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Chk1 is an evolutionarily conserved protein kinase that regulates cell cycle progression in response to checkpoint activation. In this study, we demonstrated that agents that block DNA replication or cause certain forms of DNA damage induce the phosphorylation of human Chk1. The phosphorylated form of Chk1 possessed higher intrinsic protein kinase activity and eluted more quickly on gel filtration columns. Serines 317 and 345 were identified as sites of phosphorylation in vivo, and ATR (the ATM- and Rad3-related protein kinase) phosphorylated both of these sites in vitro. Furthermore, phosphorylation of Chk1 on serines 317 and 345 in vivo was ATR dependent. Mutants of Chk1 containing alanine in place of serines 317 and 345 were poorly activated in response to replication blocks or genotoxic stress in vivo, were poorly phosphorylated by ATR in vitro, and were not found in faster-eluting fractions by gel filtration. These findings demonstrate that the activation of Chk1 in response to replication blocks and certain forms of genotoxic stress involves phosphorylation of serines 317 and 345. In addition, this study implicates ATR as a direct upstream activator of Chk1 in human cells.
Checkpoints are signaling pathways that monitor the integrity and replication status of the genetic material before cells commit to either replicate (in S phase) or segregate (in mitosis) their DNA (27). Upon activation, checkpoints interface with cyclin-Cdk complexes to block cell cycle progression or alternatively to induce cell death. The DNA replication checkpoint monitors S-phase completion and prevents mitosis in its absence. The DNA damage checkpoint monitors the integrity of the genome and arrests the cell cycle either in G1before DNA replication (termed the G1 DNA damage checkpoint), in S phase (the S-phase DNA damage checkpoint), or in G2 before mitosis (the G2DNA damage checkpoint). Eukaryotic cells activate an evolutionarily conserved set of checkpoint proteins that rapidly induce cell cycle arrest to prevent replication or segregation of damaged DNA before repair is completed.
A key component of the DNA damage checkpoint is ATM (ataxia telangiectasia-mutated), a 370-kDa protein kinase (58). The ATM gene is mutated in the human genetic disorder ataxia telangiectasia (58). Cell lines derived from patients lacking ATM are radiosensitive and exhibit defects in checkpoint responses to ionizing radiation (IR), including p53-dependent G1 cell cycle arrest and p53-independent S and G2 cell cycle arrests (31). The kinase activity of ATM is activated in response to double-stranded DNA breaks, and ATM targets several effectors of checkpoint control, including Cds1 (also known as Chk2), Brca1, p95 (nbs1), p53, and Mdm2 (2, 5, 8-10, 15, 23, 33, 34, 41, 42, 47, 69, 74). Checkpoint responses to UV light and base-damaging agents are normal in cells lacking ATM. Taken together, these findings point to a seminal role for ATM in the IR-induced DNA damage checkpoint.
ATR (the ATM- and Rad3-related protein kinase) also contributes to checkpoint control in eukaryotes (13, 32). UnlikeATM, deletion of ATR in mice results in an embryonic lethal phenotype indicating that ATR is an essential gene (6, 17). Another feature that distinguishes these two kinases is their sensitivity to different types of checkpoint signals. As mentioned above, cells lacking ATM are hypersensitive to IR, but not to UV or hydroxyurea (HU), whereas cells overexpressing a kinase-inactive form of ATR are sensitive to UV and HU, as well as to IR. This suggests that ATR plays a more prominent role than ATM during the cellular response to unreplicated DNA (induced by agents such as HU) and to certain DNA-damaging agents, including UV light (14, 68). However, overexpression of ATR complements the radioresistant DNA synthesis defect of cells lacking ATM, demonstrating that these two kinases have overlapping functions in vivo. In support of this, ATM and ATR have been shown to have similar kinase specificities (35, 52). Both prefer phosphorylating serine or threonine residues that are followed by glutamine (SQ/TQ motifs), and as such, ATM and ATR have overlapping substrate specificity in vivo. Examples of substrates shared by ATM and ATR include p53 and Brca1 (40, 62, 63).
Another potential subsrate of ATR is the human Chk1 protein kinase. Chk1 was first identified in fission yeast as an essential component of the DNA damage checkpoint (1, 66). An additional role for Chk1 in the DNA replication checkpoint was revealed when fission yeast cells lacking both Chk1 and a second checkpoint kinase, Cds1, were found to advance into mitosis with unreplicated DNA (4, 43, 72). Homologs of Chk1 have also been found in humans,Drosophila melanogaster,Caenorhabditis elegans, budding yeast, and Xenopus (20, 21, 38, 50, 55, 60). In humans, fission yeast, and Xenopus, Chk1 has been proposed to regulate the G2 checkpoint by phosphorylating the Cdc25 protein phosphatase on a residue or residues that facilitate the binding of 14-3-3 proteins (38, 54, 56, 72, 73).
Like ATR, Chk1 has been shown to be an essential gene in mice (6, 17, 44, 61). These findings unveil essential functions for both ATR and Chk1 in the absence of environmentally imposed genotoxic stress. Embryos and conditional ES cell lines lacking Chk1 also exhibit defective checkpoint responses to replication blocks and DNA-damaging agents, establishing a checkpoint function for mammalian Chk1 in mice (44, 61). Evidence that Chk1 contributes to G2 checkpoint control in human cells comes from studies showing that agents such as UCN-01 and SB-218078, which are potent inhibitors of Chk1, abrogate G2 checkpoint function in human cells (7, 25, 29).
Chk1 is a component of signaling pathways that respond to structures characteristic of DNA damage and/or incomplete DNA replication. In fission yeast, Chk1 responds to DNA damage induced by either IR or UV, and Chk1 also functions in the DNA replication checkpoint (4, 22, 43, 66, 67, 72). Xenopus Chk1 responds to DNA replication blocks and UV damage, but not to DNA with double-strand breaks, which is characteristic of gamma-irradiated DNA (26, 37). The regulation of Chk1 is perhaps best characterized inXenopus, where it has been demonstrated that Chk1 is phosphorylated and activated by ATR and that the ATR-Chk1 pathway responds to unreplicated DNA and UV-damaged DNA (26). In the case of human Chk1, there is controversy concerning not only what types of DNA structures it responds to, but also whether its kinase activity is elevated in response to genotoxic stress. Some studies have reported that the electrophoretic mobility of human Chk1 is retarded after exposure to IR (7, 44, 56), HU (7, 44), and UV (44, 46). Other studies failed to observe changes in the electrophoretic mobility of Chk1 when cells were treated with these agents (30). In addition, two studies have reported slight increases in Chk1 kinase activity in response to UV and BNP1350, a topoisomerase I inhibitor (46, 71), while other studies have reported that the kinase activity of human Chk1 is not altered in response to genotoxic stress (30, 76). Finally, human Chk1 has been shown to be phosphorylated on serine 345 when cells are treated with UV, IR, or HU in an ATR-dependent manner (44). However, it is not known if additional sites become phosphorylated during genotoxic stress, if phosphorylation contributes to Chk1 activity, or if human Chk1 is a direct target of ATR.
In this study, we investigated the regulation of human Chk1 in response to different types of genotoxic stress. We report that human Chk1 is phosphorylated on serines 317 and 345 in response to checkpoint activation. The kinase activity of human Chk1 is activated upon phosphorylation of serines 317 and 345, and the phosphorylated, activated form of Chk1 elutes more quickly on gel filtration columns. Finally, we report that ATR directly phosphorylates human Chk1 on serines 317 and 345 in vitro and that the phosphorylation of these residues is ATR dependent in vivo.
Cell lines293 and HeLa cells were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS), 100 U (each) of penicillin and streptomycin per ml, and 1 mM glutamine (culture medium). AT22IJE T cells expressing ATM (AT+) or not expressing ATM (AT−) (77) were cultured in a mixture of DMEM, 10% FBS, 100 U of penicillin and streptomycin per ml, 1 mM glutamine, and 100 μg of hygromycin per ml. U2OS cells were grown in modified McCoy's 5A medium (Gibco) supplemented with 10% FBS, 100 U of penicillin and streptomycin per ml, and 1 mM glutamine.
Antibodies.Chk1 was detected with a rabbit polyclonal antibody raised against bacterially-produced glutathioneS-transferase (GST)-Chk1 or with commercial monoclonal (SC8408) or polyclonal (SC7898) antibodies purchased from Santa Cruz Biotechnology. Chk1 fusion proteins were precipitated with either anti-c-Myc (9E10)–agarose conjugate (Santa Cruz Biotechnology) or anti-Flag M2 antibody–agarose affinity gel (Sigma Chemical Co.) and detected by Western blotting with c-myc polyclonal antibody (A-14; Santa Cruz Biotechnology). Bound primary antibodies were detected with either horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (ICN/CAPPEL) or HRP–rat anti-rabbit antibody (Zymed), and proteins were visualized by chemiluminescence with the ECL enhanced chemiluminescence reagent (Amersham). Antibodies specific for Chk1 phosphorylated on serine 317 were generated by immunization of rabbits with the phosphopeptide C-VKYSSpSQPEPRT coupled to keyhole limpet hemocyanin.
Generation of recombinant adenoviruses.Wild-type and mutant forms of Chk1 were cloned as Flag-tagged fusions into the adenovirus shuttle vector pAdTrack-CMV (cytomegalovirus) (28). Chk1 (wild type and mutants) cDNAs were amplified with forward primer 3 containing the Flag tag and a KpnI site (5′-ATAGGTACCATGGACTATAAGGACGACGATGATAAGGCAGTGC CCTTTGAGGAAG-3′) and reverse primer 4 containing a stop codon and anXbaI site (5′-ATATCTAGATCATGTGGCAGGAAGCC). PCR products were digested with KpnI and XbaI and cloned into pAdTrack-CMV. The pAdTrack-CMV-based plasmids encoding wild-type and mutant Chk1 proteins were cotransformed with pAdEasy-1 into Escherichia coli BJ5183 to achieve homologous recombination. Recombinant adenoviruses were generated and propagated with the pAdEasy system as described previously (28).
