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- Firm:Hogan LovellsRemove this filter
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Hogan Lovells co-hosts school event to mark National Voter Registration Day
Hogan Lovells has co-hosted an event with social enterprise Bite the Ballot at the Elizabeth Garret Anderson school in Islington to mark National Voter Registration Day.
Hogan Lovells lawyers achieve victory in Garden City case
Hogan Lovells' pro bono team has achieved a significant victory in a Fair Housing case.
Eduardo Ustaran joins Hogan Lovells' privacy and information management practice
Hogan Lovells has hired partner Eduardo Ustaran to join its global privacy and information management practice.
Law360 Rising Stars list recognises Hogan Lovells partner Mike McGill
Hogan Lovells has announced that Washington DC partner Mike McGill has been named on this year's Law360 Rising Stars list.
Hogan Lovells partner wins public law prize at ILO's Client Choice Awards
Hogan Lovells' Lucas Osorio is the exclusive winner of International Law Office's Client Choice Awards in the public law category for Spain.
Hogan Lovells appoints Gary Veron
Gary Veron has joined Hogan Lovells' government regulatory practice as a partner in the Washington, DC, office.


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Free Application for Federal Student Aid (FAFSA) helps determine eligibility for:
In order to receive aid from year to year, the FAFSA must be renewed annually.
Board of Governor's Fee Waiver (BOGFW):
To apply, submit the FAFSA or the BOGFW Application
Use Federal School Code: 001282
The Federal Student Aid Personal Identification Number (PIN):
To apply for aid for the 2013-2014 Academic Year, you will need the following:
The Cal Grant GPA Verification Form is available on-line. Forms must be submitted to the California Student Aid Commission (CSAC) by March 2, 2012 for the 2012-2013 Academic Year and March 4, 2013 for the 2013-2014 Academic Year.
NOTE: Students who have completed more than 16 transferable units at SJCC are not required to complete the form. SJCC will send the information to CSAC electronically.
Additional assistance
Financial Aid Workshops The Financial Aid Office hosts various workshops to help with applications and other financial aid matters. FAFSA workshops are generally held on Fridays from 10am to 11:30am in the Career/Transfer Center located on the 2nd floor of the Student Center (room SC 216). Times and locations of other workshops vary.
Students interested in:
Please check with the Financial Aid Office for more details.
Live help is available! If you are completing the FAFSA online and need help you can get live online help by clicking Live Help in the upper left hand corner. FAFSA on the Web Customer Service Chat hours: Monday – Friday 5:00 a.m. – 8:59 p.m. and Saturdays 6:00 a.m. to 3:00 p.m.
Phone assistance is available by calling: SJCC Financial Aid Office at (408) 288-3741 during office hours or: Federal Student Aid Information Center at 1-800-4FED-AID (1-800-433-3243)


text:
Style Winners & Losers
Cate Blanchett continues to conquer with her elegant style. See which other stars won and lost on the red carpets this week.Style Winners & Losers
All you need to know about wool
Top 5 best-dressed stars at the Oscars
Oscar fashion hits & misses
Who Wore It Best
Love It or Rate It
How to get bold eyebrows
Are you ignoring your eyebrows? It's time to give your face-framers some attention. -
How to brighten your eyes with coloured liner
Experiment with colour this spring. Line your eyes with electric blue, bold teal, ultra violet and sea green. -
How to get Natalie Portman's natural beauty look
Get inspired by Natalie Portman's simple but totally pretty makeup look.
All you need to know about wool
Long associated with scratchy cardigans and dowdy suiting, wool is making a fashion comeback. -
9 stylish bathing suits perfect for a sunny getaway
A sexy swim suit for everyone! 3 bikinis, 3 tankinis and 3 one-piece suits to choose from. -
5 spring trends you can wear now
Spring is just around the corner, get a head start on your spring wardrobe with these five wearable trends.
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10 affordable red carpet looks worn by celebs
Yes, all of that Gucci Couture and Atelier Versace is fun to fantasize about, but does anyone ever walk down the red carpet in something that you could actually, you know, buy? Once in a while, the answer is actually yes. These stars made fast-fashion finds look like a million bucks. By Ryan Porter


text:
Watch for Sale:
Octo 40mm Auto Black EG00.001.1.2JB
Mail program opens only if defined in browser properties
Why I think this
WATCH is interesting: |
Details about this Watch Price: Is firm, my margins are very
small, no additional discounts sorry. See the
OCTO overview table for the price
please. |
Dimensions (mm):
Case diameter (excl.crown): 40
Lug to Lug: 46
Height: 11.5
Lug width: 20
Bracelet : 20
Weight: 160 gram
Max Wrist Size: 8 inch
Crown type: NOT screw down
Crystal Material: Mineral
Water Resistance: 50 m
Specialties: ETA 2846, 25 jewels, solid link bracelet
On Helen's 13.5 cm or 5.25 inch wrist


text:
Tweet |
list price: | |
savings: | $19.97 |
GamersGate | $19.98 expiredMar 02 |
EXPIRED: | Mar 02 |
price history: |
Get price drop email alerts |
Twenty dollar SimCity shows up on GMG today. No coupon required for the discount. This ties the all time low last seen in the final days of the PC gaming winter sales.
SimCity is down to twenty bucks in GameFly's EA Origin. Unlike most of their deals, this offer is only good for 24 hours, ending tomorrow at 1PM Eastern. Ties its all time low price.
Grab SimCity for only $16 - lowest ever price (even better than Origin's Black Friday and Cyber Monady weekend sale). 24 hour deal only!
this was cancelled by gamestop shortly after ordering with no reason but a canned response.
Just wanted to let you know that I could not find this deal on Amazon, but the page doesn't mention an expiration date. I think you might need to correct this
list price: | |
savings: | $29.99 |
GamersGate | $30.00 expiredMar 02 |
EXPIRED: | Mar 02 |
price history: |
Get price drop email alerts |
list price: | |
savings: | $14.99 |
GamersGate | $15.00 expiredMar 02 |
EXPIRED: | Mar 02 |
price history: |
Get price drop email alerts |
SimCity's Cities of Tomorrow expansion dropped back down to $15 today at GamersGate. That ties its all time low and is a good 50% off the usual $30 list price tag.
Best price ever on SimCity's expansion "Cities of Tomorrow". Origin's 50% coupon code knocks the price down to $15. That beats out the previous best $18 price tag seen during the final stages of the 2013 PC gaming winter sales.
New low on SimCity 2013's latest expansion Cities of Tomorrow. GameFanShop has the price down to $20 instantly, and you can add a Dealzon exclusive coupon for an extra buck off. Previous low was spotted last week at $20, but most retailers are $30.
New low on the November 12th released SimCity: Cities of Tomorrow expansion. At $20 it even beats out the previous best $21.60 Cyber Monday price tag. Buying on the Origin Store, and anywhere else, will run you $30.
Lies or a glitch be damned, but the code doesn't work - copy/paste, type in, no matter, just all the same result.
Hey ChaosCrazed, it's a glitch. I've contacted GameFly support and our contacts with GameFly. This is an unintentional glitch with their checkout process. Check back later today.
OK< thanks!
Hey ChaosCrazed. Just FYI the code works now!
SimCity's first big expansion pack is out today: Cities of Tomorrow. The best deal online right now is at GameFly after a 20% off coupon code. That makes it six bucks less than the $30 competition.
its a crapy game


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Okanogan County Public Health |
Report Violations For Public Health emergencies after hours call 911. To report
contact us at: 509-422-7140 |
Okanogan County Public Health is conducting a Community Health Assessment. WE NEED YOUR OPINION! Please click on the following link to take the short, 11 question survey.
Prescription Drug Disposal Sites AvailableDid you know that Okanogan County has three permanent disposal sites:
Since the County's first Prescription Drug Take Back Day in September 2010, we have disposed of over 300 pounds of prescription drugs, and contributed to a nearly 70% decrease in 10th grade students reporting using prescription drugs to get high! Information on the 2014 Tire Recycling & Disposal Event Mosquito Information Pertussis (Whooping Cough) English Deer Mouse BrochureFDA Reminds Consumers of Serious Risks of Using Decorative Contact LensesNews ReleasesRabies InformationThe Latest news from: Center for Disease Control and PreventionEmergency Response/Preparedness
Okanogan Street Map for directions |
1234 South 2nd Avenue |
For more information contact: Ella Robbins, Email Web Master: Marta Reed
06/08/13 |


text:
From EatingWell: January/February 1998, The EatingWell Diabetes Cookbook (2005)
These brownies are a high-energy treat you can pack in your lunch bag or tuck into your fanny pack when you are out walking. Wrap cooled brownies individually for fast picnic assembly or lunchbox fill-ups.
