactive ERK2 (TEY-AAA), and ERK2 in which the two Ser residues within the NTS were replaced with Ala (SPS-APA). These three constructs were subjected to in vitro phosphorylation by either GST-ERK2, which has very low activity, active GST-ERK2 (Act-ERK), or inactive GST-ERK2 (ATP binding site mutation; KA-ERK). 32P incorporation into GFP-ERK2 was detected mainly when this protein was incubated with Act-ERK2. The phosphorylation of TEY-AAA was reduced by ∼25%, while that of SPS-APA was reduced by ∼80% (Fig. 7B). Next, we analyzed the ability of active ERK2 to phosphorylate GST-ERK2 and its NTS mutants. As expected, active ERK2 phosphorylated both ERK2-SPS and ERK2-SPA (Fig. 7C) but not ERK2-APS or ERK2-APA. These findings suggest that while Ser246 is phosphorylated mainly by CK2, Ser244 phosphorylation may be mediated by active ERK as well.
The ability of ERK to phosphorylate Ser244 in vitro raised the question as whether these kinases can also phosphorylate this site in vivo. To answer this question we transfected the GFP-ERK2-SPA construct into HEK-293T cells, which were then serum starved and pretreated with TBB, U0126, or both. Then, the cells were stimulated with TPA, and Ser244 phosphorylation of the construct was detected using special anti-pSPS Ab that recognizes monophosphorylated as well as dually phosphorylated NTS. This experiment revealed that the phosphorylation of ERK2-SPA was significantly increased upon TPA stimulation (Fig. 7D). TBB had no significant effect on Ser244 phosphorylation, while U0126 or the combination of TBB and U0126 abolished it. Since the ERK2-SPA construct cannot be phosphorylated by CK2, these results indicate that Ser244 is indeed phosphorylated by ERK upon stimulation in the examined cells. To explore the relative roles of CK2 and ERK in the phosphorylation of Ser244, we then transfected the cells with an ERK-SPE mutant that can be phosphorylated by both kinases, pretreated them with TBB, and stimulated the cells with TPA. As expected, NTS phosphorylation of this construct was elevated after stimulation, and this elevation was inhibited by TBB (∼70%) (Fig. 7E). Since TBB inhibited more than 95% of the CK2 activity (data not shown), the small degree of elevation of phosphorylation is likely mediated by active ERK. Indeed U0126 added to the TBB abolished the residual phosphorylation, indicating that Ser244 phosphorylation is mediated mainly by CK2 but also by active ERK.
The induction of CK2 phosphorylation is mediated by the release of hindered NTS from cytoplasmic anchors.Since CK2 is a constitutively active kinase whose activity is not changed upon stimulation, the reason for the significant increase in the CK2-mediated phosphorylation upon stimulation was not clear. One possibility was that the CK2 phosphorylation site is hindered in resting cells and is exposed only after stimulation. Indeed, in resting cells, ERK interacts with various anchoring proteins, and the interaction with many of them is reversed upon stimulation and TEY phosphorylation (47). Therefore, we undertook to study whether MEK1, which serves as one of the cytoplasmic anchors, prevents the phosphorylation of NTS by CK2. First we confirmed that MEK1 and -2 serve as anchor proteins, and we found that knocking down these kinases indeed causes nuclear translocation of about 20 to 25% of the ERK molecules (Fig. 8A). In addition, MEK1/2 knockdown elevated NTS phosphorylation in quiescent but not stimulated cells. These results indicate that endogenous MEK1 and -2 are responsible for the anchoring of a portion of ERK molecules, which upon release from MEK1/2 are phosphorylated on their NTS by CK2. To further establish this finding, we examined the ability of CK2 to phosphorylate GST-ERK2 in the presence of increasing concentrations of His-MEK1. We found that the NTS phosphorylation by CK2 was decreased upon increasing HA-MEK1 concentration (Fig. 8B). This effect was not due to inhibition of CK2 activity, because the latter had no effect on the phosphorylation of casein in a similar experiment. Thus, it is likely that MEK1 hinders the NTS and protects it from CK2 phosphorylation. Finally, we used CRS-AAA, which does not interact with most of the anchoring proteins (34). When this construct was transfected together with MEK1, its basal CK2-dependent NTS phosphorylation was much higher than that of the WT (Fig. 8C), and this phosphorylation was prevented by TBB. These data clearly suggest that the NTS is hindered by anchoring proteins and is released upon stimulation to allow its phosphorylation by CK2.