Generation of recombinant baculovirus encoding His-Chk1 and affinity purification of Chk1 antibody.Chk1 was amplified by PCR with pAdTrack-CMV-Flag-Chk1(wild type) as template with forward primer 5′-GGGAATTCGGTGGAGTCATGGCAGTGC CC and reverse primer 5′-GGGGTACCCTGTGGCAGGAAGCC. The PCR product was digested with EcoRI and KpnI and ligated into pFASTBACHTa (Gibco BRL). Recombinant baculovirus encoding His6-Chk1 was generated with the BAC-TO-BAC Baculovirus Expression System (Gibco BRL) and protocols suggested by the manufacturer. His6-Chk1 protein was purified from infected Sf9 insect cells on Ni-nitrilotriacetic acid agarose (Qiagen), and purified His6-Chk1 was eluted in a mixture of 50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 200 mM imidazole, and 1 mM dithiothreitol (DTT). Four milligrams of His6-Chk1 was coupled to 2 g of CNBr-activated Sepharose 4B (Sigma Chemical Co.) according to the manufacturer's recommendations. Antibodies generated against GST-Chk1 were applied to the column, and Chk1-specific antibodies were eluted with 0.1 M glycine (pH 2.8) and immediately neutralized with 1 M Tris (pH 8.0).
Coupling of antibody to immobilized protein A for immunoprecipitations.ImmunoPure immobilized protein A agarose (Pierce) and affinity-purified Chk1 antibodies were incubated for 1 h at 4°C in 1 ml of mammalian cell lysis buffer 1 (MCLB1: 50 mM Tris-HCl [pH 8.0], 2 mM DTT, 5 mM EDTA, 0.5% Nonidet P-40, 100 mM NaCl, 1 μM microcystin, 1 mM sodium orthovanadate, 2 mM phenylmethylsulfonyl fluoride [PMSF], 10 μg of aprotinin per ml, 20 μM leupeptin) at a ratio of 14 μg of affinity-purified antibody to 30 μl of packed beads. The antibody-bead mixture was washed once with 1 ml of MCLB1, twice with 1 ml of LiCl buffer (0.5 M LiCl, 50 mM Tris [pH 8.0]), and twice with 1 ml of MCLB1.
Treatment of cells with DNA-damaging agents and caffeineHeLa cell cultures were incubated in culture medium containing 50 μM cisplatin, 3 μM etoposide, or 3 μM 1-β-d-arabinofuranosylcytosine (ara-C) for 1 h. The medium was removed, and cells were washed once and then incubated for 6 h in culture medium. For UV irradiation, the culture medium was removed, cells were washed twice with phosphate-buffered saline (PBS), and cells were irradiated in uncovered tissue culture dishes with 254-nm UV light at a dose of 10 J/m2 with a Stratalinker (Stratagene). Fresh culture medium was added back, and cells were incubated for an additional 6 h. In some experiments, HeLa cells were incubated in 10 mM caffeine for 2.5 h prior to HU treatment.
PlasmidsChk1 was amplified by PCR with pAdTrack-CMV-Flag-Chk1(+) as the template with forward primer 1 (5′-AGGGAATCCCATGGCAGTGCCCTTTGTGG-3′) and reverse primer 2 (5′-GCTCTAGAGCTCATGTGGCAGGAAGCC-3′). The PCR product was digested with EcoRI and XbaI and ligated into pcDNA3-myc to generate pcDNA3-myc-Chk1. Alanine was substituted for aspartic acid at position 130 to generate kinase-inactive Chk1 (Chk1D130A). Phosphorylation-site mutants were made with the Quick Change mutagenesis kit (Strategene) by using pcDNA3-myc-Chk1 as a template. In each case, serine was changed to alanine. pcDNA3-myc-Chk1 was used as a template with forward primer 5′-GTGAAGTACTCCAGTGCTCAGCCAGAACCCCGC-3′ and reverse primer 5′-GCGGGGTTCTGGCTGAGCACTGGAGTACTTCAC-3′ to generate pcDNA3-myc-Chk1(S317A). pcDNA3-myc-Chk1 was used as a template with forward primer 5′-CAAGGGATCAGCTTTGCTCAGCCCACATGTCC-3′ and reverse primer 5′-GGACATGTGGGCTGAGCAAAGCTGATCCCTTG-3′ to generate pcDNA3-myc-Chk1(S345A). pcDNA3-myc-Chk1 was used as a template with forward primer 5′-CATATGCTTTTGAATGCTCAGTTACTTGGCACCCC-3′ and reverse primer 5′-GGGGTGCCAAGTAACTGAGCATTCAAAAGCATATG-3′ to generate pcDNA3-myc-Chk1(S357A). pcDNA3-myc-Chk1 was used as a template with forward primer 5′-GGCACCCCAGGATCCGCACAGAACCCCTGGCAG-3′ and reverse primer 5′-CTGCCAGGGGTTCTGTGCGGATCCTGGGGTGCC-3′ to generate pcDNA3-myc-Chk1(S366A). pcDNA3-myc-Chk1 was used as a template with forward primer 5′-GCTGATTGATATTGTGAGCGCACAGAAGGTTTGGCTTCCTGCC-3′ and reverse primer 5′-GGCAGGAAGCCAAACCTTCTGTGCGCTCACAATATCAATCAGC-3′ to generate pcDNA3-myc-Chk1(S468A). pcDNA3-myc-Chk1(S317A) was used as a template with primers described above to generate pcDNA3-myc-Chk1(S317A, S345A). pcDNA3-myc-Chk1(S357A) was used as a template with the primers described above to generate pcDNA3-myc-Chk1(S357A, S366A). Chk1 (wild type and D130A) proteins were also amplified by PCR with forward primer 5 (ATACCCGGGAATGGCAG TGCCCTTTGTGG) and reverse primer 6 (ATAGAATTCTCATGTGGCAGGAAGCC). The PCR products were digested with SmaI andEcoRI and ligated into pGEX2TN. pGEX2TN-Chk1 was used as a template to generate pGEX2TN-Chk1(S317A), pGEX2TN-Chk1(S345A), pGEX2TN-Chk1(S357A), pGEX2TN-Chk1(S366A), and pGEX2TN-Chk1(S468A) by using the methodologies and primers described above.
Purification of soluble GST-Chk1 (wild type and mutants) for kinase assays.JM109 cells were transformed with plasmids encoding GST-Chk1 (wild type and mutants). Cultures were grown at 37°C to an optical density at 600 nm (OD600) of 0.6, and isopropyl-1-thio-β-d-galactopyranoside (IPTG) was added to a final concentration of 0.5 mM. After growing for an additional 3 h at 30°C, cells were pelleted by centrifugation. Cell pellets were washed with PBS buffer and resuspended in STE (100 mM NaCl, 10 mM Tris HCl [pH 8.0], 1 mM EDTA) supplemented with 2 mM PMSF, 10 μg of aprotinin per ml, 20 μM leupeptin, and 1.0 mg of lysozyme per ml. After rocking at 4°C for 20 min, Sarkosyl was added to a final concentration of 1.5%, and lysis was accomplished by sonication. Lysates were clarified by centrifugation, and Triton X-100 was added to a final concentration of 2%. Proteins were precipitated with glutathione-agarose (Sigma Chemical Co.) and washed twice in STE, twice in LiCl buffer (0.5 M LiCl, 50 mM Tris HCl [pH 8.0]), and twice in 50 mM Tris HCl (pH 7.4). GST-fusion proteins were eluted with 20 mM glutathione in 50 mM Tris HCl (pH 7.4).
Purification of GST-Cdc25C200–256 GST-Cdc25C200–256 was produced in bacteria and purified as described previously (53). The protein was eluted from GSH agarose in buffer consisting of 50 mM Tris (pH 7.4), 20 mM glutathione, 2 mM PMSF, 10 μg of aprotinin per ml, and 20 μM leupeptin followed by dialysis in 1 liter of dialyis buffer (25 mM Tris [pH 8.0], 5 mM DTT) overnight at 4°C.
Chk1 kinase assaysTo monitor the activity of endogenous Chk1, 3 × 106 HeLa cells were untreated or treated with 3 mM HU for 17 h. Cells were lysed in MCLB2 (50 mM Tris-HCl [pH 8.0], 2 mM DTT, 5 mM EDTA, 0.5% Nonidet P-40, 100 mM NaCl, 1 μM microcystin, 1 mM sodium orthovanadate, 2 mM PMSF,10 μg of aprotinin per ml, 20 μM [5 μg/ml] leupeptin, 10 mM β-glycerophosphate, 1 mM sodium fluoride). Clarified lysates, representing 2 mg of total cellular protein, were incubated with 14 μg of affinity-purified Chk1 antibody coupled to protein A agarose overnight at 4°C. Precipitates were washed twice with MCLB2 and twice with incomplete kinase buffer (50 mM Tris Cl [pH 7.4], 1 mM DTT, 10 mM MgCl2). Fifty-microliter kinase reactions were carried out in the presence of incomplete kinase buffer containing 10 μM ATP, 10 μCi of [γ-32P]ATP, and 5 μg of soluble GST-Cdc25C200–256. Reaction mixtures were incubated at 30°C for 25 min and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and proteins were transferred to nitrocellulose membrane. Radiolabeled proteins were visualized by autoradiography. The same membranes were then subjected to Western blot analysis with anti-Chk1 monoclonal antibody. For ectopically produced Chk1, clarified lysates, representing 2 mg of total cellular protein, or column fractions were incubated with 30 μl of anti-Flag–agarose (Sigma Chemical Co.) for 2 h at 4°C. Precipitates were washed twice with MCLB2, twice with LiCl buffer, and once with incomplete kinase buffer. Kinase assays were performed as described above.
Phosphatase treatment.HeLa cells were lysed in MCLB2 (50 mM Tris-Cl [pH 8.0], 100 mM NaCl, 10 mM MgCl2, 0.5% Nonidet P-40, 2 mM DTT) containing 2 mM PMSF,10 μg of aprotinin per ml, and 5 μg of leupeptin per ml. Clarified lysates containing 150 μg of total cellular protein were incubated with 5 U of calf intestinal phosphatase (NEB) in the absence or presence of 10 mM β-glycerophosphate and 10 mM NaF at 37°C for 1 h.
