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Protein tyrosine phosphatase (PTP)-PEST is a critical regulator of cell adhesion and migration. However, the mechanism by which PTP-PEST is regulated in response to oncogenic signaling to dephosphorylate its substrates remains unclear. Here, we demonstrate that activated Ras induces extracellular signal-regulated kinase 1 and 2-dependent phosphorylation of PTP-PEST at S571, which recruits PIN1 to bind to PTP-PEST. Isomerization of the phosphorylated PTP-PEST by PIN1 increases the interaction between PTP-PEST and FAK, which leads to the dephosphorylation of FAK Y397 and the promotion of migration, invasion, and metastasis of v-H-Ras-transformed cells. These findings uncover an important mechanism for the regulation of PTP-PEST in activated Ras-induced tumor progression.
Most cancer deaths are due to tumor invasion and metastasis rather than to primary tumors. Thus, understanding the fundamental mechanisms underlying tumor cell migration, invasion, and metastasis holds the promise of improving therapeutic approaches for cancer (7). Activating mutations of Ras, a small GTP-binding protein, occur in ~30% of human cancers and promote tumor progression (32). Accordingly, considerable effort has been expended in understanding the functions of Ras proteins in tumor cell migration, invasion, and metastasis (6).
Cell migration is a highly coordinated and dynamic multistep process of leading-edge protrusion, focal adhesion turnover, tractional force generation, and tail retraction and detachment, all of which involve precise regulation of cell-cell adhesion and cell-to-extracellular matrix adhesion. Focal adhesions are specific regions of cells that make close contact with the extracellular matrix (30, 44). Functional regulation of the molecules involved in focal adhesion signaling is a key component of tumor cell motility.
Focal adhesion kinase (FAK) is a ubiquitously expressed nonreceptor protein tyrosine kinase that localizes at focal adhesions, mediates signaling induced by integrins, and plays an important role in many cellular functions (26, 27). Activated FAK, marked by autophosphorylation at Y397, recruits a number of SH2 and SH3 domain-containing proteins, including c-Src. The binding of c-Src to FAK is proposed to disrupt the intramolecular interaction between the c-Src SH2 domain and the negative regulatory carboxy-terminal Y529. Activated Src, in turn, phosphorylates FAK and further enhances FAK activity, thereby forming a positive feedback loop leading to the activation of downstream signaling molecules, such as extracellular signal-regulated kinase 1 and 2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3-K)/AKT (17, 29).
FAK can exert control over the rate of focal adhesion turnover and, therefore, cell motility (29). FAK is a positive regulator of normal cell migration, and its overexpression or activation has been shown to promote cancer cell metastasis of some tumor types (17, 29). However, accumulated evidence shows that in response to certain oncogenic signals, FAK negatively regulates cancer cell migration and invasion (4, 5, 9, 15, 16, 21, 24, 25, 31, 37, 38–40). We previously reported that the activation of epidermal growth factor receptor (EGFR) and Ras promotes cancer cell migration by dephosphorylation of FAK Y397. This dephosphorylation may facilitate focal adhesion turnover at the leading edge of cells, which contributes to the promotion of cell invasion and metastasis (21, 44). FAK Y397 dephosphorylation requires ERK1/2-mediated phosphorylation of FAK S910, which recruits protein interacting with NIMA (never in mitosis A)-1 (PIN1). PIN1, a member of the parvulin subfamily of peptidyl-prolyl cis/trans-isomerases (PPIases), specifically recognizes phosphorylated serine (S) or threonine (T) in pS/TP peptide sequences (20). PIN1 catalyzes the cis/trans isomerization of the peptidyl-prolyl peptide bond at FAK S910/P911, which is required for FAK dephosphorylation at Y397 (45).
Protein tyrosine phosphatase (PTP)-PEST is a ubiquitously expressed cytosolic PTP that is named for its proline-glutamine-serine-threonine-rich motifs (PEST sequences) (19, 35, 41, 42). The tightly regulated activity of PTP-PEST plays instrumental roles in focal adhesion breakdown, cell spreading, and cell motility (1, 14, 19). PTP-PEST dephosphorylates several protein kinases and signaling molecules involved in cell migration and transformation, including FAK, p130CAS (a FAK substrate), c-Src, EGFR, leupaxin, growth factor receptor-bound protein 2 (GRB2), and the p66SHC isoform of SHC-transforming protein 1 (SHC1) (19, 37). In response to Ras activation, PTP-PEST binds to FAK and dephosphorylates it at Y397, thereby reducing the total number of focal contacts of transformed cells and promoting migration of tumor cells (2, 44, 45). However, the mechanisms underlying activated Ras-regulated PTP-PEST dephosphorylation of FAK remain elusive.
In this report, we show that Ras-induced PTP-PEST phosphorylation at S571, which is mediated by ERK1/2, creates an interacting motif for PIN1 binding. Isomerized PTP-PEST then interacts with and dephosphorylates FAK at Y397, leading to increased tumor cell migration, invasion, and metastasis.
FAK+/+, FAK−/−, PTP-PEST+/+, and PTP-PEST−/− fibroblasts, U251 glioblastoma cells, and 293T cells were all maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% bovine calf serum (HyClone, Logan, UT).
Monoclonal antibodies against phospho-ERK (sc-7383), glutathione S-transferase (GST) (sc-138), and hemagglutinin (HA) (sc-7392) and polyclonal antibodies against mitogen-activated protein kinase kinase 1 (MEK1) (sc-219), ERK2 (sc-154), ERK1 (sc-94), and FAK (sc-558) were from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibodies against FLAG and tubulin were from Sigma (St. Louis, MO). Monoclonal antibodies against H-Ras and phospho-FAK Y397 and Matrigel were from BD Biosciences (San Diego, CA). The PTP-PEST rabbit polyclonal antibody was from Bethyl Laboratories (Montgomery, TX). Purified active ERK2 protein (P6080) was from New England BioLabs (Ipswich, MA). Purified active GST-ERK5 protein (04-146) was from Carna Biosciences (Kobe, Japan). U0126 was from EMD Biosciences (San Diego, CA). [γ-32P]ATP was from MP Biomedicals (Solon, OH).
A PCR-amplified human PTP-PEST cDNA was cloned into the pcDNA3.1-FLAG vector between the BamHI and NotI restriction enzyme sites. The FLAG-PTP-PEST S571A mutant was generated using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA).
Cells were plated at a density of 4 × 105 per 60-mm-diameter dish at 18 h prior to transfection. Transfection was performed using HyFect reagents (Denville Scientific, Metuchen, NJ) according to the vendor's instructions. The transfection efficiency for 293T cells was about 90%. Stable cell lines were selected with hygromycin (100 μg/ml) for 10 to 14 days at 37°C. After treatment with hygromycin, antibiotic-resistant colonies (more than 100 colonies) were pooled and expanded for further analysis under selective conditions.
Extraction of proteins from cultured cells using a modified lysis buffer was followed by immunoprecipitation and immunoblotting with corresponding antibodies, as described previously (22).
To assess cell migration, we conducted a wound-healing assay. Cells were plated at 70% confluence in DMEM supplemented with 10% fetal bovine serum. At 24 h after seeding, the monolayers were wounded by scoring with a sterile plastic 200-μl micropipette tip, washed, and then incubated in DMEM. After 24 h, cells were photographed using a low-magnification phase-contrast microscope as described previously (21).
Cell lysates (250 μg/sample) were incubated with 100 ng of GST or GST fusion proteins together with glutathione-agarose beads in a modified buffer consisting of 50 mM Tris-HCl (pH 7.5), 1% Triton X-100, 150 mM NaCl, 1 mM 1,4-dithiothreitol, 0.5 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 12 μg/ml leupeptin, 20 μg/ml aprotinin, 100 μM sodium vanadate, 100 μM sodium pyrophosphate, and 1 mM sodium fluoride at 4°C. The glutathione beads were then washed four times with lysis buffer and the bound proteins eluted with sodium dodecyl sulfate (SDS) sample buffer prior to SDS-polyacrylamide gel electrophoresis (PAGE).