Makes: 24 brownies
Active Time:
Total Time:
Per brownie: 93 calories; 2 g fat (0 g sat, 0 g mono); 18 mg cholesterol; 15 g carbohydrates; 2 g protein; 1 g fiber; 72 mg sodium; 60 mg potassium.
Carbohydrate Servings: 1
Exchanges: 1 other carbohydrate


text:
- Home
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Search Results for « Speechvibe »
Product Search Results: [0]Categories: [1]Companies: [1]
Speech Recognition
Speech recognition software enables the control of computer system by verbal commands. Many speech recognition titles also support dictation, the conversion of voice to text. Major titles in speech recognition include Dragon NaturallySpeaking, MacSpeech and…
Conceptual Speech Technologies
[1] Software Titles
[0] News Items
Conceptual Speech Technologies has 1 download available on Software.com. Top downloads include SpeechVibe. Exclusive discounts apply, choose a product below.


text:
Entries in 'shift' (16)
A third of all console spending to be digital by 2017 - report
IHS head of games Piers Harding-Rolls has predicted a dramatic swing to digital during the imminent console generation.
OPINION: What's the upside to this strange generation change?
Are publishers like EA, Ubisoft and Activision being smart by releasing their games as cross-generation titles this Christmas?
E3 2012: EA says E3'12 "is the opening act of next-gen cycle shift"
EA Labels president Frank Gibeau has said that E3 2012 is the first chapter in the next-gen story.
OPINION: Time to invest in the next generation
My son, only 17 months old, knows how to turn on my iPhone, he's seen me use it so much.
As triple-A 'stagnates', QA firms turn to downloads
QA and localisation firms are generating more money from social and mobile titles than triple-A boxed games.
EA's Shift set for NFS IP spin-off
EA wants to establish Shift as a new franchise when Shift 2 Unleashed races to UK shelves in March.
Shift 2 gets UK release date
EA has confirmed that racer Shift 2: Unleashed will be released in the UK on March 25th.
Ferrari returns to Need for Speed
EA has announced that seven years on from its last appearance in a Need for Speed title, Ferrari is to make a return to the series in the form of ...
Need for Speed has "turned the corner"
In an exclusive interview with MCV, EA Europe's senior VP Jens Uwe Intat has stated that after a troubled few years the Need for Speed IP is back on track.
Need for Speed tops 100m sales
Popular racing series Need for Speed has now sold over 100m copies worldwide, publisher EA has revealed, elevating it into an elite group of super-franchises.
UK CHARTS: Need for Speed victorious
Reinventing the Need for Speed franchise was always going to be a bit of a gamble for EA, but it seems that the risk has paid off with Xbox 360, ...
NFS Shift UK Championship announced
Ahead of the release of Need for Speed: Shift on September 18th, publisher EA has teamed up with the UK's leading games retailer GAME to run a nationwide competition that ...
EA: Need For Speed back on top
Electronic Arts believes Need For Speed: Shift will see the franchise reclaim its crown as king of the racers, after a disappointing showing in 2008.
Need for Speed: Shift dated
With three Need for Speed titles on the way this year following EA's decision to drastically alter the way it handles its successful racing franchise, the publisher has dated ...


text:
Bear Box at Upper Paradise North (WC04)
Generated on 30 Apr 2013 - see revision history - please send updates to the webmaster
Detail Map of waypoint WC04
in Woods Creek Northern Rae Lakes Loop:
(scroll down for directions, waypoints, and area map)
(lat,lon) = (36.8699000, -118.5171500) in decimal degrees, NAD83. (zone,east,north) = (11S, 364856.0, 4081317.0) in decimal meters, NAD27.
Map exported from TOPO! software, used with permission.
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text: HIV drug resistance in low- and middle-income nations stood at 6.8 percent in 2010, the World Health Organization said Wednesday. WHO released its first-ever report on the issue ahead of next week's 19th International AIDS Conference. According to WHO AIDS chief Gottfried Hirnschall, "That is a level we sort of expected. It is not dramatic but we clearly need to look very carefully on how this would evolve further." WHO did not recommend a change in treatment guidelines based on the study. Drug resistance can occur when HIV mutates naturally, when treatment is interrupted, or when patients take medications incorrectly or irregularly. Approximately 8 million people in low- and middle-income countries received antiretroviral drugs last year, up 20 percent from 2010, according to a separate UNAIDS report released Wednesday. High-income countries have higher rates of resistance, from 8 percent to 14 percent. Many of these nations launched widescale treatment programs years ago, often using single- or dual-drug therapies, which can encourage resistance. However, these higher rates have largely leveled off or decreased over time. In 12 of the low- and middle-income countries in the WHO study, health care facilities lost count of up to 38 percent of people who began treatment. When people interrupt or stop treatment, "this not only means that they are themselves more likely to become sick, it also increases the likelihood that drug resistance will emerge and the resistant virus could be transmitted to others," the report said. WHO called for clinics to monitor for early warning indicators of resistance, including poor treatment adherence, supply breaks, and signs of treatment failure, such as rising viral levels in the blood. Hirnschall added, "Simpler regimens using fixed-dose combinations have made it much easier for people to adhere to antiretroviral treatment, limiting the spread of drug resistance in recent years."www.aegis.org


text:
that would be flippen sweet
that would be flippen sweet
Your 14 year old disney expert!
If I go crazy, will you still call me superman?
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Copyright © 2013 The Authors All rights reserved.
Cell Reports, Volume 3, Issue 1, 103-115, 27 December 2012
S6K1 Alternative Splicing Modulates Its Oncogenic Activity and Regulates mTORC1
Vered Ben-Hur1, Polina Denichenko1, Zahava Siegfried1, Avi Maimon1, Adrian Krainer2, Ben Davidson3 and Rotem Karni1, ,
1 Department of Biochemistry and Molecular Biology, the Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel
2 Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA
3 Department of Pathology, Oslo University Hospital, Norwegian Radium Hospital, 0424 Oslo, Norway
Graphical Abstract
► The long S6K1 isoform functions as a tumor suppressor ► S6K1 short isoforms are upregulated in breast cancer and possess oncogenic properties ► S6K1 short isoforms bind mTORC1 and enhance cap-dependent translation ► Inhibition of mTORC1 can reverse the oncogenic properties of the S6K1 short isoforms
- Ribosomal S6 kinase 1 (S6K1) is a major mTOR downstream signaling molecule that regulates cell size and translation efficiency. Here, we report that short isoforms of S6K1 are overproduced in breast cancer cell lines and tumors. Overexpression of S6K1 short isoforms induces transformation of human breast epithelial cells. The long S6K1 variant (Iso-1) induced opposite effects. It inhibits Ras-induced transformation and tumor formation, while its knockdown or knockout induces transformation, suggesting that Iso-1 has a tumor-suppressor activity. Furthermore, we found that S6K1 short isoforms bind and activate mTORC1, elevating 4E-BP1 phosphorylation, cap-dependent translation, and Mcl-1 protein levels. Both a phosphorylation-defective 4E-BP1 mutant and the mTORC1 inhibitor rapamycin partially blocked the oncogenic effects of S6K1 short isoforms, suggesting that these are mediated by mTORC1 and 4E-BP1. Thus, alternative splicing of S6K1 acts as a molecular switch in breast cancer cells, elevating oncogenic isoforms that activate mTORC1.