ATR kinase assays.293 cells (2.8 × 106) were transfected with 30 μg of plasmid encoding kinase-active or -inactive Flag-ATR by using the calcium phosphate transfection system according to the manufacturer's instructions (Life Technologies). Forty-eight hours after transfection, cells were lysed in MCLB3 (50 mM Tris-Cl [pH 8.0], 150 mM NaCl, 0.5% Nonidet P-40, 2 mM DTT, 5 mM EDTA, 10% glycerol) containing 1 μM microcystin, 1 mM sodium orthovanadate, 2 mM PMSF, 10 μg of aprotinin per ml, 5 μg of leupeptin per ml, 10 mM β-glycerophosphate, and 1 mM sodium fluoride. Lysates representing 4 mg of total cell protein were incubated with anti-Flag agarose for 2 h at 4°C. Precipitates were washed twice with MCLB3, once with LiCl buffer, and once with incomplete kinase buffer (20 mM HEPES [pH 7.4], 10 mM MgCl2, 10 mM MnCl2, 50 mM NaCl, 1 mM DTT). Kinase assays (50 μl) were performed in incomplete kinase buffer supplemented with 5 μM ATP, 20 μM β-glycerophosphate, 10 μCi of [γ-32P]ATP, and 1 μg of GST-Chk1 (wild type and mutants) that had been purified from bacteria.
Adenovirus infections and gel filtration.HeLa cells were either mock infected or infected for 60 min with recombinant adenoviruses encoding Flag-tagged versions of Chk1 (wild type and mutants) at a multiplicity of infection (MOI) of 10 in 1 ml of serum-free DMEM. After 1 h, 5 ml of culture medium was added. After 15 h of infection, cells were either untreated or were incubated with 10 mM HU for 4 h. Cells were lysed in MCLB1. Lysates were passed through a 27-gauge needle 10 times and then clarified by centrifugation at 20,000 × g for 10 min followed by 50,000 rpm for 30 min. Clarified lysates containing 2 to 5 mg of total cellular protein were loaded onto a Superdex 200 10/30 column (Pharmacia) and eluted with 50 mM HEPES (pH 7.4), 150 mM NaCl, and 1 mM EDTA. Columns were run at 0.35 ml/min, and 0.5 ml was collected per fraction. For mock-infected cells, 150 μl from fractions 23 to 30 was resolved by SDS-PAGE. Chk1 was visualized by Western blotting with anti-Chk1 polyclonal antibody (SC7898). For adenovirus-infected cells, fractions were first incubated with 30 μl of packed anti-Flag–agarose. Precipitates were resolved by SDS-PAGE, and Flag-Chk1 (wild type and mutants) proteins were visualized by Western blotting with Chk1 polyclonal antibody (SC7898). When kinase assays were performed, precipitates were first incubated in complete kinase buffer containing GST-Cdc25C200–256. Kinase reactions were resolved by SDS-PAGE, and proteins were transferred to nitrocellulose. Radiolabeled proteins were visualized by autoradiography, and then the membrane was subjected to Western blotting with Chk1 polyclonal antibody (SC7898).
In vivo 32P labeling and two-dimensional peptide mapping analysis.293 cells (2 × 106)were either mock transfected or transfected with plasmids encoding myc-tagged wild-type and mutant forms of Chk1 by using the Superfect transfection reagent (Qiagen). After 16 h of incubation, cells were incubated in phosphate-free DMEM containing 2 mCi of32P-labeled inorganic phosphate per ml and 10 mM HU for 4 h. When labeling exogenous Chk1, 1 μM UCN-01 was also included during the labeling period. Cells were lysed in MCLB1. Endogenous Chk1 was immunoprecipitated from mock-transfected cell lysates with affinity-purified polyclonal antibody that had been precoupled to protein A beads. Ectopically expressed Chk1 was immunoprecipitated with anti-myc monoclonal antibody–agarose (Santa Cruz). Immunoprecipitates were subjected to SDS-PAGE on an 8% polyacrylamide gel, and proteins were transferred to nitrocellulose. Radiolabeled Chk1 was digested in a solution containing 0.2 mg of trypsin (Worthington) per ml in 50 mM ammonium bicarbonate for 17 h. Tryptic phosphopeptides were separated in the first dimension by thin-layer chromatography at pH 1.9 and in the second dimension by ascending chromatography in a buffer consisting ofn-butanol–pyridine–acetic acid–water in a ratio of 75/50/15/60 (65). Alternatively, tryptic phosphopeptides were incubated with 1.2 U of Asp-N endopeptidase (Roche) per ml in 50 mM ammonium bicarbonate for 17 h and then incubated with Ziptip C18 resin (Millipore) to purify the phosphopeptides prior to two-dimensional phosphopeptide mapping. In some cases, an unlabeled phosphopeptide of the sequence LVQGISFpSQPTCP was resolved by two-dimensional mapping.
Checkpoints induce the phosphorylation and activation of Chk1.To examine how the Chk1 protein kinase is regulated during checkpoint responses, HeLa and U2OS cells were treated with HU to activate the DNA replication checkpoint (Fig. 1A). U2OS cells contain a functional p53 protein, whereas HeLa cells lack functional p53 due to the expression of the papillomavirus E6 protein, which targets p53 for destruction (59). The electrophoretic mobility of Chk1 was observed to be retarded on SDS gels after HU treatment of both cell types, suggesting that p53 status does not impinge upon this pathway (Fig. 1A, lanes 2 and 6). Phosphatase treatment confirmed that the slower electrophoretic form of Chk1 was due to HU-induced phosphorylation of Chk1 (lanes 3 and 4). Interestingly, the reduced electrophoretic mobility of Chk1 was also observed when HeLa cells were treated with UV light and a variety of chemotherapeutic agents that induce DNA damage or interfere with DNA replication, including cisplatin, ara-C, and etoposide (Fig. 1B).
To determine whether the protein kinase activity of Chk1 was regulated by phosphorylation, immune complex kinase assays were performed in vitro (Fig. 1C). Endogenous Chk1 was immunoprecipitated from HeLa cells that had been cultured in the absence (lane 1) or in the presence (lane 2) of HU. Chk1 immunoprecipitates were monitored for their ability to phosphorylate GST-Cdc25C200–256, a protein consisting of amino acids 200 to 256 of Cdc25C fused to GST. This substrate has previously been used to monitor the activity of Chk1 (25, 56). Interestingly, the protein kinase activity of Chk1 isolated from HU-treated cells was measured to be approximately fivefold higher than that isolated from untreated cells.
Gel filtration analysis of human Chk1.Gel filtration analysis was performed to monitor the elution profile of Chk1 before and after HU treatment (Fig. 2). In the absence of HU treatment, Chk1 eluted in fractions 29 and 30, consistent with it being a monomer in cells. However, after HU treatment, Chk1 was also detected in fractions that eluted earlier (Fig. 2 [and see Fig. 7]). Interestingly, the phosphorylated form of Chk1 that migrates with a slower electrophoretic mobility on SDS gels was preferentially found in the fractions that elute earlier. These findings suggest that the phosphorylated form of Chk1 either multimerizes, binds to cellular proteins, or undergoes conformational changes causing it to elute with protein standards of 100 to 158 kDa. To determine whether ectopically expressed Chk1 behaved like endogenous Chk1, we produced a recombinant adenovirus expressing Flag-tagged Chk1. Lysates prepared from infected HeLa cells were resolved by gel filtration, and selected fractions were incubated with Flag-agarose to specifically precipitate Flag-tagged Chk1. Precipitates were analyzed for the presence of Chk1 by Western blotting. As seen in Fig. 2, in the absence of HU treatment, Flag-Chk1 eluted in the same fractions as endogenous Chk1 (fractions 29 and 30). After HU treatment, Flag-Chk1 was observed to also elute in fractions 27 and 28, as did endogenous Chk1.
Checkpoint-induced phosphorylation of Chk1 is ATM independent but caffeine sensitive.To determine whether the HU-induced phosphorylation of Chk1 required functional ATM, immortalized fibroblasts derived from a patient lacking ATM (AT cells), stably transfected with either the ATM expression vector (AT+) or an empty vector (AT−), were treated with HU. The electrophoretic mobility of Chk1 was then monitored by immunoblotting (Fig.3A). The slower electrophoretic form of Chk1 was detected in both AT+ (lane 2) and AT− (lane 5) cells, although reduced levels of Chk1 protein were observed in both cell lines after HU treatment. We also monitored the electrophoretic mobility of Chk1 after treatment of AT+ and AT− cells with ionizing radiation. As seen in Fig. 3A (lanes 3 and 6), the electrophoretic mobility of a fraction of Chk1 was found be retarded in both AT+ and AT− cells, although to a lesser extent than that observed with HU.
We also monitored the effects of caffeine on the HU-induced phosphorylation of Chk1, because both ATM and ATR are inhibited by caffeine (3, 9, 49, 57, 75). As seen in Fig. 3B, the ability of HU to induce the phosphorylation of Chk1 was completely abrogated when cells were pretreated with 10 mM caffeine (lane 4). Taken together, these observations suggest that the HU-induced phosphorylation of Chk1 is independent of ATM, but may be dependent on ATR or another caffeine-sensitive kinase.
Chk1 is phosphorylated on serines 317 and 345 in vivo.Two-dimensional phosphopeptide mapping of Chk1 was performed to identify residues that were phosphorylated in response to HU treatment. HU-treated HeLa cells were incubated with32P-labeled inorganic phosphate, and endogenous Chk1 was isolated by immunoprecipitation. In addition, HeLa cells were transfected with plasmids encoding myc-tagged Chk1. Transfected cells were incubated with 32P-labeled inorganic phosphate in the presence of both HU and 1 μM UCN-01. UCN-01 inhibits the activity of Chk1 kinase, but not the activity of the kinases that lie upstream of Chk1 (7, 25, 29). We observed enhanced phosphorylation of ectopically expressed Chk1, but not that of endogenous Chk1 when UCN-01 was included during the labeling period. Although the mechanistic basis for this is not known, UCN-01 is expected to inhibit the kinase activity of ectopically produced Chk1, thus allowing cells to overproduce Chk1 in a kinase-inactive form.32P-labeled endogenous Chk1 and myc-tagged Chk1 were digested with trypsin, and peptides were separated in two dimensions. As seen in Fig. 4, two predominant phosphospeptides (denoted 1 and 2) were evident in the case of endogenous (Fig. 4B) and ectopically expressed (Fig. 4C) Chk1. Given the caffeine sensitivity of Chk1 phosphorylation, we examined the Chk1 protein sequence for the presence of SQ and TQ residues. Serine and threonine residues followed by glutamine are preferred sites of phosphorylation for the ATM and ATR kinases (35, 52). As seen in Fig. 4A, there are five SQ residues located within the C terminus of Chk1 (serines 317, 345, 357, 366, and 468). A mutant form of Chk1 containing alanine in place of serine at position 317 was expressed as a myc-tagged fusion protein in HeLa cells. Two-dimensional tryptic phosphopeptide mapping demonstrated that mutation of serine 317 resulted in the disappearance of phosphopeptide 1 (Fig. 4D). This indicates that serine 317 is phosphorylated in HU-treated cells.