GST-PIN1 and the GST-PIN1 WW mutant were expressed in BL21(DE3) cells following induction with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for 4 h at 37°C. Protein was purified from bacterial extracts by incubation with glutathione-Sepharose beads followed by extensive washing with 20 mM Tris (pH 7.4), 150 mM NaCl, and 1 mM dithiothreitol. The GST fusion proteins were eluted with reduced glutathione (10 mM) in 50 mM Tris (pH 7.4). The eluted proteins were dialyzed in phosphate-buffered saline (PBS) for 16 h (23).
His-PTP-PEST in the pCold I vector was expressed in BL21(DE3) cells following induction with 1 mM IPTG for 24 h at 16°C. His-PTP-PEST protein was purified from bacterial cell lysate using Ni-nitrilotriacetic acid (NTA) His-binding resin and eluted with 1 M imidazole. The eluted proteins were dialyzed in PBS for 16 h.
FLAG-PTP-PEST was transiently expressed in 293T cells and immunoprecipitated with an anti-FLAG antibody. The kinase reactions were done by mixing FLAG-PTP-PEST protein with or without purified ERK2 or ERK5 in kinase assay buffer containing 10 μCi of [γ-32P]ATP, 10 mM Tris-HCl (pH 7.4), 5 mM MnCl2, 1 mM dithiothreitol, and 20 μM ATP for 30 min at 37°C. Reactions were stopped by adding an equal volume of 2× SDS-PAGE sample buffer and boiling for 5 min. Samples were then separated by 8% SDS-PAGE and transferred onto nitrocellulose membranes for exposure to X-ray film (11).
Cell invasion was assessed by the invasion of the cells through Matrigel-coated Transwell inserts. Briefly, Transwell inserts with an 8-μm pore size were coated with 100 μl of a final concentration of 1 mg/ml Matrigel in cold serum-free medium. Cells were trypsinized, and cell suspension (500 μl; 1 × 106 cells/ml) was added in triplicate to wells. After a 24-h incubation, cells that invaded the Matrigel and passed through the filter were stained with crystal violet and photographed using a digital camera mounted onto a microscope with a magnification of ×50. The membranes were dissolved in 4% deoxycholic acid and read colorimetrically at 590 nm as previously described (11, 45).
The control vector and the vector expressing FLAG-PTP-PEST or the FLAG-PTP-PEST S571A mutant were separately transfected into 293T cells. Cell lysates were prepared in 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) buffer consisting of 10 mM HEPES (pH 7.5), 1% CHAPS, 150 mM NaCl, 1 mM dithiothreitol, 0.5 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 12 μg/ml leupeptin, and 20 μg/ml aprotinin. FLAG-PTP-PEST was immunoprecipitated using the FLAG antibody, and its activity toward 3,6-fluorescein diphosphate (FDP) was examined using an Enzolyte FDP protein phosphatase assay kit (AnaSpec Co., Fremont, CA). The fluorescein signal was read using a fluorescence plate reader at excitation and emission wavelengths of 485 and 528 nm, respectively (45).
U251 cells with or without depletion of PIN1 were treated with cycloheximide (CHX) (100 μg/ml). The cells were harvested and analyzed at various time points.
FLAG-PTP-PEST immunoprecipitated from v-H-Ras-expressing PIN1−/− cells was incubated with 10 pM purified GST, wild-type (WT) GST-PIN1, or the GST-PIN1 C113A mutant and was then digested with trypsin (5 ng) for 5 min. The digested fragments were separated by 10% SDS-PAGE, followed by immunoblotting analysis with a PTP-PEST antibody.
The GelCode Blue (Thermo Fisher Scientific, Waltham, MA)-stained gel band was subjected to in-gel digestion with 200 ng of modified trypsin (sequencing grade) (Promega, Madison, WI) at 37°C for 24 h. The resultant peptides were analyzed by nano-liquid chromatography-coupled ion trap mass spectrometry with on-line desalting on a system consisting of a FAMOS autosampler, an Ultimate Nano liquid chromatography module, and a Switchos precolumn switching device on a 75-μm by 150-mm C18 column (all from Dionex Corp., Sunnyvale, CA). Electrospray ion trap mass spectrometry was performed on an LTQ linear ion trap mass spectrometer (Thermo Fisher Scientific). Proteins were identified by a database search of the fragment spectra against the National Center for Biotechnology Information nonredundant protein database (NCBInr) using Mascot (Matrix Science, London, United Kingdom) and Sequest (Thermo Fisher Scientific). Phosphopeptide matches were manually curated.
PTP-PEST+/+ or PTP-PEST−/− v-H-Ras-expressing cells with reconstituted expression of WT PTP-PEST or PTP-PEST S571A were subcutaneously injected into athymic nude mice (5 × 106 cells in 0.3 ml PBS per mouse). Three weeks later, the tumor volumes (V) were estimated by measuring two dimensions (length [a] and width [b]) and calculated using the equation V = ab2/2. For the metastasis assay, these cells were injected into the lateral tail veins of athymic nude mice (1 × 105 cells in 0.1 ml PBS per mouse). After 3 weeks, the mice were sacrificed, and lung tissues were excised and fixed with Bouin's solution. Lung surface tumors were counted under a low-power dissecting microscope. Six mice in each group were used, and the standard errors were calculated as the variation in tumor number. Differences were evaluated for statistical significance at a P value of <0.05 using the two-tailed Student t test. Experiments were done twice.
We previously showed that upon Ras activation, PIN1 binds to the complex of FAK and PTP-PEST and is required for the interaction between PTP-PEST and FAK (45). Although FAK is a direct substrate of PIN1 (45), we could not exclude the possibility that PIN1 regulates PTP-PEST simultaneously to promote the binding of PTP-PEST to FAK. To examine whether activated Ras promotes the binding of PIN1 to PTP-PEST independently of FAK, we infected FAK+/+ and FAK−/− cells with a v-H-Ras-expressing adenovirus (Fig. 1A) and performed a GST-PIN1 pulldown assay. As shown in Fig. 1B, the level of PTP-PEST associated with PIN1 was moderate in the absence of v-H-Ras but was significantly enhanced by v-H-Ras expression in both FAK+/+ and FAK−/− cells; the levels of enhanced expression were comparable. The GST-PIN1 WW domain mutant, which contains W11A and W34A mutations in the binding domain, was incapable of binding to PTP-PEST (Fig. 1B). Immunoblotting analysis of the immunoprecipitated PIN1 from FAK−/− cells with an anti-PTP-PEST antibody showed that expression of v-H-Ras resulted in an increased association of endogenous PIN1 with endogenous PTP-PEST (Fig. 1C). These results indicate that Ras activation promotes the interaction between the PIN1 WW domain and PTP-PEST in a FAK-independent manner. Similarly, expression of v-H-Ras (Fig. 1D) induced the interaction between GST-PIN1 and FAK in PTP-PEST−/− cells to a level comparable to that in PTP-PEST+/+ cells (Fig. 1E). These results indicate that both PTP-PEST and FAK can bind to PIN1 independently.
Depletion of PIN1 from U251 glioblastoma cells (Fig. 2A) largely inhibited v-H-Ras-induced FAK dephosphorylation (Fig. 2B), which was in line with our previous findings that PIN1 is required for v-H-Ras-regulated FAK phosphorylation (45). A pulse-chase experiment showed that the presence or absence of v-H-Ras or PIN1 did not significantly change the expression or half-life of PTP-PEST (Fig. 2C), suggesting that PTP-PEST-dependent FAK dephosphorylation is not due to regulation of the PTP-PEST expression level. Incubation of FAK immunoprecipitated from v-H-Ras-expressing PTP-PEST−/− cells with FLAG-PTP-PEST immunoprecipitated from v-H-Ras-expressing PIN1−/− cells did not result in increased association of FAK with PTP-PEST or FAK dephosphorylation (Fig. 2D). However, preincubation of purified WT PIN1, but not of an inactive PIN1 C113A mutant, with immunoprecipitated FLAG-PTP-PEST enabled PTP-PEST to bind to and dephosphorylate FAK (Fig. 2D). An in vitro phosphatase assay showed that PTP-PEST phosphatase activity toward 3,6-fluorescein diphosphate (FDP) was not affected by incubation with purified WT PIN1 or the PIN1 C113A mutant (Fig. 2E), which suggests that PIN1 promotes the binding of PTP-PEST to FAK without affecting its PTP-PEST activity. To test whether PTP-PEST is a direct substrate of PIN1 for cis-trans isomerization, we incubated FLAG-PTP-PEST immunoprecipitated from v-H-Ras-expressing PIN1−/− cells with purified GST, WT GST-PIN1, or the GST-PIN1 C113A mutant and then digested the samples using trypsin. Immunoblotting with an anti-PTP-PEST antibody showed that PTP-PEST incubated with WT GST-PIN1 had less full-length protein after digestion than those incubated with GST or GST-PIN1 C113A and lacked an ~75-kDa digested product (Fig. 2F). These results strongly suggest that PIN1 isomerase activity alters the conformation of PTP-PEST and leads to a change of protease susceptibility.