The induction of CK2 phosphorylation is mediated by the release of NTS from cytoplasmic anchors. (A) Knockdown of MEK1/2 induces partial nuclear translocation and NTS phosphorylation of ERK. HeLa cells were transfected with either siRNA of MEK1/2 or siRNA control and then were either stimulated with TPA (250 nM, 15 min) or left untreated as controls. The cells were next harvested, and their extracts were subjected to Western blot analysis using the indicated Abs. HeLa cells were grown in parallel on coverslips, followed by their transfection with siRNA as described above. The cells were then treated as described above, and the nuclear translocation of their endogenous ERK was analyzed in nonstimulated cells as in Fig. 1A. Scale bar, 10 μm. (B) MEK1 prevents ERK2 phosphorylation by CK2. GST-ERK2-WT (0.1 μg per sample) was mixed in the indicated proportions with 6His-MEK1 and subjected to in vitro CK2 phosphorylation. As a control, dephosphorylated α-casein was mixed with 6His-MEK1 instead of ERK2. GST-ERK2 and α-casein phosphorylation was detected by autoradiography, and the amount of the indicated proteins was detected using the appropriate Abs or by Coomassie staining. (C) ERK anchoring prevents phosphorylation of its NTS by CK2 in vivo. HEK-293T cells were cotransfected with either GFP-ERK2-WT or GFP-ERK-CRS-AAA together with RFP-MEK1. After serum starvation, the cells were pretreated with TBB (10 μM) or dimethyl sulfoxide (DMSO) control for 2 h. Cell extracts were subjected to Western blot analysis with anti-pSPS-ERK and anti-GFP Abs. The bar graph represents NTS phosphorylation measured by densitometry (three experiments). The statistical differences were analyzed by the Student t test (bar graph; *, P < 0.01).
The SPE mutant exhibits an increased electronegative patch on the protein surface.Structural comparison of ERK2-SPE and ERK2-SPS (WT) revealed no significant difference in their interlobe orientations. When we observed the Ser246 area in the WT structure, we found that two negatively charged amino acids, Glu248 and Asp249, were located on the protein surface. In the SPE mutant structure, Glu246 is in close proximity to these amino acids, making this region more electronegative (Fig. 9A and Table 1). Recently, the mechanism enabling substrate recognition in CK2 was discovered (24). The crystal structure of human CK2 (PDB code, 2PVR) was solved, and it exhibited sulfate ions in the substrate-binding pocket mimicking the P + 1 and P + 3 acidic residues. This structure reveled that the first P + 1 sulfate is stabilized by interaction with amino acids Arg191 and Lys198, while the P + 3 sulfate is stabilized by interactions with Arg155, Asn189, and Arg80. The conserved catalytic Asp156 (CK2 numbering) was positioned 11.3 Å from the P + 3 sulfate (24). Observation of the NTS studied here indicates that the P + 1 site is occupied by a polar residue, Gln247, followed by two acidic amino acids, Glu248 and Asp249, which we showed to be important for the phosphorylation by CK2. The distance between the two sulfate ions in the CK2 structure was 10.7 Å, while in the WT ERK2 structure, the distance between Gln247 and Asp249 (P + 1 and P + 3 site) was 11.3 Å and the distance between Glu248 and Asp249 (the two acidic residues) was 4.72 Å; neither of these last two distances is suitable for the substrate binding pocket. However, the distance between Gln247 and Glu248, 10.7 Å, was exactly right. We generated an initial molecular model while pair fitting Gln247 and Glu248 from WT ERK2 to the P + 1 and P + 3 sulfate ions, respectively, from the CK2 structure (Fig. 9B and Table 1). Gln247 and Glu248 were stabilized by the same interaction described for the sulfate ions in the CK2 structure. Ser246 is in close proximity to the catalytic Asp156 (a distance of 6.11 Å), indicating Gln247 and Glu248 to be the important residues for proper interaction between ERK2 and CK2.