To directly monitor the phosphorylation of serine 317 in vivo, we generated an antibody that recognizes Chk1 when it is phosphorylated on serine 317. HeLa cells were either untreated or were treated with HU or UV (Fig. 4E). Lysates were prepared and analyzed directed by SDS-PAGE (lanes 1 to 4) or were incubated with the S317 phosphospecific antibody prior to SDS-PAGE (lanes 5 to 8). As expected, HU and UV treatments caused the appearance of the slower electrophoretic (phosphorylated) form of Chk1. Interestingly, the phosphospecific antibody immunoprecipated only the slower electrophoretic (phosphorylated) form of Chk1 found in HU- and UV-treated cells (lanes 6 and 8). Preincubation of the antibody with the phosphopeptide immunogen blocked the immunoprecipitation of S317-phosphorylated Chk1 (lane 7). These results verify that Chk1 is phosphorylated on serine 317 in vivo in response to HU and UV treatments.
Because serines 345, 357, and 366 are contained within a single, large tryptic peptide (Fig. 4A), we digested myc-tagged Chk1 (wild type and mutants) with trypsin followed by Asp-N endopeptidase. As seen in Fig.5A, five phosphopeptides were visualized under these conditions. Mutation of serine 317 resulted in the loss of phosphopeptide 1 (Fig. 5B), whereas mutation of serine 345 resulted in the loss of phosphopeptide 2 (Fig. 5C [and see Fig. 8]). S345 has previously been shown to be phosphorylated in response to checkpoint activation (44). Replacement of serines 357 and 366 with alanine did not cause the loss of any of the five phosphopeptides identified for wild-type Chk1 (Fig. 5D). In addition, replacement of serine 468 with alanine did not alter the two-dimensional phosphopeptide mapping results, suggesting that this residue is not phosphorylated in vivo (data not shown). These findings also indicate that serines 317 and 345 are sites of Chk1 phosphorylation in vivo.
HU-induced activation of Chk1 requires phosphorylation of serines 317 and 345.To address the functional significance of phosphorylation of serines 317 and 345, recombinant adenoviruses expressing single- and double-phosphorylation-site mutants of Chk1 were generated. HeLa cells were infected with wild-type and mutant viruses in the presence of HU. Lysates were prepared and fractionated by gel filtration. Selected fractions were analyzed for Chk1 protein and for Chk1 kinase activity. As seen in Fig. 6and as shown earlier (Fig. 2), the phosphorylated, activated form of Chk1 eluted in fractions consistent with a molecular size of ∼100 to 158 kDa (fractions 26 to 28). The bulk of the hypo- or unphosphorylated, inactive Chk1 migrated in fractions 29 to 30, as expected for monomeric protein. Mutation of either S317 or S345 to alanine reduced the amount of Chk1 in the fractions eluting earlier (fractions 26 to 28). In addition, the kinase activity associated with Chk1(S317A) and Chk1(S345A) in fractions 26 to 28 was markedly reduced compared with that of wild-type Chk1. Finally, the mutant of Chk1 containing alanine in place of serines 317 and 345 eluted primarily in fractions 29 and 30 and was poorly activated by HU treatment. Taken together, these findings suggest that phosphorylation of both S317 and S345 contributes to the activation of Chk1 and its faster elution on gel filtration columns.
To determine whether the intrinsic protein kinase activity of Chk1 was altered as a consequence of amino acid substitutions, we monitored the kinase activity of bacterially produced Chk1 (wild type and mutants). Kinase-dead Chk1, wild-type Chk1, Chk1(S317A), Chk1(S345A), and Chk1(S317A, S345A) were expressed and purified from bacteria as GST-fusion proteins and were tested for their ability to phosphorylate GST-Cdc25C200–256 in vitro. As seen in Fig.7A, each phosphorylation site mutant of Chk1 had kinase activity similar to that of wild-type Chk1 in this assay. Wild-type and mutant forms of Chk1 expressed in HeLa cells were also examined for kinase activity (Fig. 7B). Lysates prepared from cells infected with adenoviruses encoding green fluorescent protein (GFP; lane 1), Flag-Chk1 (lane 2), Flag-Chk1(S317A, S345A), and kinase-inactive Flag-Chk1(D130A) were incubated with anti- Flag–agarose, and precipitates were tested for their ability to phosphorylate GST-Cdc25C200–256 in vitro. The double-phosphorylation mutant of Chk1 (lane 3) had kinase activity similar to that of wild-type Chk1 (lane 2) in this assay. These results indicate that the failure of the phosphorylation site mutants of Chk1 to be fully activated when cells are treated with HU (Fig. 6) reflects an inability of these mutants to be phosphorylated rather than intrinsic defects in kinase activity as a result of amino acid substitution.
Phosphorylation of Chk1 by ATR.We next asked whether Chk1 is a direct substrate of ATR in vitro. Kinase-active and -inactive forms of ATR were tested for their ability to phosphorylate Chk1 in vitro. A kinase-inactive mutant of Chk1(D130A) fused to GST was used as a substrate in these assays. As seen in Fig.8A, Chk1 was phosphorylated by kinase-active (lane 2) but not kinase-inactive (lane 3) ATR. Radiolabeled Chk1 was isolated and digested with trypsin followed by Asp-N endopeptidase. Peptides were separated in two dimensions, and phosphopeptides were visualized by autoradiography. As seen in Fig. 8B, two predominant phosphopeptides were detected under these conditions. Mutation of serine 317 caused loss of phosphopeptide 1 (Fig. 8D), whereas mutation of serine 345 caused loss of phosphopeptide 2 (Fig.8E). To further confirm that phosphopeptide 2 represented the peptide containing phosphorylated serine 345, we synthesized the phosphopeptide expected after digestion of Chk1 with both trypsin and Asp-N endopeptidase (LVQGISFpSQPTCP) and monitored its position after two-dimensional phosphopeptide mapping. As seen in Fig. 8B and C, the synthetic phosphopeptide comigrated with phosphopeptide 2, confirming that phosphopeptide 2 contains S345. These results demonstrate that Chk1 is phosphorylated on serines 317 and 345 by ATR in vitro and indicate that Chk1 may also be a direct target of ATR in vivo.
To examine the contribution made by ATR to S317 phosphorylation in vivo, we expressed either kinase-active or -inactive ATR in 293 cells and examined S317 phosphorylation after UV treatment. As seen in Fig.8F, UV-induced phosphorylation of Chk1 was attenuated in cells expresssing kinase-inactive ATR (lane 4) relative to kinase-active ATR (lane 2). Immunoprecipitation with the S317 phosphospecific antibody demonstrated that phosphorylation of Chk1 on serine 317 in response to UV was reduced in cells expressing kinase-inactive ATR (lane 8). It has previously been reported that S345 is phosphorylated in response to HU, UV, and IR in an ATR-dependent manner in vivo (44). Taken together, these results demonstrate that Chk1 is inducibly phosphorylated on S317 and S345 in response to various stimuli that cause checkpoint activation and that phosphorylation of Chk1 on both S317 and S345 in vivo is ATR dependent.
In this study, regulation of the human Chk1 protein kinase was examined after exposure of cells to agents that induce various forms of DNA damage or interfere with DNA replication. There has been controversy concerning not only what DNA structures human Chk1 responds to, but also whether the kinase activity of Chk1 is elevated in response to genotoxic stress. It has been reported that human Chk1 is inducibly phosphorylated on serine 345 in response to HU and UV and, to a lesser extent, IR (44). In addition, dominant-negative forms of ATR interfere with the phosphorylation of Chk1 on serine 345 in vivo (44). However, it has not been determined if human Chk1 becomes phosphorylated on additional sites in response to genotoxic stress in vivo, if phosphorylation regulates the kinase activity of human Chk1, or if human Chk1 is a direct target of ATR.
In this study, serines 317 and 345 were identified as sites of phosphorylation in vivo and as sites of ATR phosphorylation in vitro. Furthermore, expression of a kinase-inactive form of ATR interfered with the UV-induced phosphorylation of Chk1 on serine 317 in vivo. It has previously been reported that kinase-inactive ATR also interferes with the UV-induced phosphorylation of Chk1 on serine 345 in vivo. These findings in conjunction with the role of ATR in regulating cellular responses to UV and HU argue that human Chk1 may be directly phosphorylated on serines 317 and 345 by ATR in vivo.Xenopus Chk1 contains four SQ/TQ consensus sites within its C terminus, and all four can be phosphorylated by ATR in vitro (26). Expression of a mutant form of Chk1 lacking all four phosphorylation sites abrogated checkpoint responses ofXenopus extracts to replication blocks and UV-damaged DNA (26). These findings highlight the conserved nature of the ATR-Chk1 checkpoint pathway in eukaryotic organisms.
Human Chk1 has five ATM or ATR consensus phosphorylation sites (SQ/TQ) within its C terminus. These include serines 317, 345, 357, 366, and 468, each of which is followed by glutamine. Serines 317 and 345 are the only two found conserved in Chk1 from other species, including fission yeast, C. elegans, Xenopus, andDrosophila. In addition, residues inclusive of and surrounding serines 317 and 345 in human Chk1 comprise optimal ATM or ATR consensus phosphorylation sites (35). Interestingly deletion of the C terminus of human and Xenopus Chk1 potently activates its intrinsic kinase activity, suggesting that the C terminus functions as a negative regulator (12, 51). Phosphorylation of Chk1 by ATR may serve to relieve this C-terminal inhibitory function to activate Chk1 during the cellular response to genotoxic stress. In addition to activating the protein kinase activity of Chk1, phosphorylation also caused Chk1 to elute faster on gel filtration columns. Additional studies will be required to determine if the faster elution profile of the phosphorylated form of human Chk1 is a result of Chk1 binding to cellular proteins, multimerizing, or undergoing conformational changes. In checkpoint-activated extracts,Xenopus Chk1 has been shown to bind to claspin, a 215-kDa protein, and the interaction between Xenopus Chk1 and claspin is required for checkpoint-induced activation of Chk1 (37).