PIN1 binds to phosphorylated serine or threonine residues in pS/TP-peptide sequences in its substrates (20). To test whether PTP-PEST is phosphorylated upon Ras activation, we transfected FLAG-PTP-PEST alone or in combination with v-H-Ras into 293T cells, immunoprecipitated PTP-PEST, and performed an in vitro kinase assay. Figure 3A shows that PTP-PEST was significantly phosphorylated by PTP-PEST-associated protein kinases only in the presence of v-H-Ras expression. These results suggest that Ras expression activates a downstream protein kinase that associates with and phosphorylates PTP-PEST.
Given that ERK1/2 are downstream from Ras signaling and can phosphorylate FAK, we examined whether ERK2 is able to also phosphorylate PTP-PEST. Incubation of purified active ERK2 or ERK5 with immunoprecipitated, heat-inactivated FLAG-PTP-PEST showed that ERK2, but not ERK5, phosphorylated PTP-PEST (Fig. 3B). In addition, immunoblotting of immunoprecipitated ERK2 with an anti-FLAG antibody showed that v-H-Ras expression significantly increased the interaction between ERK2 and FLAG-PTP-PEST (Fig. 3C). Furthermore, ERK activation by coexpression of a constitutively active MEK1 mutant (MEK1 Q56P) resulted in enhanced association of PTP-PEST with HA-ERK2, which was demonstrated by immunoblotting of the immunoprecipitated FLAG-PTP-PEST with an anti-HA antibody (Fig. 3D). These results indicate that Ras-induced ERK2 activation increases the binding of ERK2 to PTP-PEST.
To test the effect of ERK1/2 activation on activated Ras-induced association between PIN1 and PTP-PEST, we treated FAK−/− cells expressing v-H-Ras with the MEK inhibitor U0126. U0126, which inhibited v-H-Ras-induced ERK1/2 phosphorylation and subsequent activation, blocked Ras-induced GST-PIN1 binding to PTP-PEST (Fig. 3E). These results suggest that ERK1/2-mediated PTP-PEST phosphorylation is required for PIN1 to bind to PTP-PEST.
Analysis of the PTP-PEST amino acid sequence revealed that PTP-PEST has 17 putative ERK1/2 phosphorylation sites. To identify the ERK1/2-mediated phosphorylation sites of PTP-PEST, we analyzed a tryptic digest of purified PTP-PEST phosphorylated by ERK2 by mass spectrometry. One candidate phosphopeptide spanning amino acids 565 to 592 was detected by nano-liquid chromatography online to mass spectrometry. S571 was identified as the likely phosphorylation site in this peptide (Fig. 4A). The S571A mutation reduced ERK2-dependent phosphorylation of PTP-PEST in vitro (Fig. 4B). These results indicate that ERK2 phosphorylates PTP-PEST primarily at S571. Consistent with the results showing that ERK activation is required for Ras-induced interaction between PIN1 and PTP-PEST, the PTP-PEST S571A mutation reduced PTP-PEST's binding to GST-PIN1 (Fig. 4C). These results indicate that ERK2 phosphorylates PTP-PEST at S571 and that this phosphorylation is required for the interaction between PTP-PEST and PIN1.
Ras activation induces the binding and dephosphorylation of FAK by PTP-PEST (45). To test the effect of ERK1/2-mediated PTP-PEST phosphorylation on FAK Y397 dephosphorylation, we stably transfected WT FLAG-PTP-PEST or the FLAG-PTP-PEST S571A mutant into PTP-PEST−/− cells (Fig. 5A). Consistent with our previous observations (45), expression of v-H-Ras resulted in FAK dephosphorylation at Y397 in WT PTP-PEST-expressing cells, and this dephosphorylation was blocked by U0126 treatment (Fig. 5B). In contrast, v-H-Ras-induced FAK Y397 dephosphorylation was largely inhibited in the cells expressing the PTP-PEST S571A mutant. These results indicate that Ras-induced PTP-PEST S571 phosphorylation is required for PTP-PEST to dephosphorylate FAK Y397.
PIN1 promotes the binding of PTP-PEST to FAK without affecting PTP-PEST activity (Fig. 2D and E), suggesting that ERK-dependent phosphorylation of PTP-PEST affects PTP-PEST's ability to bind FAK but not PTP-PEST's phosphatase activity. To further validate this implication, we immunoprecipitated FLAG-PTP-PEST and FLAG-PTP-PEST S571A from 293T cells expressing v-H-Ras and measured PTP-PEST phosphatase activity toward FDP. As shown in Fig. 5C, the PTP-PEST S571A mutant had phosphatase activity comparable to that of WT PTP-PEST, which indicates that ERK-dependent phosphorylation of PTP-PEST does not significantly alter its phosphatase activity. To determine whether the PTP-PEST S571A mutation affects the binding of PTP-PEST to FAK, we immunoprecipitated FAK from v-H-Ras-expressing PTP-PEST−/− cells that reconstituted the expression of WT PTP-PEST or the S571A mutant. Immunoblotting with an anti-FLAG antibody showed that expression of v-H-Ras dramatically enhanced the association between FAK and WT PTP-PEST and that this association was blocked by U0126 treatment. In contrast, PTP-PEST S571A failed to bind to FAK in response to v-H-Ras expression (Fig. 5D). These results indicate that ERK1/2-mediated PTP-PEST phosphorylation promotes the interaction between PTP-PEST and FAK and thus facilitates the dephosphorylation of FAK Y397 by PTP-PEST.
The reconstituted expression levels of WT PTP-PEST and the PTP-PEST S571A mutant in PTP-PEST−/− cells were comparable to the expression level of endogenous PTP-PEST in PTP-PEST+/+ cells (Fig. 6A). To examine the effect of ERK1/2-mediated PTP-PEST S571 phosphorylation on v-H-Ras-induced regulation of focal adhesion, we immunostained vinculin, a marker of focal adhesions, and showed that expressing v-H-Ras in PTP-PEST+/+ cells changed the cell morphology to spindle shaped and reduced the number of focal adhesions (Fig. 6B). In contrast, PTP-PEST−/− cells, which had more focal adhesions than PTP-PEST+/+ cells, were largely resistant to v-H-Ras-induced morphological changes and reductions in the number of total focal adhesions. However, this resistance was abrogated by reconstituted expression of WT PTP-PEST, but not of the PTP-PEST S571A mutant, in PTP-PEST−/− cells (Fig. 6B). These results strongly suggest that v-H-Ras-induced PTP-PEST phosphorylation is instrumental in the v-H-Ras-induced changes in focal adhesions and cell morphology. To examine the role of PTP-PEST in v-H-Ras-induced cell migration, we next conducted a monolayer wound-healing assay. Compared with PTP-PEST+/+ cells, PTP-PEST−/− cells showed a reduced migration rate in response to infection with adenovirus expressing v-H-Ras (Fig. 6C). This migration inhibition was rescued by reconstituted expression of WT PTP-PEST but not of the PTP-PEST S571A mutant. Similarly, a Matrigel invasion Transwell assay showed that PTP-PEST deficiency blocked v-H-Ras-induced cell invasion, and this effect was abrogated by reconstituted expression of WT PTP-PEST but not by expression of the PTP-PEST S571A mutant (Fig. 6D). These results indicate that ERK1/2-mediated PTP-PEST S571 phosphorylation promotes v-H-Ras-induced cell migration and invasion.
We did not find any obvious changes in cell proliferation when growing v-H-Ras-expressing PTP-PEST+/+, PTP-PEST−/−, or PTP-PEST−/− cells with reconstituted expression of WT PTP-PEST or the PTP-PEST S571A mutant in 1% or 10% serum (Fig. 7A). All these cell types induced similar tumor growth rates when subcutaneously injected into athymic nude mice (Fig. 7B). These results indicate that ERK1/2-mediated PTP-PEST S571 phosphorylation does not affect v-H-Ras-promoted cell proliferation or tumorigenesis.