Structural analysis of SPE-ERK2. (A) Electrostatic surface presentation of WT ERK2 and SPE-ERK2 of the C′ lobe. The SPS region (circled) displays increased electronegativity due to the S246E mutation, which could have an effect on Imp7 recognition. (B) ERK2-CK2 binding model. (I) The available crystal structure of human CK2α (PDB code, 2PVR) (blue) with two sulfate ions in the P + 1 site (green) and P + 3 site (red) are located 10.7 Å apart. Amino acids important for the substrate recognition pocket are shown and labeled. (II) The MAP kinase insert of the SPS region of ERK2 and the side chains of Ser246, Gln247, Glu248, and Asp249 are shown in orange. The distance between the side chains of Gln247 and Glu248 is measured to be 10.7 Å, similar to that of sulfate ions in the CK2 active site. (III) The structures of ERK2 and CK2 were pair fitted on Gln247 and Glu248 (ERK2) to the P + 1 and P + 3 sulfate ions from CK2, which exhibit an almost perfect fit, introducing Ser246 into the active site (note that no calculation was conducted). In this regard, Ser246 Oγ is located 6.11 Å from Asp156 Oδ1, yet with minimal conformational change in the side chain conformation, a distance acceptable for polar interaction could be reached.
Recently, we have elucidated a novel mechanism of stimulated nuclear translocation, which involves NTS phosphorylation and binding to Imp7 (6). To date, this NTS has been identified in ERK, MEK1, SMAD3, and the Drosophila protein Drosha (43), but it is likely to mediate the nuclear translocation of other signaling proteins as well (53). Moreover, the role of Imp7 in the translocation of signaling molecules was shown in stimulated SMAD (49) and JUN (46). In the current study, we extended our knowledge of the NTS by showing that its phosphorylation is mediated by CK2 and to some extent by transautophosphorylation. We also found that in resting cells, this phosphorylation is prevented due to sequestering interactions with cytoplasmic anchoring proteins, which are released upon stimulation. Our results best fit a model in which ERK is localized in the cytoplasm of resting cells due to interaction with various proteins (7). These proteins anchor ERK by interaction with a region that contains the NTS of ERK, which is therefore hindered. Upon stimulation, the regulatory Thr and Tyr within the activation loop of ERK are phosphorylated, which induces their release from the anchoring proteins (47). Consequently, the NTS of ERK is exposed (Fig. 8) and can undergo phosphorylation on its two Ser residues (Fig. 2). Ser246 seems to be phosphorylated mainly by CK2, while Ser244 phosphorylation is mediated by both CK2 (∼70%) and activated ERK (∼30%; Fig. 7). Phosphorylation of Ser246 is sufficient to induce full (but slower) nuclear translocation of ERK, while the phosphorylation of Ser244 accelerates it but cannot induce significant translocation by itself (Fig. 5). The phosphorylation then forms an acidic patch (Fig. 9) that induces binding of ERK to Imp7 and consequently allows nuclear shuttling through the nuclear pores.
The determination of the signaling specificity of the ERK cascade has attracted much attention over the past years (39). One of the main parameters that control the ERK selectivity is the subcellular distribution of this kinase as well as its upstream regulators (4). In this sense it has previously been shown that the nuclear activity of ERK is important for the activation of transcription factors necessary for relevant gene expression (29). Other roles of the nuclear activities, such as chromatin remodeling, direct induction, or suppression of transcription and regulation of the cell cycle, have been demonstrated as well. Therefore, nuclear translocation of ERK is essential for the induction of many ERK-dependent processes, and indeed, specific abrogation of ERK nuclear translocation blocks the progression of proliferation and oncogenic transformation (2, 10, 44). It should be noted, however, that the nuclear activity is probably not sufficient to induce all ERK-dependent processes, as their activity in the cytoplasm (5) and mitochondria (12) as well as ERK1c activity in the Golgi apparatus (37, 38) is also necessary to induce proliferation and survival. Therefore, ERK translocation seems to be regulated by a variety of methods, such as cytoplasmic anchoring by interaction with specific proteins (7, 16), changes in NPC's number in different cell types (40), interaction with Imp7 (21), and phosphorylation by CK2 (the study presented here). All of these mechanisms are likely to play important roles in governing the specificity and efficiency of ERK signaling in health and disease.