Although Chk1 is highly conserved throughout evolution, the signals that Chk1 responds to have diverged in eukaryotic organisms. In fission yeast, Chk1 responds to DNA damage induced by either IR or methyl methanesulfonate as well as UV (66, 67). InXenopus, Chk1 is activated by DNA replication blocks and UV-damaged DNA, but not by double-stranded DNA breaks of the type induced by IR. In this study, we report that human Chk1 is activated by HU, UV, etoposide, cisplatin and ara-C, but poorly by IR. HU blocks DNA replication by inhibiting ribonuceotide reductase, but may subsequently induce DNA damage. ara-C is a nucleoside analog that incorporates into DNA and interferes with DNA polymerases, which in turn interferes with DNA chain elongation during both DNA replication and DNA repair (48, 64). UV and cisplatin perturb the helical structure of the DNA duplex and as such inhibit DNA replication (19). Etoposide is a DNA topoisomerase II inhibitor that causes double-stranded DNA breaks (11). IR also induces double-stranded DNA breaks, but does not signal efficiently to human Chk1. One function of DNA topoisomerase II is to disentangle intertwined daughter chromatids after DNA replication and prior to mitosis. Thus, inhibition of topoisomerase II by etoposide also leads to activation of the chromatid catenation checkpoint (18), and this may explain in part why etoposide but not IR leads to Chk1 activation. Thus, human Chk1 is activated when DNA replication is directly blocked (i.e., by agents such as HU) or disabled (ara-C, UV, cisplatin), but is poorly activated by double-stranded DNA breaks induced by IR. In addition, Chk1 may be activated when cells recognize mismatches that result from attempted replication across modified or damaged bases. An essential role for Chk1 in genome surveillance during DNA replication may explain why disruption of Chk1 results in early embryonic lethality in mice (44, 61).
Data reported in this study and elsewhere indicate that ATR lies directly upstream of Chk1 in pathways that respond to unreplicated DNA and certain forms of DNA damage. There are likely to be several downstream cellular targets of Chk1 in these pathways as well. In humans, fission yeast, and Xenopus, Chk1 has been shown to phosphorylate Cdc25 on a residue or residues that facilitate the binding of 14-3-3 proteins (38, 54, 56, 72, 73). The functional significance of 14-3-3–Cdc25 interactions has been demonstrated in several organisms by expression of mutants of Cdc25 that cannot bind to 14-3-3 proteins. Perturbations in the DNA replication checkpoint and/or the G2-DNA damage checkpoint have been observed in cells expressing the non-14-3-3 binding mutants of Cdc25 (39, 54, 72, 73). Loss of 14-3-3 binding leads to the nuclear accumulation of Cdc25 in fission yeast,Xenopus, and human tissue culture cells (16, 24, 25, 36, 45, 70, 73). Thus, one function of Chk1 may be to keep Cdc25C out of the nucleus as part of the cellular response to checkpoint activation. The Cdc25A protein phosphatase has also been proposed to be a target of human Chk1. A recent study reported that the G1 cell cycle arrest induced by UV-damaged DNA requires ubiquitin-mediated proteolysis of Cdc25A, and this in turn requires Chk1 (46). Chk1 has also been shown to directly phosphorylate Cdc25A in vitro, although the functional significance was not examined (56). Further studies will be required to identify additional substrates of human Chk1.
We are grateful to J. Schwarz and C. Ryan for assistance with production of recombinant adenoviruses and J. Gales for production and purification of antibodies. We thank K. Cimprich and R. Abraham for providing ATR reagents and M. Linder for helpful discussions. We thank J. Hurov, C. Lovly, and M.-S. Chen for comments on the manuscript.
This work was supported by a grant from the National Institutes of Health. H.P.-W. is an Investigator of the Howard Hughes Medical Institute.
- Received 7 February 2001.
- Returned for modification 19 March 2001.
- Accepted 23 March 2001.
- Copyright © 2001 American Society for Microbiology


text:
Hello All!
I hope you are having a wonderful week.
I'm finally feeling a little better. The blahs seem to be passing somewhat.
Thank you, God!
I was truly getting on my own nerves.
With this new found energy, I went to work on a couple of projects.
One of them was repainting the cupboard in my Mud Room.
The black paint was way too dark for the small space.
I had noticed several pins on Pinterest about chalk paint and thought I would give it a try.
I went with what seemed to be cheapest and easiest for me.
I have been reading that some people also apply a wood wax finish. I am not familiar with wood wax.
I do believe the cupboard does need some type of sealant on it and I don't want the shine of polyurethane.
I would love to know if you all have any suggestions on the type of wood wax I need.
Here are a couple of before and after photos…
I wanted to give the cupboard an aged, worn, appearance.
I made sure when painting it that some of the black did show through in areas.
I also gave it a light sanding once everything was dry.
I am pleased with how it turned out.
The white paint does help brighten up the dark corner.
Here are a few more photos of the cupboard…
And just because I love them, here's a photo of my small collection of early blue and white clothing. :)
The other thing I have been working on is the wooden house my Mom made for Christmas.
I almost have the exterior finished.
All I need to do is add the wood shingles to the roof, just waiting on payday, then Hobby Lobby here I come. :)
Here is a Before of the house….
And now an After…
*Not the best photos.
I promise to take better ones when it is completely finished and put in its permanent spot.
I decided to add little wooden legs to the bottom of the house.
I found these at Home Depot for a couple of dollars each and knew they would look great.
They really set the house off.
It is coming along pretty good. I am excited to see it with the wood shingle roofing.
Hurry up Friday!! Hehee!
Mom did a pretty awesome job!
Especially since this was the first one she had ever created and she did it all out of her head with no pattern.
I will treasure it always!
Well, It has been a long and very busy day for me.
Work was NuTs today!!
I am exhausted and heading off to bed early tonight.
Yep, my pillow is calling my name.
Thanks so much for stopping by to visit with me.
Until Next Time…
Take Care and Prim Filled Blessings To You All,
I am linking up to:

subset: commoncrawl


text:
New York City-based Health Recovery Solutions raised $1 million from undisclosed investors, the company's COO Rohan Udeshi told MobiHealthNews. This brings the company's total funding to $1.8 million.
The company's product, PatientConnect, aims to help providers prevent costly patient readmissions by engaging them with a tablet-based program. The hospital provides patients with a cellular-enabled Samsung tablet, with PatientConnect preloaded. Providers pay Health Recovery Solutions a licensing fee for the package.
Patients can use the system to communicate with their physician via video chat. The tablet software also encourages patients to record medication, weight, activity, and symptoms. Clinicians and caregivers can monitor this data through their own apps, ClinicianConnect and CaregiverConnect. The patient tablet, which is equipped with a 4G data connection, will also offer educational materials about the conditions, which take the form of quizzes, pamphlets, and videos. The system can also collect data from A&D and Nonin home health devices.
When clinicians notice that a patients is not improving, they can reach out to those specific patients to help them stay on track and reduce readmissions.
"Right now we have about 15 [paying customers]," Udeshi said. "We've proved out some great results, all of our clients are averaging between 4 and 8 percent 30-day readmission rates. Now our goal is to use that funding to expand our sales and marketing efforts as well as expand our offerings to more diseases and reach a larger population of patients."
PatientConnect currently offers programs for patients with congestive heart failure, COPD, and diabetes. But Health Recovery Solutions has developed and is rolling out offerings for stroke recovery, post cardiac surgery, and hip and knee joint replacements. Towards the end of the year, Udeshi said the company plans to develop an offering for pediatric monitoring too.
Some of the company's customers include Johns Hopkins, Mass General, University of Pennsylvania Health System, AtlantiCare, and FirstHealth of the Carolinas. Health Recovery Solutions' newest client is Home Health Visiting Nurses, which is part of MaineHealth.
Health Recovery Solutions uses Samsung's tablets, but the company is also in talks with the technology giant to conduct a trial that makes use of Samsung's Simband.
"The goal at the end of the day is to be an 'all-cause' readmission platform," Udeshi said. "Right now Medicare is only penalizing certain conditions for readmissions. Eventually we're going to want to do away with all avoidable readmissions. [PatientConnect] is software-based: It can be completely customizable for any type of condition. We want to get to the point where the software can handle any patient you put in regardless of what the diagnosis is, what their demographic is, what the age is. [We want to be] the platform that can be tailored for any type of patients and keep them healthy and empowered."
Health Recovery Solutions was also in accelerator Blueprint Health's summer 2012 class.


text:
Astor Dining Table
Items Included
- Dining Table
The Astor extendable dining table is the perfect solution for everyday and entertaining table use. It easily transforms from a medium size (71-inch, with 8-person seating) to an extended (94-inch, with 10-person seating) table. The table top easily extends from both ends revealing a hidden centre leaf. The German-made mechanism within the table allows the tops to glide smoothly for perfect alignment every time. A weighted chromed steel base serves as the counter balance to the expansive top.
71"W (94"W extended) x 40"D x 30"H
Grey, Walnut, Wenge: Composite Wood, Natural Wood Veneer, Polished Steel
White: Composite Lacquered Wood, Polished Steel


text:
Ibuybeauti is looking for 10 Beauty Bloggers1:42 AM
If you have been lurking on my blog then you probably already know that Ibuybeauti is one of my blog sponsors. They became my sponsor last May 2013 which is a long time already.
If you are not familiar with Ibuybeauti then click THIS to see my
By the end of the month, Ibuybeauti will be officially launching a review blog (Beautifan) and their main goal is to help their current and future customers choose which products are perfect for them.
In order to provide reviews for their customers, they are currently looking for 10 beauty bloggers to review some of the products in their shop. Chosen reviewers will get to choose what products they want to try and they must write their honest opinions on the products.
The product review should be posted in their review blog, Beautifan. Beautifan will be divided in certain categories to make navigation easier. I can't link you to Beautifan right now because it's still under construction. I will let you all know the exact date of the launch of Beautifan soon. They will also have a giveaway there so stay tuned! *-*
Here are some of my reviews of the products that Ibuybeauti sent me:
Blog Address
Skin Type
Best Beauty Review link
Brief self introduction
Application Period:
Feb 3 - Feb 18
Announcement of winners:
Feb 20
Check out ibuybeauti.com to see their wide selection of Korean beauty products.