To examine the effect of PTP-PEST S571 phosphorylation on v-H-Ras-induced cell metastasis, we injected the same cell types into the tail veins of athymic nude mice. Dissection of the mice 3 weeks later revealed that PTP-PEST−/− cells expressing v-H-Ras had generated fewer metastatic nodules in the lungs of the mice than PTP-PEST+/+ cells that expressed v-H-Ras (Fig. 7C). Compared to the reconstituted expression of WT PTP-PEST, expression of the PTP-PEST S571A mutant largely inhibited Ras-induced metastasis. In addition, immunoblotting analysis of tumor tissues showed that the phosphorylation levels of FAK Y397 and p130CAS were increased in tumors induced by injecting mice with v-H-Ras-expressing PTP-PEST−/− cells with or without expression of the PTP-PEST S571A mutant. Conversely, tumors induced by injecting v-H-Ras-expressing PEST+/+ or PTP-PEST−/− cells with reconstitution of WT PTP-PEST did not show increased FAK Y397 phosphorylation (Fig. 7D). These results indicate that ERK1/2-mediated PTP-PEST S571 phosphorylation promotes v-H-Ras-induced metastasis.
Ras is either mutated or activated in many types of human cancer (6, 8). However, the mechanism by which Ras regulates focal contacts and consequently promotes tumor cell motility remains to be further elucidated. We have demonstrated here that activated Ras regulates the binding of PTP-PEST to FAK via ERK1/2-mediated phosphorylation and PIN1-dependent isomerization of PTP-PEST. The increased interaction between PTP-PEST and FAK leads to FAK dephosphorylation at Y397 in v-Ras-transformed cells and promotes cell migration, invasion, and metastasis.
PTP-PEST has been implicated in regulation of focal adhesion disassembly and cell migration (1, 14). PTP-PEST−/− mouse fibroblasts have more focal adhesions, spread faster on fibronectin substrate, and have slower migration than PTP-PEST+/+ cells (1), all of which suggest that PTP-PEST promotes focal adhesion turnover and cell migration. Mutations of PTP-PEST that alter its catalytic activity have been found in both breast and squamous carcinoma cells (34), which implies a role for misregulation of PTP-PEST in tumor cell motility. PTP-PEST can be posttranslationally modified, which affects its catalytic activity or its access to substrates. It has been shown that treatment of HeLa cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), forskolin, or 3-isobutyl-1-methylxanthine results in phosphorylation of the S39 and S435 residues of PTP-PEST, which is regulated by cyclic AMP-dependent protein kinase A (PKA) and protein kinase C (PKC) (13). Phosphorylation of S39 in vitro reduces PTP-PEST's affinity for its substrate. In addition, PTP-PEST immunoprecipitated from TPA-treated cells displays reduced phosphatase activity (13). In line with the fact that PTP-PEST can be regulated by phosphorylation, we have shown here that activation of ERK1/2 by Ras results in phosphorylation of PTP-PEST at S571. Phosphorylation of S571, which by itself does not alter the catalytic activity of PTP-PEST toward phosphopeptide substrates, recruits PIN1 for the cis-trans isomerization of PTP-PEST. The conformational changes of PTP-PEST induced by PIN1, which may alter the structure of the protein-interacting domains adjacent to S571 (10, 13), increased PTP-PEST's association with FAK and led to FAK dephosphorylation. The fact that mutation of S571 in PTP-PEST inhibits v-H-Ras-induced FAK Y397 dephosphorylation and cell migration, invasion, and metastasis highlights the significance of ERK1/2-mediated PTP-PEST phosphorylation in v-H-Ras-enhanced tumor cell motility.
PIN1 isomerizes the phosphorylated pS/TP bonds in its substrate proteins and catalytically induces their conformational change, which can be involved in regulating protein-protein interactions (18, 46). We previously showed that activation of Ras and ERK1/2 results in the colocalization and interaction of PIN1, PTP-PEST, and FAK at the lamellipodia, the front leading edge of cells (45). ERK1/2 phosphorylate FAK S910 and recruit PIN1 for FAK cis-trans isomerization, which leads to interaction between FAK and PTP-PEST (45). However, Ras can still induce the interaction between PIN1 and ERK1/2-phosphorylated PTP-PEST in FAK−/− cells. In addition, Ras promotes the association between PIN1 and ERK1/2-phosphorylated FAK in PTP-PEST−/− cells. These results indicate that ERK1/2- and PIN1-mediated phosphorylation and the isomerization of both FAK and PTP-PEST occur simultaneously in a mutually independent manner. Nevertheless, both events are coordinated in the regulation of the interaction between FAK and PTP-PEST and subsequent FAK Y397 phosphorylation.
Increased FAK expression and activity, both of which promote tumor growth and metastasis, have been detected in several types of human cancer (17, 26, 29, 33). In contrast, paradoxical evidence shows that FAK expression was reduced in metastatic tumors compared to their corresponding primary tumors from patients with colorectal adenocarcinoma, cervical cancer, and intrahepatic cholangiocarcinoma (3, 12, 28). Previously, we and other groups showed that activation of the ErbB family members EGFR, ErbB2, ErbB3, and ErbB4 and of the insulin-like growth factor 1 (IGF-1) receptor induced Tyr dephosphorylation of FAK, inactivation of FAK, and increased tumor cell motility, invasion, and metastasis (4, 5, 9, 15, 16, 21, 24, 25, 36, 38). In response to IGF-1 signaling, SHP2 is instrumental in tumor cell invasion (25), and SHP2 activity is required for FAK Tyr dephosphorylation (25, 43). Notably, activation of EGFR and Ras results in a reduction of total focal adhesions, with limited focal contacts remaining at the leading-edge lamellipodia and tail regions (21, 44, 45). Moreover, FAK phosphorylation can be dynamically regulated because EGFR- and Ras-induced FAK dephosphorylation is overridden by activated integrin signaling during cell attachment. However, integrin-induced FAK phosphorylation occurs only during the process of adhesion and is downregulated by EGFR and Ras signaling thereafter (21, 44). On the basis of all these findings, we proposed that overexpression of FAK in human tumor cells might contribute to malignancy by promoting survival through the activation of a positive FAK-c-Src feedback loop and subsequently of downstream tumor-promoting molecules, such as ERK and AKT (17, 29, 45). However, some other tumors that have aberrant Ras activity, which does not require Src for its transforming ability (data not shown), can activate cell proliferation molecules such as ERK and AKT in a FAK-c-Src-independent manner. Activated Ras signaling inhibits FAK activity, which leads to reduced cell adhesion and increased cell migration. Importantly, FAK activity can be dynamically regulated by integrated Ras and integrin signaling, which may play an instrumental role in the regulation of lamellipodial dynamics and turnover of focal adhesions at the leading edge of the cells during migration (44, 45). Thus, FAK might have different roles in different tumors and at different stages of tumor progression.
In summary, we have uncovered an important mechanism of PTP-PEST-regulated FAK dephosphorylation in response to oncogenic Ras signaling (Fig. 8). Activation of Ras results in phosphorylation of FAK S910 and PTP-PEST S571 by ERK1/2, which leads to the recruitment of PIN1 and the cis-trans isomerization of both FAK and PTP-PEST. The conformational alterations of FAK and PTP-PEST enable them to bind each other to facilitate subsequent FAK Y397 dephosphorylation and enhanced tumor cell migration, invasion, and metastasis.
We thank Tony Hunter (The Salk Institute for Biological Studies) for FAK knockout cells, Anthony Means (Duke University) for PIN1 knockout cells and constructs, Michel L. Tremblay (McGill University) for PTP-PEST knockout cells, Steven Reed (The Scripps Research Institute) for a vector expressing pGEX-4T GST-PIN1 WT and the WW mutant, Kevin Pumiglia (Albany Medical Center) for the v-H-Ras adenovirus, and Zach Bohannan for critical reading of the manuscript.
This work was supported by National Cancer Institute grants 5R01CA109035 (Z.L.), 5 P50 CA127001-03, and CA016672 (Cancer Center Support Grant), research grant RP110252 (Z.L.) from the Cancer Prevention and Research Institute of Texas, American Cancer Society Research Scholar Awards RSG-09-277-01-CSM (Z.L.) and RSG-08-288-01-GMC (D.X.L.), an institutional research grant from The University of Texas MD Anderson Cancer Center (Z.L.), and Department of Defense grant W81XWH-09-1-0349 (D.X.L.).
Published ahead of print on 29 August 2011.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