The PI3K/Akt/mTOR pathway is one of the major signaling pathways hyperactivated in many cancers and leads to uncontrolled proliferation, increased survival, motility, and invasiveness of cancer cells (Mamane et al., 2006; Manning and Cantley, 2007; Yuan and Cantley, 2008). mTOR resides in two distinct complexes: mTOR complex-1 (mTORC1) and complex-2 (mTORC2) (Zoncu et al., 2011). mTORC1 core contains mTOR, Raptor, and G-β-L and is considered to be sensitive to rapamycin. mTORC2 contains Rictor, as the mTOR partner instead of Raptor, and, depending on the cell type, is less sensitive to rapamycin (Sarbassov et al., 2004, Sarbassov et al., 2006). The best-characterized substrates of mTORC1 are S6 Kinase 1(S6K1) and eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1), while Akt is a substrate of mTORC2 (Sarbassov et al., 2006; Shiota et al., 2006). Several components of the mTOR signaling cascade have been identified as oncogenes or tumor suppressors that activate or repress this pathway, respectively (Zoncu et al., 2011; Shaw and Cantley, 2006). Among the two well-characterized mTORC1 substrates, S6K1 and 4E-BP1, the latter has been shown to be important for efficient protein translation, proliferation, and oncogenic transformation (Hsieh et al., 2010; Ohanna et al., 2005; She et al., 2010). S6K1 has been implicated in the regulation of cell size (Ohanna et al., 2005), and its role in transformation is controversial; in some reports, it seems dispensable for transformation (Hsieh et al., 2010), while in others it appears to be required (Alliouachene et al., 2008). A link between S6K1 function and cancer was suggested by the finding that RPS6KB1, the gene encoding for S6K1, resides in the chromosomal region 17q22-17q23, which is often amplified in breast and lung cancers (Bepler and Koehler, 1995; Monni et al., 2001). However, direct evidence that S6K1 expression or activity is sufficient to lead to cellular transformation is lacking. We have previously reported that the splicing factor oncoprotein SRSF1 is upregulated and in some cases amplified in several types of tumors, including breast and lung (Karni et al., 2007). SRSF1 modulates the splicing of RPS6KB1, which encodes S6K1 in both mouse and human (Karni et al., 2007). Furthermore, SRSF1 activates the mTORC1 pathway, which phosphorylates S6K1 downstream of Akt (Karni et al., 2008). The spliced variants of S6K1 yield a long active kinase p85/p70 S6K1 (will be referred to as Iso-1) and shorter splicing variants (will be referred to as Iso-2 in mouse and h6A and h6C in human). We have shown previously that SRSF1 increases the expression of the shorter S6K1 isoform, and that this isoform possesses oncogenic activity and can transform immortal mouse fibroblasts (Karni et al., 2007).
In this study, we examine the oncogenic and signaling activities of S6K1 splicing isoforms and their expression in cancer. Our findings suggest that while Iso-1 is tumor suppressive in vitro and in vivo and can block Ras-induced transformation, the short kinase inactive S6K1 splicing isoforms possess oncogenic properties. We show that the short isoforms of S6K1 bind mTOR and activate mTORC1, leading to increased 4E-BP1 phosphorylation, cap-dependent translation, and upregulation of the antiapoptotic protein Mcl-1. Furthermore, mTORC1 activation is critical for the oncogenic activity of S6K1 short isoforms as the mTORC1 inhibitor rapamycin or expression of a phosphorylation-defective mutant of 4E-BP1 (Hsieh et al., 2010; She et al., 2010) partially inhibit the oncogenic properties of these isoforms. Taken together, our results suggest that S6K1 alternative splicing acts as a switch between a tumor suppressor protein and an oncoprotein, which is deregulated in breast cancer and modulates mTORC1 activity.
S6K1 Short Isoforms Are Upregulated in Breast Cancer Cell Lines and Tumors
The gene RPS6KB1 encoding for p85/p70 S6K1 can be alternatively spliced to form a number of truncated isoforms. In mouse cells, the splicing factor SRSF1 induces the inclusion of three additional exons (a-b-c) located between exon 6 and 7 (Figure 1A). By PCR, cloning, and sequencing, we have discovered that in human there are two alternative exons in this region: a and c, which can be included together or individually generating two protein isoforms that we have termed h6A and h6C (Figures 1A; Figures S1D and S1E; Table S1. List of Primers, Related to Figure 1). All of these isoforms in mouse or human that include combinations of exons 6 (a–c) are termed S6K1 short isoforms. Inclusion of the alternative exons mentioned above results in exposure of alternative polyadenylation sites and alterations in the reading frame that, in turn, generate a stop codon in exon 6c in mouse and exons 6a or 6c in humans. The presence of these stop codons creates transcripts containing approximately half of the original S6K1 coding sequence (Iso-1), and lacking more than half of the conserved kinase domain (Figure 2A).
In all of these alternative splicing events, the presence of a polyadenylation sequence, and in the case of h6A also a premature stop codon (PTC) located less then 55 bp from the next exon junction complex, prevents degradation of the generated transcripts by the Nonsense Mediated Decay (NMD) mechanism (Figures 1A and S1E) (Schoenberg and Maquat, 2012). We found that while in immortal breast cells (MCF-10A, HMLE) the expression of S6K1 short isoforms is relatively low, in breast cancer cell lines inclusion of exons 6a and 6c is significantly increased, especially in metastatic breast carcinoma cell lines (Figures 1B and S1A). Indeed, while in both primary and immortal breast cells S6K1 short protein isoforms were hardly detected at the protein level, in breast cancer cell lines elevated protein levels of S6K1 short isoforms were detected (Figure S1C) (Karni et al., 2007; Rosner and Hengstschläger, 2011). In human breast tumor samples, we found elevated expression of S6K1, h6A, and h6C isoforms compared to the immortal breast cell lines (Figures 1D, S1F, and S1G). Interestingly, whereas most analyzed breast cancer cell lines and tumor samples presented high expression of S6K1 short isoforms, we did not find elevated expression of the full-length isoform Iso-1 in most tumors (Figures 1B–1D). Two of the cell lines that showed elevated Iso-1 expression (MCF-7, BT474) possess amplification of the RPS6KB1 gene, and, except for MCF-7, all tumors and cell lines showed an increase in short isoforms/Iso-1 ratio (Figures S1F and S1G), indicating that an alternative splicing switch in S6K1 occurs in breast cancer.
All S6K1 protein isoforms are identical in their N terminus but share only partial homology in their kinase domain and differ from each other in their C terminus. Iso-1, Iso-2, h6A, and h6C contain distinct sequences in their C terminus consisting of 330, 121, 12, or 24 amino acids, respectively (Figure 2A).
S6K1 Short Isoforms Enhance Motility and Anchorage-Independent Growth
We sought to examine the oncogenic activity of S6K1 isoforms in human breast epithelial cells. We found that Iso-2, h6A, and h6C were able to transform human immortal breast MCF-10A cells and mouse NIH 3T3 cells enabling them to form colonies in soft agar despite the relatively low expression of the short isoforms in comparison to Iso-1 (Figures 2B, 2C, and S2A–S2C). The kinase dead version of Iso-1 (Iso-1 K123 > A) enhanced transformation and increased the ability of MCF-10A and NIH 3T3 cells to form colonies in soft agar as well (Figures 2C and S2C). An in vitro kinase assay using S6 as a substrate shows that the short isoform Iso-2 and the kinase dead K123 > A version of Iso-1 have no kinase activity (Figure S2G). The oncogenic effects of S6K1 short isoforms did not require a functional kinase activity since all short S6K1 isoforms share truncated kinase domains and cannot phosphorylate ribosomal protein S6, or mTOR, both known S6K1 substrates (Figures S2E–S2G and 5A) (Chiang and Abraham, 2005; Holz and Blenis, 2005). In contrast to the expression of S6K1 short isoforms, overexpression of Iso-1, the long active kinase, did not enhance colony formation in soft agar and even reduced the basal level of colony formation (Figure 2C).
One characteristic of cellular transformation is enhancement of cell motility (Hanahan and Weinberg, 2011). MCF-10A cells expressing the shorter isoforms or the kinase dead version of Iso-1 (Iso-1 K123 > A) showed accelerated migration rate compared to cells transduced with S6K1 Iso-1 or the empty vector in a tissue culture wound healing assay (Figures 2D and 2E). In order to exclude the possibility that these effects are the result of changes in proliferation, we measured the proliferation rate of the cells. Cells overexpressing the shorter variants, empty vector, or Iso-1 K123>A did not show enhanced proliferation rate in comparison to Iso-1 (Figure S2D). Altogether, these results suggest that S6K1 short kinase inactive isoforms promote transformation of MCF-10A cells.