For more information/inquiries, don't hesitate to contact them at firstname.lastname@example.com
If you love Korean cosmetics or you just simply want to try it then I think you should join Ibuybeauti's sponsorship program now! :D
Join now and be a part of Ibuybeauti family! :)
26 comments
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Please follow my blog 'Heylinni' at:
I will gladly follow you back!!! Us bloggers need to support each other :) x
I love all your beauty and skin tips! Thank for sharing this.ReplyDelete


text:
Gave them away, didn't like flavor
I received the Chai flavor in my box, and I normally love chai tea so I was excited. However, these tasted almost entirely like cinnamon, which is something I like in moderation but can't stand on its own. These were just shy of being hot cinnamon flavored, and even though I tried to give them a chance, I just couldn't stand the flavor and gave them away to a friend.1


text:
Police said they averted a "massacre" after foiling a suicide bombing and gun attack yesterday on one of Egypt's most popular ancient attractions, in a rare assault on the country's vital tourism sector.
Egypt has been shaken by a tide of attacks claimed by Islamist militants since the army toppled the democratically elected government two years ago, but the incident in the southern city of Luxor was the first in years targeting tourists.
This has added to fears that unrest could scare off the visitors who are crucial to the economy in a country where tourism accounts for more than a tenth of GDP.
Police said two attackers were killed and another seriously wounded near the Karnak temple in Luxor, a popular site close to the famed Valley of the Kings.
No tourists were hurt in the mid-morning incident and visitors were kept inside Karnak's ruins, an antiquities ministry official said.
The incident unfolded after a taxi carrying two passengers attempted to enter the parking lot and was stopped by a suspicious policeman, Tourism Minister Khaled Ramy said in a statement.
Police asked to search the trunk, where they found two large bags that made them suspicious, a general from Luxor police said.
When they asked to open the bags, one of the men jumped out of the car, ran away and detonated his explosive vest.
In the ensuing confusion, the other passenger emerged from the car and, joined by an accomplice who had come in on foot, pulled assault rifles from the bags and started shooting.
The police responded, killing one and seriously wounding the other.
Health ministry official Nahed Mohamed said two civilians and two policemen were wounded in the shootout, but not seriously.
Police said the taxi driver was not a suspect.
"If they had managed to enter the temple, it would have been a massacre," said the general, as 19 fully loaded magazines for the rifles were found in the bags and there were 604 tourists were in the temple.
"We have enhanced security measures in place at all our sites and we continue to take every possible measure to ensure that no harm comes to anyone visiting Egypt," Ramy said.
No group had yet claimed responsibility for the attack.
But Mathieu Guidere, professor of Arab geopolitics at France's University of Toulouse, said the incident indicated a change in strategy in militants choosing targets.
The aim is "to weaken the Egyptian economy by destroying the tourism industry" and to have "the maximum media impact" as attacks on local targets were not drawing international attention, he said.
Karnak in central Luxor was built on the ruins of Thebes, the capital of ancient Egypt.
The huge temple dedicated to the god Amon lies in the heart of a vast complex of religious buildings in the city, 700km south of Cairo.
Tourism in Egypt has faltered since early 2011, when a popular uprising toppled longtime strongman Hosni Mubarak after three decades in power.
Years of instability scared off visitors from overseas, damaging the economy and sending Egypt's foreign currency reserves plunging.
Last year, 10mn tourists visited Egypt, bringing with them $7.5bn in revenues. That generated 19.3% of currency reserves.
World Bank President Jim Yong Kim, who happened to be in Luxor on business yesterday, strongly criticised the attack.
"We abhor violence of all types, which runs completely counter to our work to end extreme poverty in the world," he said in a statement, adding that "we must confront extremism by relentlessly creating hope."
Militants have carried out bombings regularly since the army ousted Islamist president Mohamed Mursi, mainly in the Sinai Peninsula, where a local affiliate of the Islamic State group has claimed a spate of attacks on security forces.
A government crackdown has left hundreds of Mursi supporters dead, thousands imprisoned and dozens sentenced to death after speedy trials, described by the UN as "unprecedented in recent history".
The militants claim their attacks are in retribution for the crushing of dissent under the administration of President Abdel Fattah al-Sisi.
Police avert 'massacre' at tourist site in Egypt
Police said they averted a "massacre" after foiling a suicide bombing and gun attack yesterday on one of Egypt's most popular ancient attractions, in a rare assault on the country's vital tourism sector.


text:
Heads Up for Android and iOS is now available for PC as well. If you have not played this game before, here is an overview of what the game is about before we move on to the method to download Heads Up for PC.
Heads Up Gameplay:
Heads Up is a funny guessing game by Warner Bros. International Enterprises. The game has been used on the famous Ellen DeGeneres show where Ellen and the Hollywood Stars had a blast playing this game.
Heads Up is designed with amazing user interface and excellent visuals. The game is very easy to play with some simple rules. Minimum of two people can play this game. One player has to the device in front the forehead; he is the guesser so he will not be allowed to look at the device's screen. The other player has to help the guesser guess the correct answer by giving some clues but the will not be allowed to speak. If you answer correctly, tilt the device's screen upwards. If you are not able to guess, you can pass by tilting the device downwards and a new card will be shown.
Heads Up will show various cards on the screen and each round of game will last for a specific time period. There are different categories to choose from; Act it Out involves Mime where you have to act the word out and not speak. Just for Kids requires you to give certain clues that leads to word without saying out the word itself. Pitch Perfect is for music lovers where you will have to hum the tune to help your friend guess the song. Animals Gone Wild is about guessing various animals shown on the card.
Heads Up is perfect to kill time and boredom and have long laughter sessions with your friends and family. The game is hilarious and fun to play.
Download Heads Up for PC:
To download Heads Up for PC, you will need an Android emulator like Bluestacks. If you do not have it already, you can download it from the website Bluestacks.com. Search for Heads Up using the Bluestacks search tool and click on the install button to automatically download and install the game. Once the game has been installed, go to My Apps in Bluestacks homepage to open Heads Up.
Download: Heads Up for PC


text: Ethiopia, the ancient nation where, some rumors said, the direct descendants from King Solomon can be found. Others said Queen of Sheba was from the land of Ethiopia. Ethiopians have their own unique language, Amharic. The Ethiopians can engage in BoyleSports Bingo, and the company will give 300% from the initial €30 wagered.


text:
Finn Kollerup has a long history of teaching and mentoring people who want to learn more about creativity and innovation, and those who want innovation skills in-house.
We offer this in different forms:
Both the keynote and the innovation training builds on breakout sessions, dialogues and hands on experience with different methods and tools – you won't be bored..
Our goal is to expand your understanding of the concepts of creativity and innovation, by using metaphors, stories and humour in a high energy presentation. We are strong believers in dialogue and action-based work, which means that we often use small breakouts during the presentation where the participants discuss what they have been hearing.
Read more about Finn Kollerup as a keynote speaker
Creativity and innovation skills are not something that belong to a certain personality or type. We know that these qualities can be learned and trained. By giving people the chance to work with the right tools and methods they can accomplish great results within your company.
We tailor your training program so that it fits your needs both in terms of content and time. This means that we can either plan an accelerated process where we are together for a couple of days, or design a program where we meet up once a week for a couple of months, in which we create the opportunity to work with the new tools and methods at home and get feedback.


text:
Penn State is the "Big Daddy" of tailgating in the northeast. It is the "Flagship" of the football rich state of Pennsylvania and has a large student body (about 45,000) that really gets into football weekends. Being a rural campus it has plenty of parking and you definitely know that the college and the game is the biggest event around central Pennsylvania.
Penn State, a land grant school, is the most eastern school of the Big 10 and can be hard to get to by car depending on which direction you're coming from due to the Appalachian Mountains (more precise the Alleghenies). The closest Big 10 school (Ohio State) is 5.5 hours away. The good news is Penn State is RV friendly and allows parking starting on Thursday evening for RVs. The school is located in the small city of State College in Centre County (Pop. 150,000). State College however lately has become a popular destination for retirees that enjoy the four seasons and outdoor activities.
Pattee Mall on the campus
Flying and Riding
State College is served by the small regional University Park Airport (SCE). Delta, United and US Airways offers limited service. The airport is conveniently located just a couple miles from the campus. Be advise airfare into State College can be high and you also can suffer long waits at hubs if you miss your connection. A better option might be to fly into Harrisburg (1 3/4 hours away), Pittsburgh (2 3/4 hours away), Baltimore-Washington (BWI) (3 1/4 hrs)or even Philadelphia (3 1/2 hours away) and rent a car. Train service is not available into State College. However, it is a short trip away (about an hour) from several Amtrak train stations, located in Altoona, Lewistown, and Huntingdon. Greyhound Bus Service is another option for those living close by as it offers service to the University. An easy to read map showing Penn State and State College is here.
Hotel State College on Allen Street in State College, PA a nice place to spend a weekend
Picture from Nittany Memories check it out
A printable map of the campus
Directions to the Stadium
Lots of different printable maps here.
Most info taken from the 2011 Penn State Information Guides. The newest 2010 stadium brochure is smaller and suitable for printing.
A nice overview map to use--centered on Beaver Stadium.
Driving Directions to Beaver Stadium
The Campus is located within driving distance of many major cities including Harrisburg (1.5 hrs., 90 mi), Pittsburgh (2.5 hrs., 150 mi), Philadelphia (3.5 hrs., 200 mi), Baltimore (3 hrs., 170 mi), Washington D.C. (3.5 hrs., 210 mi), New York City (4 hrs., 250 mi) and Toronto (6 hrs., 300 mi).
These directions are suggested routes only and do not account for construction delays, detours, recent road closures, etc...
University Park Campus Map
From New York City-
From I-280 west, merge onto I-80 west. Continue for 200 miles and take exit 161 for PA-26/US-220 south toward Bellefonte. Turn left at Jacksonville Rd/PA-26 S/US-220 south. Continue onto I-99 south and about 10 miles later take exit 74 (Innovation Park/ Penn State University).
From Philadelphia-
Merge onto I-76 heading west. About 80 miles later, take exit 247 to merge onto I-283 N toward PA-283/Harrisburg/Hershey. Merge onto I-83 N and continue onto US-322 W and merge onto I-81 S. Take exit 67B to merge onto US-22 W/US-322 W toward Lewistown/State College. Continue on US-322 and take the exit toward Innovation Park/Penn State Univ/Beaver Stadium/Bryce Jordan Center.
From Pittsburgh-
Follow I-376 east to Monroeville, then merge onto US-22 by exiting at Murrysville. About 75 miles later, merge onto I-99 north towards Altoona. Go less than 50 miles, then take the US-322/Mt. Nittany Expressway via Exit 73 towards Penn State/State College.
From the West or Ohio area-
Take I-80 to exit 123 (Woodland) just east of Clearfield, then US Route 322 east to the ramp onto I-99 N and take the State College exit. **NOTE--This is the quickest route; however, it is a two-lane road for the majority of the trip.