text: NewsBusiness Lonrho considers African float Thursday 22 June 1995 23:02 BST Click to follow Lonrho is considering a flotation of most of its African trading businesses within the next 12 months, Dieter Bock, chief executive, said yesterday. The company, which is also considering a flotation of its Metropole and Princess hotel chains, announced a 53 per cent rise in underlying profits for the six months to March to pounds 52m. Earnings per share jumped 113 per cent to 3.2p and an interim dividend of 2.25p was declared.Investment Column, page 26 More about: Africa Hotels Investment Savoy Hotel Reuse content Comments

subset: commoncrawl


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- About Behind the Badge Foundation
- Roll Call of Honor
- Resources & Support for Families
- Donate
- Washington State Law Enforcement Memorial
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Germain, Officer Frederick A.
End of Watch:
Friday, July 21, 1922
Spokane P.D.
On Friday, July 21, 1922, Officer Frederick Germain of the Spokane Police Department was killed during a pursuit.
Officer Germain was in pursuit of a speeding car being driven by someone later identified as a repeat offender well known to local police.
As Officer Germain was pursuing the car, a large truck swerved to avoid a collision and it drove into a ditch. The truck spun around and then struck Officer Germain, who was riding a motorcycle.
Officer Germain died instantly.


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Obama's New Year Resolution: More Middle East War - by Stephen Lendman
Syria and Iran are targeted for regime change.
Syria and Iran are targeted. Regime change is planned. At issue is replacing them with client ones, controlling the region's strategic resources, and depriving key rivals China and Russia from access.
Pressure keeps building relentlessly. For months, Syria's been ravaged by externally generated violence. Its economy's also suffered enormously. According to a Damascus University assessment:
"The general financial situation of the country is suffering from the inability of the state budget because of the inability of the general revenue to cover expenses."
Moreover, conditions ahead look worse because tax revenues are half what's needed. Economic sanctions also impede oil revenues. As a result, the estimated 2012 budget deficit will be about 529 billion Syrian pounds ($9 billion dollars) out of a total $1,316 billion budget. A 40% revenue shortfall amounts to 18% of GDP.
Combined with accumulated deficits, financial strain is hugely disruptive. Syria's been cut off from credit markets. Traditionally, Central Bank loans funded deficits. However, sufficient reserves are lacking. Prices keep rising. Purchasing power further erodes. In the past six years, it's fallen 40 - 50%.
The solution requires banks and corporate Syria to help. They're able to provide considerable government funding from their "enormous profits during the economic reform and liberalization process." Doing this can be done through laws specifying amounts, rates and terms.
Syria can only raise 60% of budget needs. It can't borrow so needs other ways to cover the shortfall. Currently, 59 Syrian pounds = one US dollar. So far, economic collapse has been prevented. However, it's coming without help, and with it greater pressure on an already beleaguered regime.
Washington Intervention Looms
In mid-December, State Department official Fred Hof called Syria a "dead man walking," saying it can't hang on much longer. More serious are reports of Washington preparing to intervene.
Options include a no-fly zone, a humanitarian corridor or safe zone along the Syrian/Turkish border, increasing aid to insurgent forces, bring in greater numbers, tightening sanctions further, perhaps a blockade, and if other measures fail, then war.
In fact, under international law, blockades are acts of war. They're variously defined as:
- surrounding a nation or objective with hostile forces;
- measures to isolate an enemy;
- encirclement and besieging;


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As you're celebrating motherhood this weekend, take a few moments to kick your feet up, enjoy a drink and pat yourself on the back for all you do. Moms work hard – damn hard – every single day. Sure, there are nights we hit our pillows in sheer exhaustion (every night), but parenting brings joy.
Kay Warren, wife to the popular Christian pastor Rick Warren writes …
We tend to think that life comes in hills and valleys. In reality, it's much more like train tracks. Every day of your life, wonderful, good things happen that bring pleasure and contentment and beauty to you. At the exact same time, painful things happen to you or those you love that disappoint you, hurt you, and fill you with sorrow. These two tracks — both joy and sorrow — run parallel to each other every single moment of your life.
So motherhood brings happiness, sadness, frustration, anticipation … joy.
Some might argue Mother's Day is just one more holiday created by the greeting card companies to get us to spend and consume. Perhaps. But moms deserve a day – more than a day – to receive some special attention and thanks.
So Happy Mother's Day, and to give you even more reason to smile at the magnitude of moms, check out this cool infographic below. We kind of rock. Just saying!
image courtesy of Personal Creations


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The KITRIS Webclient is the centrepiece of the KITRIS Tennis System providing you with statistics, analysis and much more.
The KITRIS Webclient provides web-based access to your tennis diary. It contains all your tennis activities. Here, you will find each stroke tracked with your KIT, and each minute you have spent on the court is documented. Each break point you have won and all your forehand winners are visualized in the Webclient.
You will find further information on the product under FAQ and in the shop area of the company website.