Expression of S6K1 Short Isoforms Enables Growth-Factor-Independent Three-Dimensional Acini Formation and Elevates 4E-BP1 Phosphorylation
The immortal breast epithelial cells MCF-10A possess the ability to grow into spheroid structures (acini) when grown on matrigel. To study the effects of S6K1 isoforms on growth of three-dimensional (3D) reconstituted basement membrane cultures, we seeded MCF-10A cells transduced with S6K1 isoforms in the presence or absence of growth factors necessary for acini formation in MCF-10A cells (Arias-Romero et al., 2010). Cells overexpressing S6K1 truncated isoforms, as well as the kinase-dead version of Iso-1, formed large, hyperproliferative 3D structures (Figures 3A–3C) with slight morphological disruption (Figure S3A), even when grown in the presence of only one of the growth factors. This suggests that S6K1 isoforms can replace the strong proliferative signal provided by either epidermal growth factor (EGF) or insulin/insulin growth factor (IGF)-1. This observation is further underscored by the fact that the kinase active isoform of S6K1 (Iso-1) failed to support growth-factor-independent acinus formation and may even inhibit basal proliferation in matrigel (Figures 3A, 3B, and S3A).
Insulin/IGF-1 and EGF activate their corresponding receptors leading to activation of several mitogenic signaling cascades, among them the Ras-Raf-MAPK-ERK and the PI3K-Akt-mTOR pathways (Shaw and Cantley, 2006; Yuan and Cantley, 2008; Mendoza et al., 2011). We hypothesized that S6K1 short isoforms affect acinus proliferation by activating the insulin/IGF-1 and EGF signaling downstream of the receptors, bypassing the need for growth factor activation (Figures 3A and 3B). Thus, we sought to investigate the activities of these signaling pathways. We measured the phosphorylation state of known downstream effectors of these pathways: Akt, ERK, and 4E-BP1 (Shaw and Cantley, 2006; Yuan and Cantley, 2008; Mendoza et al., 2011) in MCF-10A cells overexpressing S6K1 isoforms with and without growth factor activation. We found that overexpression of S6K1 kinase inactive isoforms stimulated the phosphorylation of 4E-BP1, even under serum starvation conditions (Figures 3D, 3E, and S3D). Under these conditions, we observed only a slight and inconsistent activation of Akt or ERK (Figures S3B–S3D). Notably, while overexpression of S6K1 Iso-1 failed to increase 4E-BP1 phosphorylation under serum starvation conditions compared to cells transduced with empty vector (Figures 3D, 3E, and S3D), knockdown of this isoform in MCF-10A cells increased 4E-BP1 phosphorylation (Figures 3F and 3G), supporting the notion that Iso-1 plays an opposite role than S6K1 kinase inactive isoforms. Thus, the increased level of 4E-BP1 phosphorylation we observed, even in serum- and growth-factor-deprived cells, may contribute to the transforming phenotype of cells harboring S6K1 short isoforms (Figures 2C–2E, 3A, 3B, and S2C). Moreover, 4E-BP1 phosphorylation is higher in most breast cancer cells compared to immortal nontransformed cells (Avdulov et al., 2004) in correlation with elevated levels of S6K1 short isoforms (Figures 1B, 1C, S1A, and S3E).
S6K1 Iso-1 Inhibits Ras-Induced Transformation In Vitro and In Vivo
Our results, unexpectedly, indicated that the full-length S6K1 Iso-1 did not support growth-factor-independent acinus formation, as opposed to the short isoforms (Figures 3A–3C). To further study the possibility that S6K1 Iso-1 acts as a tumor suppressor, we silenced its expression in the immortal MCF-10A cell line using Iso-1-specific small hairpin RNA (shRNAs) (Karni et al., 2007) (Figure 3F). We measured reduced phosphorylation of mTOR S2448, a known substrate of p70 S6K1 (Holz and Blenis, 2005; Chiang and Abraham, 2005) in these cells (Figure 3F). Knockdown of S6K1 Iso-1 in MCF-10A cells increased both colony formation in soft agar and acinus number and size in matrigel (Figures 4A and S4C). Similar results were obtained using NCI-H460 lung carcinoma cells (Figures S4A and S4B). Even though overexpression of Iso-1 did not significantly decrease cell motility (Figures 2D and 2E), its knockdown in MCF-10A cells was sufficient to increase motility (Figure S4D).
To examine if Iso-1 can suppress the oncogenic potential of transformed cells, we coexpressed S6K1 Iso-1 with an active RAS mutant (H-RasV12) in MCF-10A cells (Figure S4E). Cells coexpressing the kinase active S6K1 isoform (Iso-1) and oncogenic RAS formed fewer and smaller colonies in soft agar compared to cells coexpressing Ras and empty vector, suggesting that S6K Iso-1 possesses tumor-suppressive activity (Figure 4B). Moreover, cells coexpressing Iso-1/Ras did not gain the spindle/fibroblastic shape that is characteristic of cells transformed with an oncogene such as Ras (Janda et al., 2002) (Figure 4C). The same cell pools were injected subcutaneously into NOD-SCID mice. Supporting the results in vitro, we found that RAS-transformed cells coexpressing Iso-1 did not form tumors in vivo (zero tumors formed of eight injections) as opposed to the empty vector (eight of eight tumors formed) (Figures 4D and S4F). 4E-BP1 phosphorylation levels were lower in cells coexpressing Iso-1 and H-RasV12 than in cells expressing H-RasV12 alone (compare the γ bands in Figure 4E). Moreover, cells coexpressing Iso-1 and RasV12 showed about 20% decrease in Cap-dependent translation in correlation with the decreased phosphorylation of 4E-BP1 (Figure S4G). Taken together, these data suggest that S6K1 Iso-1 is a putative tumor suppressor.
S6K1 Short Isoforms Bind mTORC1 and Enhance Cap-Dependent Translation and Mcl-1 Expression
We hypothesized that S6K1 short isoforms might directly bind and activate mTORC1. To test this possibility, we cotransfected myc-tagged mTOR and T7 tagged Iso-1 or Iso-2 (as a representative of the short isoforms). Immunoprecipitation of transfected S6K1 with anti-T7 antibody revealed that only S6K1 Iso-2 interacts with mTOR (Figures 5A and 5B). The fact that we did not see an interaction between S6K1 Iso-1 and mTOR might be explained by T389 phosphorylation of S6K1 (Figure 5B). This phosphorylation activates S6K1 Iso-1 (Kim et al., 2002), leading to its release from mTORC1 (Holz et al., 2005).
Cells overexpressing S6K1 short inactive isoforms showed elevated levels of cap-dependent translation under serum starvation conditions as measured by a reporter gene measuring Cap- versus IRES-mediated translation from the same transcript (Figures 5C and 5D). Indeed, Iso-1 overexpressing MCF-10A cells exhibited only a slight decrease in 4E-BP1 phosphorylation that was consistent with a slight decrease of cap-dependent translation, compared to cells expressing empty vector (Figures 3D, 3E, and 5D). Moreover, on the background of H-RasV12 transformation, Iso-1 decreased both 4E-BP1 phosphorylation and cap-dependent translation (Figures 4E and S4G), suggesting that Iso-1 tumor-suppressive effect can be clearly detected on the background of a strong oncogene such as mutant Ras but not in a nontransformed cell system such as MCF-10A cells.
We next examined if S6K1 short isoforms alter the expression of proteins known to be controlled by mTORC1-4E-BP1 translational regulation and can contribute to cell transformation. One such protein is Mcl-1 (Hsieh et al., 2010), a key antiapoptotic protein that was shown to be translationaly controlled by mTORC1 (Mills et al., 2008). We found that Mcl-1 protein levels are elevated in MCF-10A cells overexpressing S6K1 kinase inactive isoforms, but not Iso-1 or empty vector (Figure 5E). Interestingly, Mcl-1 was also elevated by Iso-1 knockdown (Figure 5F). S6K family consists of two kinases, S6K1 and S6K2 (RPS6KB1 and RPS6KB2, respectively) (Lee-Fruman et al., 1999), that are known to have redundant activities (Nardella et al., 2011; Pende et al., 2004). We did not detect any changes in S6K2 protein levels in either Iso-1 knockdown or S6K1 kinase inactive isoforms overexpression (Figures S5A and S5B), indicating that the biological effects we observe are due to up or downregulation of S6K1 Iso-1 or the short S6K1 isoforms.