ERK plays a key role in regulating cell cycle progression upon extracellular stimulation, which enhances proliferation and may lead to oncogenic transformation (31). Since CK2 is the main kinase that phosphorylates the NTS of ERK, it was reasonable to assume that it participates in the regulation of these processes as well. Indeed, it was clearly shown that CK2 participates in the regulation and progression of several stages of the cell cycle (41). This was verified in several systems, including mainly mammalian cells, in which reduction of CK2 expression or activity inhibits G0/G1, G1/S, and G2/M transitions (28). Indeed, addition of CK2 inhibitors or knocking down CK2 in our cellular system attenuated cell proliferation. Although much information on the involvement of CK2 in proliferation is already available, the regulatory mechanisms coordinating its numerous functions are not clear enough. In this sense it is possible that our identification of ERK-CK2 cooperation might be one of the ways by which CK2 exerts its function on cell proliferation. In addition to the role of CK2 in proliferation, CK2 was also shown to participate in the induction of oncogenic transformation and tumor maintenance (8). However, studies in experimental transgenic mice models suggested that CK2 itself is not an oncogene but cooperates with oncogenes as well as protumorigenic and signaling molecules, thus increasing their oncogenic potential (35, 48). Some studies indicate that CK2 may cooperate with the ERK cascade in the promotion of tumorigenicity. For example, it was shown that the CK2α′ catalytic subunit synergized with H-Ras in BALB/c 3T3 fibroblast transformation (26). In addition, CK2 promoted a transformed phenotype and survival through Her-2/neu signaling in NF639 breast cancer cells (33). Although the mechanisms suggested in the above studies did not include ERK, the well-known involvement of the latter in oncogenic transformation may suggest that at least part of the effect might involve the CK2-regulated nuclear accumulation of ERK described here.
The involvement of CK2 in the phosphorylation of both Ser residues in the NTS has another implication in our understanding of both passive and active translocation of ERK (51). Since CK2 is a constitutively active kinase, it may phosphorylate the NTS whenever this region is not hindered. Although in resting cells most of the ERK molecules are attracted to anchoring proteins that hinder the SPS, some of them attached through distinct sites (32) that probably do not cover the NTS. These free NTS regions may be phosphorylated by CK2 at any time, and this therefore may explain the relatively high basal NTS phosphorylation in resting cells, which results in a relatively small induction in this phosphorylation. In addition, this phenomenon may explain the free nuclear shuttle of overexpressed proteins that are mostly free of anchoring interaction (34), leaving their NTS accessible for constitutive phosphorylation by CK2. Another interesting issue that resulted from our study is related to the consensus phosphorylation site of CK2. This site was traditionally thought to include an acidic amino acid, 3 residues C-terminal to the phosphorylated Ser/Thr (position +3), while other acidic residues in the vicinity of the Ser/Thr were thought to accelerate the rate of phosphorylation (18, 36). Here we show that aside from the canonical Glu at position +3, a phosphorylated amino acid or acidic residue at position +2 may dictate the phosphorylation by CK2 (Ser244). Therefore, these results may expand the knowledge on CK2 phosphorylation in various unknown substrates.