-OR- Exit I-80 at exit 161 (Bellefonte) and follow Route 220 south to I-99 S and take the State College exit.
From Washington, D.C.-
Take Route 270 north then take exit 32 to merge onto Route 70 west to Hagerstown. Turn left at I-70 E/S Main St/US-30 W. Continue to follow US-30 W. Take I-99 north to Route 220 to Route 322 East to State College exit #74.
Merge onto I-495 W/I-95 N and continue to follow I-95 N. Take exit 49B for I-695 W toward Towson. Merge onto I-695 N. Slight right at I-83 N and continue for 65 miles. Take exit 43 towards Capitol/2nd St. Continue onto US-22 W for 55 miles and then onto US-322 W. Take Innovation Park/Penn State Univ/Beaver Stadium/Bryce Jordan Center exit.
Persons without parking passes should follow the football directional signs from their route of arrival. Parking fee payment will be made at the lot entrance. Day of the game cost is $40 for parking instead of $10 + $2 Ticketmaster Fee if you prepay. So Plan ahead! You have been warned.
You will be directed to lots# 11 and 26.
Okay you are a visiting fan, how do I get this prepaid parking pass for $10? Well from my experience you have to check Ticketmaster. PSU allows season pass parkers the option to purchase much earlier. Single Game day buyers are allowed starting around late July. Be advised you can not buy your prepaids 10 days prior by phone. Also, single game prepaids do sellout vii8a Ticketmaster. You need advanced planning. If you arrive on Friday before your game you can try to buy a prepaid at the Joyce Athletic Center. If not they gotcha for $40. Nice, huh?
Here is the link with the current parking info.
Limited overnight parking for recreational vehicles ONLY is permitted Thursdays after 6 p.m. in the designated area (see Parking Map--Lots# 19+20 NE of the stadium). A $60 (for $2011???) per-night fee (for Thursday and Friday) is charged for this service or $120 from Thurs-Sun. There will be separate charges for towing and towed vehicles.
All your RV parking info is here.
A Printable Parking Map is here.
Disabled Parking
From the PSU ADA Guide
In accordance with Pennsylvania law, parking is available near the stadium for persons with disabilities. Ticket holders with a Handicap License Plate or a state issued Handicap Placard may use parking lot #9 behind the Intramural Building. Patrons must arrive from the west on Park Avenue to access this lot. Shuttle Busses will circulate this lot for two hours prior to kickoff and will stop at Gate B west of the stadium.
Shuttles leave from Gate B as soon as they are full, and operate approximately two hours following conclusion of the game.
Fans Requiring Wheelchair Parking
For fans requiring a wheelchair for mobility, please contact Lisa Clark at 814-863-1000 in advance to purchase the proper parking pass for parking areas south of stadium.
Driving to Beaver Stadium--Can be a slow process
RV's are a Common Site at Penn State--photo from Tailgater's Handbook
If you plan ahead you usually can buy tickets for games through Ticketmaster. The early season OOC games usually have seats. Additionally at the Ticketmaster website, PSU runs a secondary market for ticket resell by season ticket holders. If you waited and can't get any through Ticketmaster then you have to scalp. From what I could tell Pennsylvania allows you to resell tickets to football games over the Internet (if you are instate) for whatever price the market will bear. The buyer of course can be out of state. It appears to be illegal to do it in person to sell more than $5 above face value. The linked article above stated they arrest between 50-100 people each season. Penn State fans have told me tickets are easy to come by right in front of the stadium along Curtin Road. 'Pro' scalpers and regular fans are selling and buying tickets in that area. Earlier in the day tickets are also easy to find anywhere along College Ave especially between McLanahans (Grocery Store) and the book store. With that warning above (about arrest) in place if you still want to buy tickets above face value the following techniques usually have worked for me in other states: I have always found the best spot to find extra tickets is at the tailgates. I would troll the tailgate lots before game time. As game time approaches you can find tickets leading to the stadium. It is always preferred to buy extras from fellow fans.
If you are willing to risk arrest the following techniques have served me well where it is legal. If you run into a professional scalper as opposed to a fan some off the following tips are key. A few tried and true techniques to get the best price buying I have used from both scalper and fan: 1. Know what a legit ticket looks like 2. Buy from your own fan base first 3. The smaller the group the smaller the price--single tickets are cheapest 4. Prices usually peak 1-2 hours before kickoff--inside an hour the closer to game time the cheaper the ticket, right after kickoff they plummet to near nothing 5. A fan walking to the stadium in with a group of friends and still holding extra tickets to sell will usually dump them for a cheap price (they do not want to miss the game and often they are doing a favor for a buddy to sell them) usually you'll spot them holding a finger or two out to their side or over their head or saying selling one, etc... 6. If you have to deal with a professional scalper--NEVER give him what he asks, have a seating chart with you, check to make sure all tickets are really together and for that game, offer him lower and be prepared to walk away, after you turn and walk away more than half the time in my experience they'll give in and take your offer, if you can afford to wait...wait inside 20 minutes to kickoff as see the prices drop (assess the situation for the number of tickets available, how many you need before employing that strategy) 7. Don't be embarrassed to go back to a scalper to buy ticket(s) 10 minutes after you said no to his previous offer 8. Don't fall for their tactics to make you feel like you're ripping them off by offering $20 less per ticket, etc...they love to play games with your self-image and ego.
Beaver Stadium Seating Chart--don't forget your binoculars
Penn State has been argued by many to be among the best tailgating schools in the country. They come early and stay late. From the Penn State Parking and Stadium Guide:
Parking lots generally open at 8:00 a.m. For Noon games lots open at 7:00 a.m. For late afternoon or night games lot openings will be posted on the Web site: Plan to arrive at Beaver Stadium after the lots open in order to avoid standing traffic prior to opening.Fans have told me it is best to arrive early before the lots open. Park your car in queue and wait.
Most of the tailgating is done in fields. As long as it isn't pouring rain this creates a much nicer environment than compared to most other northeastern schools who heavily rely on paved parking lots.
Also new for 2009: NO GLASS BOTTLES IN THE TAILGATE LOTS! Also as before, No Charcoal Grills!
PSU fan, "psufleish23" said:
There are green dumpsters spread around the tailgate areas and on them are rolls of Trash/Recycling bags. At the very least put all your trash in bags in a neat pile so the crews working overtime on Sunday can easily pick them up.
Lots of port-a-potties but the lines are usually pretty long at them. A tip is that most of the public campus buildings within walking distance are open on weekends, especially the Law Bldg, Forestry Bldg, Business Bldg, Findley Commons, and IM Building. They have nice restrooms in them and the lines are not nearly as long.
Wearing The Other Team's Colors
Most of our fans are pretty nice to opposing team's fans, but we have had a few bad apples in some high-profile incidents recently. Most of those were with major rivals in town. For most games, don't be surprised to have people invite you over for some food or a beer. Some tips to avoid any trouble (applies to all sporting events): Try not to provoke. Avoid the larger tailgates with lots of students/drinking at them. Night games tend to be worse.
Also, a lot of fans like to give some good-nature ribbing to each other on game days. I've found that if you can be a good sport with them about it when you're at their venue, then they are usually pretty nice to you after that.
Photos from: Straight outta Johnstown
Tailgating Alternative
If you are downtown or along South Atherton (Business US 322) in one of the hotels...try setting up in your hotel parking lot and take the shuttle to the stadium. From the CATA Website
The Game Day Football Shuttle service consists of two separate routes that provide continuous service between many area hotels, Downtown State College, and Beaver Stadium.
Downtown Shuttle: serves primarily the Downtown hotels and the three Downtown parking garages.
South Atherton Shuttle: serves the many hotels along South Atherton St. along with several other areas where you can park and ride including:
The rear parking lot at Hills Plaza (not Hills South-see Hills Plaza parking map)
Along the street in Windmere Park (Rolling Ridge Dr, & Windmere Dr.)
Both shuttles will stop only at the specially marked designate shuttle stop locations along the routes. In addition to the areas noted above, riders may take advantage of on-street parking along and close to South Atherton Street, University Drive and Easterly Parkway and the fact that the three large Downtown parking garages are within two blocks of the Schlow Library stop on the Downtown shuttle. To see the actual routing and specific stop locations check the 2011 Game Day Shuttles schedule.
The South Atherton shuttle run is quite extensive and ends at Hills Shopping Plaza and also a nearby street area and Easterly Elementary School is about a half mile away from Hills Plaza but is walkable to a shuttle stop. Parking spots are offered but be sure to read the website as to where to park. This might be a nice option for visiting fans. Another route is the Downtown Shuttle Route. Current game day fares are located here and current for 2011 for both routes.
NOTE: I went to a game in Sep 09. I did this option. Parked in backside of the Hills Plaza. It worked out well. The parking lot isn't really conducive to good tailgating. It is best if you have a friend who is hosting in a lot near the stadium. The shuttle bus drops you off about a 1/4 mile from the stadium (near the basketball arena). It was well run. A great option instead of paying $40 to park on game day.
A Few More Parking Tips
From PSU fan, psufleish23:
Parking on campus in most lots is free on the weekends after 9 pm Friday. If you can get their early enough on Saturday, you get free parking and it's usually a short walk to the stadium. But, this DOES NOT apply to the parking garages on campus.
A lot of the streets in the Highlands Area (the residential area basically bordered by Beaver Ave, University Drive, Easterly Parkway, and Atherton Street) all say that parking is prohibited from 2am-6am, and it's usually enforced pretty well based on all the yellow envelopes on windshields come Sunday morning. If you come in for the weekend, you can call the State College Police Department before 10pm at 814.234.7510 with your license plate # and the location of your vehicle and they won't ticket it for the weekend.
One last note: Do Not bring a backpack to the stadium...they will not let you in.
Hope this helps.
Last Minute Tailgate Supplies
A couple things make this more of an ordeal than many other schools. Pennsylvania has archaic alcohol laws and doesn't do the consumer any favor. Beer can not be bought in grocery stores or convenient markets. Hard Liquor and Wine is only available the Pennsylvania Liquor Control Board Stores. Centre County has several shops listed. Beer is not available at PLCB stores. For beer in State College you'll need to go to one of the larger distributors (Pletchers is one and is located near the largest PLCB store) or to a smaller 6-pack bottle or take away shops. The 6-pack shops are always attached to a restaurant - for example Hooter's has one and there is another attached to Sharkies. At the larger distributors you can get only get full cases and kegs (kegs are illegal in the tailgating lots). The smaller shops sell smaller packs but have a limit on the volume of beer purchased and are much more expensive. Wine and liquor are only available at the PLCB stores. So either bring it from home or get severely inconvenienced by finding one of these stores on gameday.