Loss of S6K1/2 Enhances Cap-Dependent Translation, Mcl-1 Expression, and Transformation
In order to establish definitive proof that S6K1 Iso-1 is a tumor suppressor and to rule out any possible compensation or interference by S6K2, we analyzed S6K1 and S6K2 double-knockout (DKO) mouse embryonic fibroblasts (MEFs) (Figure 6A) (Dowling et al., 2010; Pende et al., 2004). Consistent with our previous results, serum-starved S6K DKO MEFs showed elevated phosphorylation levels of 4E-BP1 (Figure 6A). In addition, S6K DKO MEFs formed significantly higher number of colonies in soft agar and also presented 5-fold increase in cap-dependent translation, indicating that these cells are transformed (Figures 6B and 6C). Furthermore, western blot analysis of S6K DKO MEFs revealed high levels of expression of Mcl-1 (Figure 6D) in agreement with what was observed in Iso-1 knockdown in MCF-10A cells (Figure 5F). The fact that the DKO cells show minor/negligible mTOR phosphorylation (Figure 6A) and no S6 phosphorylation (Figure S2F), but were transformed, supports the notion that mTOR S2448 phosphorylation is not essential for its ability to phosphorylate 4E-BP1.
Expression of Iso-1 in S6K DKO MEFs (Figure S2E) partially reduced cap-dependent translation, colony survival, and growth of colonies in soft agar showing that Iso-1 harbors tumor-suppressive ability (Figures 6E–6G). The transformed phenotype of S6K DKO MEFs was not completely reversed by the ectopic expression of S6K1 Iso-1, probably due to the absence of S6K2 in this cell system that might contribute to the tumor-suppressive phenotype of S6K1.
The Oncogenic Activities of S6K1 Short Isoforms Are Mediated by mTORC1 Inactivation of 4E-BP1
S6K1 short isoforms activated mTORC1 as measured by 4E-BP1 phosphorylation (Figures 3D, 3E, and S3D). We next examined, using two complementary strategies, if this activation is required for their oncogenic activities: (1) inhibition of mTORC1 by the pharmacological inhibitor rapamycin, and (2) expression of a phosphorylation-defective 4E-BP1, which is mutated in its phosphorylation sites and cannot dissociate from eIF4E upon mTORC1 activation (Avdulov et al., 2004; She et al., 2010). We transduced MCF-10A cells expressing the mouse S6K1 short isoform (Iso-2) with phosphorylation-defective 4E-BP1 (4E-BP15A or 4E-BP14A), where each of the five (or four) insulin and rapamycin-responsive 4E-BP1 phosphorylation sites have been mutated to alanine (Hsieh et al., 2010).
Coexpression of both Iso-2 and mutant 4E-BP1 decreased colony formation in soft agar 2-fold relative to cells expressing Iso-2 alone (Figures 7A, 7B, S6A, and S6B). Similarly, rapamycin reduced the number of colonies formed in soft agar in cells overexpressing Iso-2 to the background level of cells expressing empty vector (Figure 7B). These results suggest that mTORC1 activation and 4E-BP1 phosphorylation play a major role in the oncogenic capabilities of S6K1 short isoforms.
Expression of mutant 4E-BP1 together with Iso-2 also partially decreased cell motility compared to cells expressing Iso-2 alone (Figures 7C and 7D).
Next, we investigated whether 4E-BP1 phosphorylation is important for Iso-2-mediated growth-factor-independent 3D proliferation in matrigel. Expression of 4E-BP15A strongly inhibited Iso-2's ability to induce growth-factor-independent acinus formation in matrigel (Figure 7E). Taken together, our results suggest that the mechanism of action of S6K1 short isoforms is mediated mostly by 4E-BP1 phosphorylation through mTORC1 activation.
We have previously shown that mouse S6K1 is alternatively spliced to form a short isoform (Iso-2), which is essential for the oncogenic activity of the splicing factor oncoprotein SRSF1(Karni et al., 2007). Here, we report that in human cells S6K1 has two alternatively spliced short isoforms that are overproduced in breast cancer cell lines and tumors. Furthermore, all of S6K1 short splicing variants lack an autoinhibitory C terminus domain (Ali and Sabatini, 2005) and half of the kinase domain and do not exhibit kinase activity, at least on the known S6K1 substrate rpS6 (Figures 2A and S2E–S2G). Overexpression of mouse or human S6K1 short isoforms enhanced transformation, anchorage-independent growth, cell motility, and growth-factor-independent 3D acinus formation of human breast epithelial cells (Figures 2, 3A–3C, S2C, and S3A). Surprisingly, the long, kinase active S6K1 isoform (Iso-1) inhibited 3D acinus formation and reduced 4E-BP1 phosphorylation, cap-dependent translation, and transformation in vitro and in vivo, demonstrating many properties that characterize a tumor suppressor. Our results suggest that only S6K1 short isoforms, but not Iso-1, interact with and activate mTORC1 leading to elevated 4E-BP1 phosphorylation, enhanced cap-dependent translation, and upregulation of the antiapoptotic protein Mcl-1. Inhibition of mTORC1 or 4E-BP1 phosphorylation can partially reverse the oncogenic activity of S6K1 short isoforms, suggesting that their oncogenic properties are at least in part mediated by this pathway.
A Switch in RPS6KB1 Alternative Splicing Upregulates Oncogenic Isoforms in Breast Cancer
We found that many breast cancer cell lines and tumors switch the splicing of RPS6KB1 to elevate the human short isoforms of S6K1 h6A and h6C (Figures 1B–1D, S1F, and S1G). There are several documented examples of an alternative splicing switch that induces a prooncogenic isoform at the expense of a tumor-suppressive isoform. In many cancers, tumor-suppressive isoforms are downregulated, while prooncogenic ones are upregulated (David and Manley, 2010). We found that while all the short S6K1 isoforms, as well as the kinase-dead form of Iso-1, induced anchorage-independent growth, enhanced motility, and growth-factor-independent 3D acinus formation in matrigel, the active S6K1 isoform (Iso-1) did not (Figures 2, 3, and S2C). Moreover, in most of these assays Iso-1 showed an opposite effect, indicating that these splicing isoforms possess antagonistic activities. Several lines of evidence suggest that Iso-1 acts as a functional tumor suppressor. (1) When coexpressed with an active RasV12 mutant, Iso-1 inhibited Ras-induced transformation in vitro and in vivo (Figure 4), suggesting it possesses an antitumorigenic activity. (2) Iso-1 knockdown in MCF-10A cells induced transformation, colony formation in soft agar, acinus formation, increased motility, and elevated Mcl-1 levels (Figures 4A, 5F, S4C, and S4D). (3) Loss of S6K1 and S6K2 had dramatic effect on cellular transformation, as immortalized MEFs from S6K1/2 DKO mice were transformed, formed large numbers of colonies in soft agar, and showed increased levels of the antiapoptotic protein Mcl-1 (Figure 6), which is translationally regulated by mTORC1(Hsieh et al., 2010). (4) Reintroduction of S6K1 Iso-1 partially inhibited the transformed phenotypes of S6K1/2 DKO cells indicating that Iso-1 possesses tumor-suppressive activities, but might also require the presence of S6K2 in these cells to restore its full capability as an antitumorigenic protein (Figure 6). It has been shown previously that S6K1 is required for insulinoma development in a mouse model where an active Akt mutant (Myr-Akt) was overexpressed in pancreatic beta cells under the insulin promoter (Alliouachene et al., 2008). Similarly, S6K1 deletion on the background of PTEN+/− and low expression of S6K2 in pheochromocytoma cells impaired adrenal tumorigenesis (Nardella et al., 2011). In both of these reports, the cancer was induced by activation of the same pathway (PI3K-Akt) and in a specific tissue. It is conceivable that under other experimental settings the outcome might be different, as already demonstrated for S6K1 deletion in other tissues (Nardella et al., 2011). The lack of an effect on tumor development in the latter cases is possibly due to redundancy between S6K1 and S6K2. Moreover, to the best of our knowledge, no study has directly shown that overexpressed or mutated S6K1 or S6K2 acts as an oncogene. Hence, the present report provides direct evidence for the pro- and antitumorigenic effects of stable knockdown or overexpression of S6K1 long isoform (Iso-1), respectively. It should be noted that the pro-oncogenic effect of Iso-1 knockdown does not involve S6K2, as the latter remained unchanged (Figure S5). Notably, the catalytic activity of S6K1 is essential for its tumor-suppressive activity, as the kinase-dead point mutant completely abrogated this activity. In addition, the fact that Iso-1 inhibited Ras transformation, but only partially inhibited cap-dependent translation raises the possibility that Iso-1 phosphorylates additional substrates other than those in the mTOR pathway, that contribute to its tumor suppressor activity independently of cap-dependent translation. Importantly, S6K1 short isoforms are catalytically inactive and did not induce phosphorylation of the known S6K1 substrates, the ribosomal protein S6 or mTOR itself in an in vitro kinase assay and upon transfection into cells (Figures 5A, 5E, S2E–S2G, and S3C). Altogether, these results suggest that Isoform-1 of S6K1 has tumor-suppressive properties, while the short isoforms are pro-oncogenic. Our results suggest that the gain of h6A and h6C observed in breast cancer cells and tumors is a mechanism to switch off a tumor-suppressive isoform and to turn on an oncogenic one.