In summary, we found that the ERK NTS is phosphorylated by CK2, demonstrating for the first time the cross talk between them. Unexpectedly, CK2 phosphorylates not only Ser246 within its consensus site but, after initial Ser246 phosphorylation, also Ser244. Ser244 is phosphorylated by activated ERK as well. We further found that Ser246 phosphorylation is sufficient to induce slow nuclear translocation, while pSer244 cannot induce this translocation by itself but may accelerate the pSer246 effect. Binding of inactive ERK to anchoring proteins (e.g., MEK1) hinders the NTS, and upon stimulation, the NTS is released to allow their phosphorylation by CK2 and ERK. Finally, we crystallized the phosphomimetic mutants of ERK2 and found that they form a strong electronegative patch in the KID of ERK2, which was shown by mutations to participate in the interaction of ERK with Imp7. Overall, our results provide a new insight into three distinct signaling problems. First, we shed light on the mechanism of ERK translocation into the nucleus. Second, we provide new understanding of CK2 activity by (i) demonstrating cooperation of CK2 with ERK in regulating nuclear activities and (ii) identifying an unexpected phosphorylation site of CK2 (Ser244). Finally, we provide new data regarding the binding of Imp7 to its cargo proteins.
We thank Tamar Hanoch and Eldar Zehorai for their help in performing various experiments, David Litchfield (University of Western Ontario, London, Ontario, Canada) for the CK2 constructs, and the staff of ESRF, Grenoble, France, for their help in maintaining and upgrading the facility.
This work was supported by the ISF research center of excellence number 180/09 awarded to R.S. and O.L. R.S. is an Incumbent of the Yale S. Lewine and Ella Miller Lewine Professorial Chair for Cancer Research.
Returned for modification 19 April 2011.
Accepted manuscript posted online 5 July 2011.
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Q: ZF - dynamically change default controller I need to load different controller as the default one in case request comes from mobile device. I was trying to get front controller instance in plugin and set default there based on device detection but it doesn't work.
Is there any way of dynamic change to FC settings?
EDIT:
plugin is registered and working but rest of the code is not
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Olympic medalist breaks down barriers
Ibtihaj Muhammad is about as American as they come. "I ate Cheerios, my dad was a policeman and my mom was a teacher. I'm the middle of five children. My parents pushed us to play sports and keep active. It was just integrated into our family as African Americans."
Ethics Bowl Team successful in regional competition
This year's Ethics Bowl Team, sponsored by the School of Business & Technology (BAE219), sent seven undergraduates of various majors to the regional competition at Oakland University last weekend. The Heidelberg team fared well, ranking third out of 11 regional schools. This third-place rank earns the 'Berg team an alternate spot at national competition in Atlanta in February.
Competing for Heidelberg were: Zak Hahn, Penelope Christian, Emma Baughman, Katie Bauman, Rebekah Marstaller, Em Swain and Reece Schmidt.
Heidelberg enhances PlusOneAdvantage® MBA program
A deal that's hard to beat just got a little bit sweeter.
Heidelberg University students, regardless of their major, have the opportunity to graduate with two degrees – a bachelor's degree and an MBA – for the price of one through the PlusOneAdvantage® MBA Tuition-Free Scholarship Program. Since the PlusOneAdvantage® was launched in 2015, students entered the MBA program immediately following graduation.
But now, undergrads can take up to a two-year gap between their graduation date and starting the PlusOneAdvantage® program.
37 student-athletes earn Fall Academic All-OAC
On Tuesday morning, the Ohio Athletic Conference announced the Fall, 2019 Academic All-OAC selections. Included on that list were the names of 37 student-athletes from Heidelberg University.
To be eligible for Academic All-OAC status, a student must be of sophomore class standing or higher, maintain varsity status, and have a cumulative GPA of 3.50.
Of the 37 students recognized, 14 belonged to the women's soccer program, seven to volleyball, seven to football, six to men's soccer, two to men's cross country, and one to women's cross country.
'Berg honors all veterans, past and present
Heidelberg saluted the service and sacrifices of all veterans, past and present, during the annual Veterans Day ceremony this morning. The ceremony was organized by Student Senate.
The ceremony began with the singing of the National Anthem by junior music major David Carey. Those in attendance observed a moment of silence for alumnus Jim Roberts, '65, former sheriff, city councilman and United Veterans Council representative, who died in September. Jim had been a mainstay at past Veterans Day ceremonies for years, leading the UVC color guard.


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