PSU fan "psufleish23" said:
The distributors usually carry all major brands, they generally align themselves with certain brands, giving better prices for that brand than its competitors. For example, Nittany Beverage gives better deals on Miller products, and W.R. Hickey gives better deals on Anheuser-Busch. I'm not sure about Pletcher's alliances, but I've heard they have a better overall selection, including micros. If you're looking for a semi-local beer, PA-brewed Yuengling Lager is a pretty good choice.
Although State College has an ordinance against open alcohol containers, it is relaxed during game day in the tailgate areas around the stadium. Also, they made a new rule a few years ago that prohibits drinking at the tailgate during the game. Once the game starts, cops patrol the lots and this will be enforced.
The closest store for all the other tailgating and food supplies is Mc Lanahans Penn State Room. They sell PSU merchandise and all sorts of tailgating supplies. Note: They don't sell beer, wine or liquor.
McLanahan's--a must for any one looking for PSU Merchandise and Tailgate Supplies.
If you are looking for a regular grocery store State College has several options. State College has a Wegman's Market on the west side. Wegman's is huge and competes with the best grocery chains in the US. Giant has two locations in State College. One on the east side and one on the west side. Weis Super Markets have four stores in the State College Area. Finally, Walmart has two Supercenters. One on the west side and one on the east side. Wegmans, Giant, and Walmart are all open 24 hours. Finally, if you are buying food for your tailgate be sure to pick up some Seltzer's Lebanon Bologna...it is one of those must try local foods.
Money Saving Tip: Weis, Wegmans and Giant all use loyalty cards. Either apply for one online or at customer service and maybe ask someone to scan their card for you to get the discounts.
State College boasts over 3000 hotel rooms and they need all of them for football weekends. The key thing is to book early if you want to be close. Here are some of the more popular motels in State College. Of course if you do some detective work you can find some hidden gems like the Nittany Budget Motel on the budget end or the Carnegie House on the higher end. The Hotel State College only offers 11 rooms but can't be beat for location. If you are a Bed and Breakfast fan this link will sure to please. This is the most comprehensive list I found for the listing of all lodging in the State College area.
Te Penn Stater Inn and Conference Center is located on campus and is usually very popular. Occasionally for out of conference opponents you can still find rooms a few weeks out.
Some out of town options: Clearfield, PA is about 45 miles NW located next to I-80. It has a nice assortment of motels. Altoona, PA is about 45 miles SW and also has quite a few motels. Another resource for Altoona showing all types of lodging options. About 70 miles east is Milton/Lewisburg and they offer options for those coming from the east coast. Finally, this guide is good for giving you phone numbers for various "Mom and Pop" Motels.
Famous Berkey Creamery
Okay let me get this out of the way first before talking about food: you need to go get ice cream at the University's Berkey Creamery (the largest college creamery in the US). They open early and close late on the weekends. Parking is available in the East Deck Parking Garage on Bigler Road located directly behind the Creamery Store and the Food Science building. The first 30 minutes is FREE. Just go you won't be disappointed.
FINE DINING: The Carnegie House, a few miles west, is a lodge but offers meals in a nice setting. The Atherton Hotel boasts the Tarragon Restaurant and a varied menu. The Tavern Restaurant is a long standing tradition and is very popular. Mario and Luigi's offers up the finest in Italian...they have an extensive menu. Spat's Cafe and Speakeasy offers Cajun Food on their varied menu. Zola's Bistro has a varied menu and popular.
CASUAL DINING: The Diner is just that but they offer the Grilled Sticky. Sounds interesting. The Diner is also a popular breakfast place. If you want something different try Korean at the Korean Kimchi Restaurant. Herwig's Austrian Bistro has fine food in a casual,cozy setting. Try Penn State Sub Shop#1 for great subs, cheese steaks and wraps. If you want steak, seafood and don't want to spend a fortune then Hoss's Steak and Sea House is for you. It offers alot of food for the money. Try Kelly's Steak and Seafood...it offers a nice menu at reasonable prices but is more upscale than Hoss's. Barbecue is hard to find in State College but west of town (about 10 miles) is Clem's Wood Fired BBQ. Clems also sell BBQ on the weekend in State College. See Bar Blue (nee: Buelah's) below for another BBQ option. If you are looking for Mexican Food--State College offers the fast food chain Qdoba Mexican Grill. Burritos are their specialty. Chipotle is also in State College if you need a burrito fix. The Rathskeller offers lunches by day and lots of beer and coeds by night. The Saloon is another college bar. Okay where do I go on Sunday morning after a hard night of partying? The Original Waffle Shop is outstanding. Expect a wait on Sunday mornings.
The Newly Renamed Bar Blue (nee: Beulah's BBQ:
Sports Bar Alert Bar Blue serves Kansas City Style BBQ and offers lots of HDTVs to watch your sporting events. It was formerly known as Buelah's. The Arena offers lots of TVs and is a full fledged sports bar--they should be able to find your game. Bill Pickle's Tap Room is Bar/Restaurant that also has TV's. Champs Sports Grill is another larger sports bar. It might be worth a look. One national chain also has a sports bar in State College. Damon's offers their famous ribs as well as a full setup of TVs.
State College has a ton of bars catering to the younger crowd. Check out your specials here: Bar Guide If you are looking for a slightly older crowd, I heard the Rathskellar, Adam's Apple (at the Tavern), or Allen Street Grille should be to your liking.
If you have a spouse that doesn't want to go to the game the Nittany Mall should keep them busy. The Woolrich Clothing Store is a must see if you love their outdoor wear. It is located about 45 minutes NE off US 220. Other shopping possibilities are here.
Penn State Golf Course--Blue and White Courses
Blue Course--from the official website:
The Blue Course finished renovations in 2006. We added three ponds and lengthened the course over 640 yards. The front nine, par 36, measures 3691 yards off the New Nittany tees. Its layout is quite different than you will find on the back nine. Tee shots require close attention to placement. A rewarding tee shot puts you in position to try your luck at carefully guarded greens. Twenty seven strategically placed green side bunkers on the front nine will challenge your course management skills, and patience. The front nine provides a challenge to the most experienced player by demanding not only length but accuracy.
The Blue Course back nine awaits your arrival. The back nine, par 36, is a newly constructed nine holes completed in 1994 and new additions in 2006. It measures 3481 yards off the New Nittany tees and provides a quite different experience than the front nine. The back nine not only includes 16 green side bunkers, but adds to the challenge by including 11 strategically placed fairway bunkers. Water hazards on the 14th and the 18th can add pressure to a seemingly easy tee shot. The back nine will provide you with large undulating greens and a links style atmosphere. Bring your "A" game. The Blue Course is the more expensive of the two courses.
White Course--from the official website:
The White Course, renovated in 2005, provides a challenging alternative to the Blue Course. With holes dating back to 1922, the White Course will test even the most skilled player. The front nine, par 36, measuring 3245 yards from the blue tees. The older course provides smaller crowned greens adding to the character and demanding a creative short game. Nearly 40 bunkers on the front side alone force you to manage your game, in your efforts to shoot a low score.
The back nine, par 36, measures 3099 yards and starts your journey with a straight forward par 5. Take advantage of these first few holes and you will be in good shape as you approach the home stretch. Elevation changes and deceptively placed fairway and green side bunkers will challenge your distance control and depth perception. Your finishing hole is a 492 yard, uphill slight dog leg right, that can make a strong case of being one of the toughest finishing holes in the area. The White Course par 3's will prove to be some of the most challenging you have played. This course will test your creativity. When in doubt, bump and run. Greens Fees here.
Toftrees Golf Resort and Conference Center
This might be the best choice for the Football Fan who also wants to play alot of golf. They offer stay and play packages. Five different tee settings allow you to play easy or play hard. Toftrees - meaning "Home among the trees" - is nestled amid 1500 scenic wooded acres in beautiful Centre County, Pennsylvania.
Toftrees Golf Resort has recently been rated 4 1/2 stars by Golf Digest magazine's "Places to Play" and one of Pennsylvania's Best Resort/ Public Golf Courses. Toftrees was selected by Meetings and Conventions magazine as one of the Top 75 Golf Conference Center Resorts in the U.S.A.
Finally, a link to a few other golf courses in the area.


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ORLANDO—Putting a league-record $22.2 million in dead salary cap money on the books eventually would never be part of a team's long-term plan. Considering the Dolphins just absorbed that hit by cutting Ndamukong Suh three seasons into a six-year deal, it's clear something went sideways.
Miami originally signed Suh to $114 million deal with $60 million guaranteed in the 2015 offseason, marking the largest contract ever for a defensive player. That deal was restructured the following year, though, because Suh was set for an untenable $28.6 million cap hit that season.
One of those two moves must be a regret for the Dolphins, but vice president Mike Tannenbaum didn't want to discuss that this morning at the NFL's annual meeting.
"Ndamukong gave us three years, and obviously we wish we won more games with him," said Tannenbaum, who took his current post in February 2015. "Our charge is to try to put a competitive team on the field each year and try to create as much flexibility as possible. I'm comfortable with where we are with our long-term plan.
"We put ourselves in position to go out and get somebody like Robert Quinn, which was an unexpected opportunity. We've planned for cap charges appropriately over the next couple of years. We try to maintain as much flexibility as possible."
When pressed a second time on whether he mishandled Suh's contract situation, Tannenbaum said, "I wish we had won more games with him. He was a good player. We just made the decision that it was time to move on."
Suh put up 181 tackles, 15.5 sacks and two forced fumbles in three years with the Dolphins. He made the Pro Bowl in 2016 and was voted team MVP last year.
He signed a one-year deal with the Rams this week to join Aaron Donald to form decisively the best defensive tackle combination in the NFL.
The upside in cutting Suh was that it frees up $17.1 million of the $26.1 million cap number he was scheduled to cost the Dolphins. That space becomes available to them in June.
The big-name free agents have already been signed this month, but Miami is prepared to go after anyone who becomes available at that point.
"We still have some pieces to add," coach Adam Gase said. "We're looking really to kind of see how it plays out. We'll see what goes on after talking with other teams in the league. You just never know who else is going to shake free from now until the start of the season."
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