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The slow form (慢拳 man quan) comprises defined sequences of certain movements which are executed in a slow and smooth manner. By learning and exercising the form one trains the power of concentration, the internalization of principles of movement, and calming of the mind. Altogether the slow form includes 100 movements and postures which are subdivided in 6 parts. Running through the whole form takes about 30-40 minutes. To shorten and simplify the form for beginners, Ma Yue Liang and Wu Ying Hua developed a short form which is easier to learn and only takes about 10 minutes to run through while still comprising the most important movements and postures.
Beside these two slow forms there is also a fast form (快拳 kuai quan) whose movements are faster and more powerful than the ones of the slow or short form. The solo weapon forms of the sabre, spear, and sword are carried out with similar speed and power.
As the forms consist of well defined sequences of movements which are not easy to learn by heart as a beginner, many questions arise about why it has to be like this. Why is it important to follow and copy the exact movements from a teacher? The answers to these questions can only be found piece by piece in continuous practice of the forms, because the inner logic of the movements has to be encompassed by the feeling for ones own body and mind. Naturalness plays a central role, but this does not to equate with an individual discretionary attitude. Quite the contrary, the term naturalness comprises the following of certain principles of movement according to the bodys physiology. One of these principles is to keep focus and balance central to the process. The first aspect of this principle highlights the physical middle of a bodys centre of balance which changes while moving. A second aspect includes the mind as pictures and thoughts that should be hold together and not easily drift away while practising.


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Jump to navigation
Quickly identify the key themes from Wayne Swan, Tony Abbott and Christine Milne's 2013 budget speeches.Ryan Sheales
The Liberals claim power prices have gone up 94% under Labor. Is that true? Dylan McConnell
Preferential and compulsory voting are uniquely Australian traditions.Angus Ferguson
Elections are won or lost in marginal seats. This analysis examines the electorates that will decide who reigns supreme on September 7th.Dr Scott Brenton.
No surprise - the Coalition won. But here's your one-stop-shop for each House of Representatives seat. Maria Rae
Our resident cartoonist explains how Kevin Rudd went about fixing his cabinetWes Mountain
"I'm Instagraming, I'm Tweeting, I'm eating with Keating..."Melbourne University
Law Review
Join University of Melbourne students as they look at the best and worst campaign ads and discuss the chances of Peter Beattie and the Greens.The Farrago Team
Voters deserve an honest appraisal of the party platforms. Nicholas Reece
The Coalition might find dealing with the new Senate is like herding cats. Dr Damon Alexander
The 'average' member of cabinet/shadow cabinet is very different to the 'average' Australian.Sally Young
The Federal Government could save hundreds of millions of dollars each year without affecting patient outcomesTerence Cheng
Find out what happened when group of Australia's top political journalists gathered to discuss election reporting.Ryan Sheales
Sometimes the only thing that will get noticed in an election campaign is an orange fish costume. Cameron Wheatley
Think you know where you stand on the various parties' policies? Test yourself using Vote Compass, an interactive tool to really get you thinking about policy.
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Mark Wahlberg Vs. San Diego?
Wed, 11 Aug 2010 11:27:23
Mark Wahlberg Videos
From our "WTF" files: Mark Wahlberg is considering taking legal action against bosses of a public aquarium in San Diego, California, after a "giant" captive octopus attacked his young son.
The actor and his wife Rhea were on family holiday with their four kids, but the trip took a frightening turn at the Sea Life Aquarium in Carlsbad, when an aggressive sea creature latched on to Rhea, their four-year old son Michael and the family's tour guide. Wahlberg recalled the incident during an interview on David Letterman's late night TV show on Thursday.
He said: "We have a tour guide so she's like, 'You have a special treat today, we'll be able to take you behind the tank and you'll be able to see the octopus...' So this woman pulls out this gigantic octopus, the thing latches on to my son's arm. It's got my wife, (it's) wrapped completely around the lady, and another guy's just standing there. They can't get this octopus off.
"My son is freaking out but no sound is coming out of his mouth. So finally we rip the thing off of him. He's got all these suction marks that look like about 30 little hickeys. The lady can't get out, she's yelling for help... He (the aquarium manager) said it's never been that aggressive."
It was Letterman who urged the actor to take legal action and Wahlberg admitted he just might take the host's advice.
Should Mark Wahlberg sue?
—Amy Sciarretto


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Historic hotel
12 Tran Phu St, Da Lat T: (063) 382 5444 F: (063) 382 5666
Perched atop an impressive sweeping lawn and set of stairs that runs down to the lake, Dalat Palace Luxury Hotel (formerly Sofitel Dalat Palace) is a keystone building of the town.
Check rates at Dalat Palace Luxury Hotel on Agoda.com
Check rates at Dalat Palace Luxury Hotel on Booking.com.
By Cindy Fan
Last updated on 11th February, 2016.
The Travelfish newsletter is sent out every Monday and is jammed full of free advice for travel in Southeast Asia. You can see past issues here.


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This is a stout powerful filly that should have a lot of athletic ability and cow sense. She's good looking too. A real nice prospect that will really…SOLD
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Offered for Sale- Hesa Premium Skip 2001 AQHA Palomino All around Show Gelding standing at 16.2 he's a is a beautiful show gelding, with fantastic presence…SOLD


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2017April 28-30
June 2-4
July 28-30
September 15-17
October 20-22
November 17-19
SUPER GIVERS 2017Quaaout Resort
Chase, BC
April 7-9, 2017
October 27-29, 2017
2017April 26-30
May 31-June 4
July 26-30
September 13-17
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November 15-19
SUPER CHOICES 2017Quaaout Resort
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April 3, 2017
October 23, 2017
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River's Edge, Cremona, Alberta
View Informational Meeting Calendar
All Calgary Choices, Givers I and Givers II Seminars are held at:
Clarion Hotel (formerly Radisson Hotel) Calgary Airport
2120- 16th Avenue NE
Calgary, Alberta, T2E 1L4
Phone: 403-291-4666
For more information or to make your reservations by phone contact:
Choices Seminars
Bay 117, 10555 - 48th Street SE
Calgary Alberta, T2C 2B7
Phone:403-289-5019 Fax: 403-220-1280
Toll Free: 1-877-707-7074
Email: firstname.lastname@example.com
I attended Choices in January 2005 and I now have the tools to use each day so I can live the life I want. I became an employee in September 2009 because every day I have the opportunity to love and touch other people so they can be the best they can be. I am humbled when I have the opportunity to listen to people's life stories and encourage them. I understand why they want to come to Choices and I want to be the person who helps them begin this journey. The reward is to watch them go through the processes and see the light in their eyes and their life on fire again.
"I am a strong and confident woman loving and touching other people lives to be the best they can be" Barb Michalko
(403) 289-5019
I became a member of the Choices staff in October 2015 and feel blessed every day to be part of such an amazing team. Working with people has always been where I am most comfortable and helping them on their Choices journey helps me on my journey.
"I love myself, nurturing the souls of others"
Reluctantly we agreed to go and are so thankful we did. It has made a good marriage that much better and God has used Choices to improve my relationship with myself, my wife and children, my relationships with my extended family and friends and my relationship with Him.
What I love about the seminar is that they are not there to tell you what to do; instead they present processes designed to allow you to discover things about yourself and for me, something discovered sticks. As well, we all know there are very few of us who like to "be told" what to do.
It was a gift to go to Choices and I encourage anyone to give themselves this gift and discover how it can bless not only themselves but those around them. It's a gift that can keep on giving.
(403) 700-7140
Brian Balson attended Choices in July 1996 in BC. He then attended Super Choices and Super Givers. After moving to Alberta in 2005 and keeping involved with Choices, Brian now hosts information meetings in various locations in Alberta and Saskatchewan.
Brian welcomes your calls, emails, or any opportunity to discuss any questions you may have about Choices.
Please come to the Choices information meetings and hear what Choices has done for others, as well as what it can do to enhance your life.
(403) 369-9003
" I am an open woman giving HOPE through encouragment and inspiration so others may heal" Katrina Chaytor(403) 289-5019