RPS6KB1 Splicing Isoforms Modulate the Activity of mTORC1
Surprisingly, we found that most S6K1 short isoforms activated mainly 4E-BP1 phosphorylation without a significant activation of Akt or ERK and in a growth-factor-independent manner (Figures 3 and S3). In accordance with increased 4E-BP1 phosphorylation, cells expressing S6K1 short isoforms showed elevated cap-dependent translation and upregulation of Mcl-1, while Iso-1-expressing cells showed low or basal cap-dependent translation, similar to the control cells expressing empty vector or reduced cap-dependent translation in Ras-transformed cells (Figures 5 and S4G). Moreover, loss of S6K1 and S6K2 enhanced cap-dependent translation in MEF cells that was partially inhibited by S6K1 Iso-1 reintroduction (Figure 6), indicating that S6K1 and S6K2 activities suppress cap-dependent translation and transformation (Figure 6). Although surprising, previous studies support this result: Cells from a knockin mouse of a mutant S6 gene (rpS6P−/−), where all five phosphorylation sites were replaced to alanines, showed a 2-fold increase in global translation as measured by methionine incorporation and a 2-fold increase in proliferation rate as compared to MEFs from WT littermates (Ruvinsky et al., 2009). Combining these two findings, both inactivation of the "kinase" (S6K1/2 DKO) and inhibition of phosphorylation of the substrate (rpS6P−/−) gave similar results of increased translation. This suggests that S6 phosphorylation might inhibit rather than increase translation. Other studies did not find reduced translation upon S6K1/2 loss, even though they did not observe increased translation (Mieulet et al., 2007). These results suggest that the growth-factor-independent growth observed in matrigel (Figure 3) could be explained by the ability of S6K1 short isoforms to activate mTORC1 that, in turn, inactivates 4E-BP1 even under low nutrient conditions. Enhanced cap-dependent translation contributes to cancer development by elevating the translation of several oncogenes and antiapoptotic genes such as Mcl-1, Bcl-X, MDM2, HIF-1α, β-catenin, c-myc, cyclin D1, and others, which are translationally regulated by cap-dependent translation and the mTORC1 pathway (Hsieh et al., 2010; Karni et al., 2002; Karni et al., 2005; Mamane et al., 2004). We found that, at least in the case of the antiapoptotic protein Mcl-1, overexpression of S6K1 short isoforms, Iso-1 knockdown or S6K1/2 knockout enhanced its expression (Figures 5E, 5F, and 6D). To examine if S6K1 short isoforms can interact with mTOR, we coexpressed mTOR with S6K1 Iso-1 or Iso-2 and found that only the short isoform could pull down mTOR, whereas Iso-1 did not, even though its expression was much higher (Figures 5A and 5B).
The Oncogenic Activities of S6K1 Short Isoforms Are Partly Mediated through 4E-BP1 and mTORC1 Activation
We examined the contribution of mTORC1-4E-BP1 axis to S6K1 short-isoform-mediated oncogenesis and found that inhibition of eIF4E/4E-BP1 dissociation greatly inhibited motility, anchorage-independent growth, and growth-factor-independent acinus formation in matrigel (Figures 7 and S6). These results suggest that most of the oncogenic effects of S6K1 short isoforms are mediated by mTORC1 activation. However, we cannot rule out the possibility that S6K1 short isoforms affect other oncogenic signaling pathways as suggested by the fact that expression of a dominant-negative 4E-BP1 did not fully suppress colony formation in soft agar and motility induced by S6K1 short isoforms. These results also suggest that S6K1 is not only a substrate of mTOR, but that it may also modulate the activity of mTOR increasing 4E-BP1 phosphorylation when S6K1 short isoforms are elevated (Figures 3 and S3D). In addition, S6K1 Iso-1 can phosphorylate mTOR at serine 2448 (Figures 3F, 5A, and 6A), although the consequences of this phosphorylation on mTOR activity are not fully understood (Chiang and Abraham, 2005; Holz and Blenis, 2005). S6K1 short isoforms cannot be phosphorylated by mTOR at threonine 389 and by phosphoinositide-dependent kinase 1 (PDK1) at threonine 229, as they lack both phosphorylation sites. However, both Iso-1 and the short isoforms should be able to bind mTORC1, since all of them contain the Raptor binding motif called mTOR-signaling (TOS motif) (Schalm and Blenis, 2002). Thus, while Iso-1 activity responds to mitogenic and nutritional stimuli, as well as energy status, the short isoforms are refractory to any of these signals, yet they can still affect mTORC1. Indeed, our data suggest that these short isoforms transmit through mTORC1 activation, a constitutive mitogenic/metabolic signal, even in the absence of growth factors (Figures 3 and S3). In addition, S6K1 short isoforms cannot induce mTOR S2448 phosphorylation, as they lack a functional kinase domain and are catalytically inactive (Figures 5A and S2E–S2G). Thus, our current hypothesis is that Iso-1 phosphorylates mTOR and inhibits cap-dependent translation and transformation. Iso-1 also phosphorylates other cellular substrates that might lead to suppression of transformation. In contrast, the short isoforms bind to the same or other cellular targets and thereby induce opposite effects. S6K1 short isoforms can bind directly to mTORC1 and enhance its activity possibly by competing with Iso-1 for mTOR binding (Figure 7F). Nevertheless, the mechanism by which S6K1 short isoforms activate mTOR is yet to be determined.
We have discovered alternatively spliced variants of the gene encoding S6K1. Our findings provide additional insight into the oncogenic switch that can be caused by alternative splicing. Furthermore, another mode of mTORC1 activation is identified in this study, which may open a new direction in manipulating mTORC1 activity for cancer therapy. In addition, we show that, the balance between S6K1 short isoforms and Iso-1 is tipped toward those short isoforms in most tumors examined, suggesting that this splicing event can be a useful marker for breast cancer development. Moreover, since S6K1 short isoforms activate mTORC1 and this activation is important for tumorigenesis, we expect that the newly approved active site mTOR inhibitors will act more efficiently on tumors where the S6K1 alternative splicing switch has occurred.
Experimental Procedures
NIH 3T3 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) calf serum (BS), penicillin, and streptomycin. Human breast cells, MCF-10A, were grown in DMEM/F12 supplemented with 5% (v/v) horse serum, 50 ng/ml EGF, 10 μg/ml insulin, 0.5 μg/ml hydrocortisone, 100 ng/ml cholera toxin, penicillin, and streptomycin (Arias-Romero et al., 2010). MCF-7, MDA-MB-231, and HEK293 cells were grown in DMEM, HMLE cells were grown in MEBM/DMEM/F12, and MDA-MB-468 cells were grown in Leibovitz-F12, supplemented with 10% (v/v) fetal bovine serum, penicillin, and streptomycin. SUM159 cells were grown in Ham's F12 with 5% (v/v) calf serum, 5 μg/ml insulin, and 1 μg/ml hydrocortisone. S6K1/2 double-knockout MEFs were generated as described in Pende et al., 2004 and were immortalized by the 3T3 protocol as described in Dowling et al., 2010.