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> Date: Thu, 15 Nov 2012 17:05:02 -0500
> From: UNNews
> Subject: UN CHIEF WELCOMES SWEARING-IN OF NEW SOMALI CABINET MINISTERS > To:
> UN CHIEF WELCOMES SWEARING-IN OF NEW SOMALI CABINET MINISTERS > New York, Nov 15 2012 5:05PM
> United Nations Secretary-General Ban Ki-moon welcomed the swearing-in today of the cabinet of ministers put forward by Somali Prime Minister Abdi Farah Shirdon and endorsed by the country's parliament. >
> "This marks another important step in the determined efforts by Somalia's new leadership to bring about a positive change in Somalia," Mr. Ban's spokesperson added in a <"http://www.un.org/sg/statements/index.asp?nid=6429">statement. >
> Prime Minister Shirdon announced the composition of cabinet – known as the Council of Ministers – on 4 November and the parliament endorsed the nominations on 13 November. The 10-member cabinet includes Deputy Prime Minister and Minister of Foreign Affairs Fauzia Yusuf Haji Adan, reportedly the first woman to hold such a senior position in the Horn of Africa nation. >
> Earlier this week, the Secretary-General's Special Representative for Somalia, Augustine Mahiga, welcomed parliament's endorsement of the cabinet ministers as a "historic milestone" for the county, which has been undergoing a peace and national reconciliation process in recent months that led to the end of a nine-year political transition period. >
> Mr. Mahiga, who also heads the UN Political Office for Somalia (UNPOS), commended Mr. Shirdon as well as President Hassan Sheikh Mohamud for their consultations with a wide cross-section of Somali society in the lead-up to the appointment of the cabinet, "which bears all positive hallmarks of a potential good government." >
> The Secretary-General and his Special Representative look forward to working with the President and his Government to implement the six-pillar policy that will guide the country to democratic elections in the course of the next four years, today's statement added. >
> That policy aims to secure progress in the areas of stability, economic recovery, peacebuilding, service delivery, international relations and unity. > ________________
> For more details go to UN News Centre at http://www.un.org/news >
> Follow us on Facebook (http://www.facebook.com/UN.News.Centre) and Twitter (http://twitter.com/UN_News_Centre) >
> To change your profile or unsubscribe go to: http://www.un.org/apps/news/email/
> Date: Thu, 15 Nov 2012 17:05:02 -0500


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Your browser does not support iframes.
To the editor: To residents of Carolina Shores: I wish to offer a formal apology for what appears to be an error in judgment with regards to last month's closed session. I was under the impression everything talked about was connected to the personnel issue.
The issues that have faced this town over the past months are so intertwined it's hard to figure out what is a personnel issue and what is not. The last thing we as a council want to do is have personal issues as well as personnel issues talked about in open session.
To the editor: Regarding Norm Harding's editorial tribute to his best friend Bob: While I'm sorry for his loss I wonder if his "free spirit" best friend also visited my yard, depositing the results of his feedings in my flowerbeds and/or driveway.
I also wonder if Bob parked himself by my bird feeders and birdbath much to the consternation of my feathered friends.
To the editor: I recently attended the Brunswick County Health Department meeting to witness discussion of the recent purchase of a "refurbished" gas chamber.
The mere word "gas chamber" angers most civilized people. After all, it has been outlawed as inhumane for the most monstrous of human criminals.
To the editor We would like to begin this letter by saying we are disappointed in the actions of one or more individuals that vandalized a home on Governors Road in Winnabow on Saturday, Dec. 19.
To let those of you know a little about this particular home, every holiday of the year is celebrated with a slew of inflated yard ornaments. This family spends a great deal of time and money every year to make all of our holidays a little brighter.
To the editor: In last week's Beacon, Rich Cerrato charged the old council in Sunset Beach as being arrogant in their vote to appoint Bob Bobinski as chairman of the town's ABC Board.
What we did was appoint the most qualified candidate after interviewing each one. Don't believe me? Just ask the other people, including new council members who were present at the interviews.
For many, the holiday season is a time filled with joy. From gatherings of friends and family to participation in religious and civic activities, the months of November and December provide much to do.
But the holidays can also be an unfortunate time—a time when ne'er-do-wells are all too intent on dashing holiday spirits.
To the editor: It is hard to believe here in America I have to fear oppression and theft of my property rights from a local government. A government that was formed in fear they would be annexed is now in the process of performing that same atrocity on its less prosperous neighbors.
The town that already brags they have more than $100 million in amenities feels the need to step on a handful of people who have little more than a will to fight.
Carolina Shores Mayor Stephen Selby said recently the town is at a point of "forgiveness and forgetfulness…We're starting new and afresh now. Those days are behind us."
Seems to us those days are yet behind the folks at Carolina Shores. Their antics of discussing things in closed session that should be discussed openly continue.
Copyright © 2017
The Brunswick Beacon is your source for local news, sports, events and information in Brunswick County, North Carolina and surrounding areas.


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Ships in 1 week
From the vaults of the world's most esteemed jewelers, this lavishly illustrated treasury features gem-laden blooms that will never fade away. Throughout history, flowers have been the inspiration for jewelers and the people who wore their creations.
For more ideas check out 5 creative ways to organize your bookshelves


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Probiotics form a defensive barrier inside protecting us from free radicals and bad bacteria
Could Your Weight Gain be Caused by an Underactive Thyroid?
Want to live a healthy and natural life while keeping your body in the best possible shape?
Bad Behavior has blocked 152 access attempts in the last 7 days.