Stable Cell Lines
NIH 3T3 and MCF-10A cells were infected with pBABE-puro retroviral vectors encoding T7-tagged S6K1 isoforms human and mouse cDNAs as described (McCurrach and Lowe, 2001). Infected cells were selected with puromycin (2 μg/ml) or hygromycin (200 μg/ml) for 72–96 hr. In the case of double infection with pWZL-hygro-Ras (McCurrach and Lowe, 2001), cells were treated with hygromycin for 72 hr after selection with puromycin. In the case of infection with MLP-puro-shRNAs vectors, MCF-10A cell transductants were selected with puromycin (2 μg/ml) for 96 hr.
Cells were lysed in Laemmli buffer and analyzed for total protein concentration as described (Golan-Gerstl et al., 2011). Primary antibodies were as follows: Sigma: β-catenin (1:2,000) phospho-ERK (1:10,000 T202/Y204); Santa Cruz: β-actin (1:200), total Akt (1:200 sc1619), GAPDH (1:1,000 sc25778), Myc (1:1,000 sc40); Novagen: T7 tag (1:5,000); BD Trunsduction Laboratories: total S6K1-anti-p70; Cell Signaling Technologies: total ERK1/2 (1:1,000); phospho-4E-BP1 Thr70 (1:1,000 #2855), 4E-BP1 (1:1,000 #9452), phospho-S6K1 Thr389 (1:1,000 #9205), p-S6 ser240/244 (1:1,000 #2215), total S6 (1:1,000 #2217), phospho-Akt ser473 (1:1,000 #9271), RAS (1:1,000 #3339), p-S2448-mTOR (1:1,000 #2972), secondary antibodies: HRP-conjugated goat anti-mouse, anti-rabbit, or anti-goat IgG (H+L) (1:10,000 Jackson Laboratories).
HEK293 cells were transfected with myc-mTOR and pCDNA3-T7-Iso-2/h6A/h6C/pCDNA3 or pCDNA-T7-Iso-1 S6K1 with empty pCDNA. Forty-eight hours after transfection, cells were lysed in CHAPS buffer as described (Sarbassov et al., 2004). After protein quantitation, 1 μg of anti-T7 antibody bound to 40 μl of 50% protein G-Sepharose was incubated with 800 μg of total protein lysate overnight. After washing four times with CHAPS buffer and beads were incubated with 50 μl of 2 × Laemmli buffer and separated by SDS-PAGE.
Colony-Survival Assay
S6K1/2 DKO MEF cell populations were transduced with retroviruses encoding for empty pB(−) vector or T7-Iso-1 S6K1. After selection, cells were seeded sparsely (200 cells/well of six-well plates) and were grown for 14 days. Colonies were fixed and stained with methylene blue as described (Karni et al., 2007).
Dual Luciferase Reporter Assay
MCF-10A cells were cotransfected using Fugene 6 (Roche) with the dual reporter vector pLPL (Cap-Renilla-IRES-Luciferase) (Gerlitz et al., 2002) and the indicated S6K1 isoforms. Cap-dependent translation (Renilla luciferase activity) and IRES-mediated translation (Firefly luciferase activity) were measured with the Promega Stop and Glow assay kit according to the manufacturer's instructions.
EGF and IGF-1 Activation
MCF-10A cells (3 × 105) were seeded in six-well plates. Twenty-four hours later, cells where washed with PBS, and the medium was replaced with a serum and growth factor-free media for 24 hr. Cells were stimulated with 50 ng/ml IGF-1 or 50 ng/ml EGF for 4 hr. After 4 hr, cells were lysed in Laemmli buffer for western blot analysis.
Anchorage-Independent Growth
Colony formation in soft agar was assayed as described (Karni et al., 2007; McCurrach and Lowe, 2001). After 14–21 days, colonies from ten different fields in each of two wells were counted for each transductant pool, and the average number of colonies per well was calculated. The colonies were stained as described (Karni et al., 2007; McCurrach and Lowe, 2001).
Growth Curves
MCF-10A cells were infected with the indicated retroviruses. Following selection, 2,000 or 3,500 cells per well were seeded in 96-well plates and grown in DMEM/F12 media with 5% or 0.2% horse serum. Cells were fixed and stained with methylene blue as described (Karni et al., 1999), and the absorbance at λ = 650 nm of the acid-extracted stain was measured on a plate reader (Bio-Rad).
3D Morphogenesis of Mammary Epithelial Cell Assay
MCF-10A cells were infected with the indicated retroviruses. Following selection, 1,500 cells per well were seeded in 96-well plates in growth-factor- reduced matrigel (BD Bioscience, Cat# 25300), as described in Zhan et al., 2008. The method is also described in Debnath et al., 2003. In some cases, 2.5 or 5 ng/ml of EGF or 10 μg/ml of insulin was added to assay medium of seeded cells.
Wound-Healing Assay
MCF-10A cells were seeded in a six-well plate until formation of a confluent monolayer, and a "wound" was created by scratching the monolayer with a p200 pipette tip as described in Liang et al., 2007. Cells were washed with PBS and growth medium was replaced to DMEM/F12 media with 5% horse serum without EGF and insulin. The wound was photographed (×100 magnification) after matching the reference point in a phase contrast microscope and wound area was measured using Photoshop record measurement analysis tools.
Xenograft Tumor Formation in Mice
Pools of Ras-MCF-10A cells expressing the indicated S6K1 isoforms were injected subcutaneously into the rear flanks of NOD-SCID mice (2 × 106 cells per site in 100 μl of serum-free media containing 0.25 v/v matrigel (BD Bioscience) using a 26-gauge needle. Tumor growth was monitored as described (Karni et al., 2007). Veterinary care was provided to all animals by the Hebrew University animal care facility staff in accordance with AAALAC standard procedures and as approved by the Hebrew University Ethics committee (no. MD-08-11517-4).
Statistical Analysis
All data presented as histograms refer to a mean value ± SEM of the total number of independent experiments. An unpaired, two-tailed t test was used to determine p values for Figures 2C and 5B.
In Vitro Kinase Assay
HEK293 were seeded on 10 cm plates (3X106) and 24 hours later were transfected with 20μg of empty pCDNA3, 20μg pCDNA3-T7-Iso-2, 3μg pCDNA-T7-Iso-1 (and 17μg of empty pCDNA3) or 4μg pCDNA-T7-Iso-1 K123A S6K1 (and 16μg of empty pCDNA3) (different isoform amounts were transfected due to different expression levels). Forty-eight hours after transfection, cells were lysed in CHAPS buffer as described (Sarbassov et al., 2004). After protein quantitation, 1 μg of anti-T7 antibody bound to 40 μl of 50% protein G-sepharose was incubated with 800 μg of total protein lysate over night. Beads were then washed 3 times with CHAPS buffer and once with DB buffer (12.5 mM HEPES pH 7.5, 100 mM KCL, 0.5 mM DTT, 6.25% Glycerol). The supernatent was removed and beads were resuspended with reaction mix containing recombinant 200 ng GST-S6 (AbnovaCat.no. H00006194-P01) in 50 μl reaction buffer containing: (20 μM ATP, 30 mM MgCl, 10 mM HEPES pH 7.5, 50 mM EGTA, 10 mM β-glycerophosphate, 5 mM NaVO4, 50 mM β-mercaptoethanol and 0.5 mM DTT). Reactions were shaken for 1 hour at 300C. Reactions were stopped by the addition of 50 μl of cold DB (12.5 mM HEPES pH 7.5, 100 mM KCL, 0.5 mM DTT, 6.25% Glycerol) followed by the addition of 30 μl 4X Laemmli buffer. 50 μl of the final volume was separated by SDS-PAGE, transfered to nitrocellulose by Western blotting and the membrane was probed with the indicated antibodies to view the phosphorylation levels and total levels of the recombinant protein GST-S6 as well as the levels of S6K1 isoforms in the reaction.
Total RNA was extracted from cells at 80% confluency with Trizol reagent (Sigma) and 2 μg of total RNA was reverse transcribed using the AffinityScriptII (Stratagene). RT-PCR was performed on 1/10 (2 μl) of the cDNA, in 50-μl reactions containing 0.2 mM dNTP mix, 10× PCR buffer with 15 mM MgCl2 (ABI), 2.5 units of TaqGold (ABI) and 0.2 mM of each primer; 5% (v/v) DMSO was included in some reactions. PCR conditions were 95°C for 5 min, then 33 cycles of 94°C for 30 s, 60°C for 30 s and 72°C for 45 sec, followed by 10 min at 72°C. PCR products were separated on 1.5% agarose gel. Primer list is included in Table S1. List of Primers, Related to Figure 1.
Quantitative RT-PCR
Total RNA was extracted with Tri reagent (Sigma) and 2 μg of total RNA was reverse transcribed using the AffinityScript (Stratagene) reverse transcriptase. We determined the mRNA levels of S6K1 Iso-1, and Iso-2 in breast cell lines or tumors by performing quantitative PCR with SYBER Green (SYBR Premix Ex Taq # RR041A) using the CFX96 (Bio-Rad) machine. Unknown samples were compared to a standard curve, which is established by serial dilutions of a known concentration of cDNA. The standard that was used is β-actin. Ct- Threshold cycle from β-actin and unknown samples were inserted into the standard curve formula and the final value was the ratio between the unknown sample divided by the β-actin standard gene. Primers are listed in Table S1. List of Primers, Related to Figure 1. The PCR reaction is composed of the following steps: 1 cycle at 95°C for 10 seconds; 40 cycles of 95°C for 5 seconds and 48°C for 20 seconds.
The authors wish to thank Nahum Sonenberg and Jerry Pelletier (McGill University) for 4E-BP1 mutant constructs; Orna Elroy-Stein for the bi-cistronic Cap-Renilla-IRES-firefly luciferase reporter gene construct; Mario Pende (INSERM, Paris) for the S6K1/2 DKO MEF's; Oded Meyuhas and Ittai Ben Porath for comments on the manuscript; members of the Ben Porath lab for technical help, and members of the Karni lab for discussions. This study was supported by the US-Israel Bi-national Science Foundation (BSF) (grant no. 2009026 to R.K. and A.K.) and Israel Science Foundation (ISF) (grant no. 780/08 to R.K.). V.B.-H. and R.K. designed the experiments, V.B.-H., P.D., Z.S., and A.M. performed experiments, B.D. provided breast tumor samples, A.K. provided reagents and comments on the manuscript, and V.B.-H. and R.K. wrote the manuscript.
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Publication Information
Received: April 2, 2012
Revised: October 9, 2012
Accepted: November 27, 2012
Published: December 27, 2012


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How do you change the default window manager on Red
Hat? Let me clarify. I log in with text, and when I
type startx, it starts GNOME, but I'd like to use KDE.
You can change the content of /etc/sysconfig/desktop
from DESKTOP="GNOME" to
DESKTOP="KDE" or
DESKTOP="WINDOWMAKER", and X will start the
corresponding window manager. The file that controls
which window manager it starts is /etc/X11/xinit; take
a look at it.
Felipe Barousse Boué
Hardware Specs for Laptop?
I am attempting to load Red Hat 7.3 on a Sony VAIO notebook,
model no. PCG-GRT 250P. I need to know what horizontal and
vertical sync values must be entered for the LCD screen.
Noel Haynes
From what I have found, your VAIO
should have a horizontal range of 27–115 and a vertical range of
50–160. My last suggestion would be
to install, say, Fedora Core 1 instead of Red Hat 7.3,
which has newer X versions.
I do not own a VAIO, but in looking around I
found this page that may be a good reference for
you: www.prongs.org/linux/sony/sony.html.
The page www.linux-on-laptops.com links to
configurations for many laptop models.
Felipe Barousse Boué
Displaying an Image in an X Application
I'm working on digital image processing on Linux. I have read
a .bmp image successfully. Now I have to display the image using X11. I have
used XCreateImage() and XPutImage(), but the figure is not being
displayed. I feel the problem is with the arguments I pass to these
functions. How do I do it?
Try using the sample code from this X tutorial:
Christopher Wingert
Encrypted Cross-Platform Remote Desktop?
Does Linux come packaged with something similar to Windows 2000 Advanced
Server's Terminal Services? I have a Windows XP workstation that needs
to be configured to have remote access to a Linux server's applications.
The connection would be over the Internet—a VPN, perhaps?
Yes, there is a cross-platform remote desktop
solution. VNC for Linux and Microsoft Windows is
available under the GNU GPL from RealVNC (realvnc.com), and
there may also be VNC packages available for your
Linux distribution. If your distribution supplies
a VNC package, you should probably use it so that
the software will be easier to update if necessary.
See the RealVNC FAQ for how to encrypt a VNC
session using SSH.
Don Marti
Mysterious Freezing Hardware
I recently purchased a new computer for the boss,
an AMD 2500+ with a Biostar M7NCG motherboard,
1.5GB of RAM and two IDE hard drives. After a
couple of months, it began freezing once or twice
a day. No single application is to be blamed. I've
tried replacing hardware and even went so far as to
put the hard drives in a totally different computer,
which did not resolve the issue. What are the best
ways of troubleshooting such a problem?
Leroy Powell
First, this looks like a hardware problem, not necessarily Linux-related
though. Without specifics, it is hard to guess what is going on. Let's
suppose you have Linux in it, then when you say "freezing", is it the
whole machine that freezes or only the graphical user interface? Those
are quite different situations. I'd first look at the system logs. If
it is the whole machine locking, probably switch memory banks,
take some memory out and try the system. If the GUI is what freezes,
I would inspect the video card. Also, check for the latest drivers, or
a newer Linux distribution may solve your problem.
Felipe Barousse Boué
I hate to say it, but in a business, a PC that's
out of warranty and has an intermittent problem is
worth less than the time required to diagnose it.
Assuming your boss pays you a fair wage, he's better
off buying a new box with a longer warranty. Put this
time-sucker out on the sidewalk.
Don Marti
Binding a Key to Send a Signal
What should I do to generate a SIGHUP signal when I
press Ctrl-K?
Narayanan Iyer
You can use the bash bind command to assign certain
behavior to key sequences. The .inputrc file is
related as well. Do a help bind from within bash
or see the "shell builtin commands" section of the
bash man page for further information. Take a look at
for more information, or go to
for a hot key creation tutorial.
Felipe Barousse Boué
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text:
Common words appear frequently in written and spoken language across many genres from radio to academic journals.
Informal words should be reserved for casual, colloquial communication.
anticipated return of jesus
Synonyms for Second Coming
anticipated return of jesus
Roget's 21st Century Thesaurus, Third Edition Copyright © 2013 by the Philip Lief Group.
Cite This Source


text:
An analyst or academic who believes that China poses no military threat, particularly to the United States (usu. derogatory).
Example Citation:
"The very name of Bill Gertz's new book ['The China Threat'] is an affront to the panda-huggers in our nation's capital. They deny the existence of a threat from China, suggesting that this view is a mindless holdover from the reflexive paranoia of the Cold War."
Steven W. Mosher, "The new cold war," The Washington Times, January 2, 2001
Steven W. Mosher, "The new cold war," The Washington Times, January 2, 2001
Earliest Citation:
"Fisher...describes himself as a member of the 'Blue Team' a loose alliance of members of Congress, congressional staff, think tank fellows, Republican political operatives, conservative journalists, lobbyists for Taiwan, former intelligence officers and a handful of academics, all united in the view that a rising China poses great risks to America's vital interests. ... Blue Team allies also speak derisively of 'panda-huggers' and 'the Relationship Police,' referring to those who seek a close and cooperative U.S. relationship with Beijing."
Robert G. Kaiser, Steven Mufson, "'Blue Team' Draws a Hard Line on Beijing," The Washington Post, February 22, 2000
Robert G. Kaiser, Steven Mufson, "'Blue Team' Draws a Hard Line on Beijing," The Washington Post, February 22, 2000
Today's phrase is based on tree-hugger, "a person who cares deeply about trees in particular, or the environment in general." The idea behind this usually derogatory term is that such a person would cling to a tree to prevent its destruction. In fact, this is exactly how the term originated. Back in 1973, villagers from the state of Uttar Pradesh in India protested the destruction of Himalayan forests by embracing trees to prevent them from being felled. Inspired by this, tree-hugger entered the language around 1977.
Panda-hugger is much newer, with the earliest citation I could find dating only to February, 2000.
Related Words:
chicken hawk
do tank
slide-rule general
chicken hawk
do tank
slide-rule general


text:
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