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|Message view
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|James Todd <james.t...@eng.sun.com>
|request for review: server/config discussion
|Mon, 19 Jul 1999 08:41:42 GMT
hi - attached is a first cut at collecting the various thoughts surrounding a "pure java http server configuration" project. this is not meant to be a holy relic or artifact or even the truth but is more a collection of thoughts and decission points, which are not final, expressed to date presented in a format which should allow for peer review and editing (althought this later part will be a bit sticky till we have a bit more infrastructure to help out). anyways, i welcome any/all feedback and anticipate subsequent drill-downs on some of the topics which are barely touched upon. the area i'd really like to see some active drill down on is: 2. Configuration Data Consumer Model or "entity data model" i'm a big fan of tufte and uml ... so, in order to convey some of these design thoughts i'm likely to start stitching together some graphics ... although this'll be a bit rough to archive and reference till these docs have a web home. note: i was fortunate enough to attend a day long seminar presented by edward tufte (http://www.cs.yale.edu/people/faculty/tufte.html) ... he's the man in my book when it comes to "conveying thought." attending one of his seminars is almost a religious experience. also, i happen to really like martin fowler's "uml distilled" as a guide map. it is a great light-weight read. i'm also impressed with "use case driven object modeling with uml" by rosenberg and scott. very practical. wow, i guess i should've inlined some amazon associate links in here to see if i could retire on these references :) house-keeping details: i put forth the motion to continue the design details of this specifi discussion under the firstname.lastname@example.com list when providing feedback, it is recommended to inline the comments/corrections/questions amongst a complete and existing document i will rev the document in order to maintain content integrity (this item should go away when we have a bit more infrastructure) as modifications are published any/all comments are welcome including: format/fields scope credits this doc isn't done by a long shot so do feel free to contribute hope this helps, - james
|View raw message


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Extremely rewarding is how many people aspire to describe their careers. But a group of CSU dental science students have already realised such satisfaction in the final year of their degree.
Text books donated by a CSU academic are set to make a huge difference to dental students in Cambodia.
Scholarships designed to support Indigenous participation in higher education have been awarded to six motivated CSU students.
The figures from an international study trip to Cambodia by 12 CSU students are impressive. The dental science and oral health students treated 557 patients, mainly children, with another 95 consultations during the international study experience in June and July.
CSU lecturer Dr Rahena Akhter has been recognised with an award at the world's biggest dentistry conference.
CSU School of Dentistry and Health Sciences will focus on increasing research capacity and the number of regional, rural and indigenous students under new Head of School Professor Boyen Huang.
Improving the oral health of pregnant women in the Riverina is the aim of a new program being coordinated by the Murrumbidgee Local Health District and CSU.
CSU is seeking to enhance rural and regional health services by forming partnerships with health care providers who would operate from state-of-the-art community health clinics in Albury-Wodonga and Bathurst.
A group of Charles Sturt University dentistry students have completed the first step in a pilot program designed to improve oral health in the remote NSW community of Brewarrina.
Charles Sturt University (CSU) will celebrate a significant milestone in rural health on Tuesday 17 December, when 32 new dentists graduate from CSU in Orange.


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10 Terrible Grammy Album of the Year Snubs
Paste Magazine
... Dismantle an Atomic Bomb in 2006) over the year's biggest and most important albums (American Idiot by Green Day, Confessions by Usher, The Diary of Alicia Keys by Alicia Keys, and Kanye's The College Dropout in 2005 and Late Registration in 2006).
The 10 biggest Grammys upsets
... album released just two months after his death. But Genius Loves Company winning Album of the Year over Green Day's American Idiot, Alicia Keys' The Diary of Alicia Keys, Usher's Confessions and Kanye West's The College Dropout was just ludicrous.
Alicia Keys: 5 Fast Facts You Need to Know
That record sold over 12 million copies worldwide and earned her five Grammy Awards in 2002. Her follow-up album blasted what we know as the sophomore slump. The Diary of Alicia Keys was released in 2003 with the singles "You Don't Know My Name", ...
Alicia Keys @ The Belasco Theater 2/8
Keys would go on to release her sophomore album The Diary of Alicia Keys which spawned numerous singles that sold a collective 8 million records world wide. These notable accolades have brought her up to this point, with numerous other awards and a ...


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Awning Windows
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Heath resistorPosted By Anoniem (not verified) On Thursday, 9 May 2013
Wednesday, May 8, 2013 - 14:00
Roland Model A
Attribution — You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work).
Noncommercial — You may not use this work for commercial purposes.
Share Alike — If you alter, transform, or build upon this work, you may distribute the resulting work only under the same or similar license to this one.
Lasercutten van deze figuren
bestandje maken en laseren ;-)
Laser cutter


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8576Varied Thrush
Expand Messages
- Mar 2, 2014Date: 03/02/2014Time: 2:30 p.m.Species: Varied thrush, Ixoreus naeviusSiting Description:I was walking my dog on the sidewalk through the the Cobble Creek Golf Course Community in Montrose. At 2:30 p.m., I noticed a medium-size gray bird drinking water from the gutter in front of a house at 577 Cobble Drive. The bird was slate gray, smaller than a robin, with a medium length tail. When it stopped drinking, it flew directly towards me. It did not have the bright orange breast of a robin. It landed in a shrub-size pine on the property of 586 Cobble Drive. I turned and walked back toward the pine. The bird was fairly well hidden in the pine. When I was only a few feet away from it, I pished several times. The bird took flight, flying away from me, across the street, to small trees on the property of 581 Cobble Drive. I had a perfect view of the dorsal side of the bird. It was slate gray. It also had a VERY obvious orange stripe down the center of each wing, where the greater secondary coverts meet the secondaries and primaries. The bird always stayed fairly close to the ground. I walked home to retrieve my camera. I spent a considerable amount of time trying to relocate the bird but was unsuccessful.I lived in western Washington State for 13 years and saw dozens of these birds every year.Maureen Briggs487 Cobble DriveMontrose, CO 81403970-240-4108skelligmichael@...
- Next post in topic >>


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Clay Paky Scores Winning Performance At The Central American and Caribbean Games
June 2015
They may be on a smaller scale than the Olympic Games but the 22nd Central American and Caribbean Games packed just as much glitz and glamour into their opening ceremonies in Veracruz, Mexico last November. Lighting designer David Grill, of David Grill and Associates, Inc. (DGA) in Mahwah, New Jersey, used Clay Paky Sharpys and A.leda B-EYE K20s to illuminate the festivities.
The Central American and Caribbean Games are held every four years with athletes from the region competing in a variety of sports, some of which qualify them for the Pan American Games. The 2014 Games were staged in the newly refurbished Estadio Luis 'Pirata' Fuente in Veracruz where more than a million people watched approximately 5,700 participating athletes.
The opening ceremonies featured several different segments in which the event producers used a large amount of projection. "The main challenge was how to use projection as a cyc element with performers in front and light the performers from 450ft away without eradicating the projections," Grill explained.
A 30ft high, three-tiered pyramid-shaped stage was the centrepiece of the opening ceremonies. The all-white structure, which featured three pools, acted as a platform for performances and a projection surface for images.
On the east side of the stadium about 400ft of the lower balcony rail was rimmed with 108 Clay Paky Sharpys and 32 B-EYE K20s. Eight more K20s lined two of the stage's pools and 18 Sharpys lined the other.
"We used the Sharpys not only for beam, canopy, and texture effects but also to light the acrobatic dancers flying through the air," said Grill. "The Sharpys were the only lights small enough and competent enough to capture things far away: We could side light the acrobats, who represented the heart and soul of the world, like a dance presentation from 200ft away. We could really have the Sharpys concentrated, focused and bright without a lot of overshoot."
Grill deployed the versatile B-EYE K20s in many different ways. "We used them as task lights, to light people, for aerial effects and for the face of the camera. We used the rotators for scenic texture projected onto various surfaces. They were better and more interesting than gobos. With the availability of the moving front face we could layer a colour over a colour. And the B-EYEs consumed virtually no power."
Grill and his team also lit the Games' closing ceremonies, which took the more conventional form of a rock concert.
Montreal-based Solotech provided the gear.
Francesco Romagnoli, Clay Paky Area Manager for North and Latin America, added, "This was an amazing looking show and we were proud to have been a part of it. Congratulations to Mr. Grill for such a great design."
A.C.T Lighting is the exclusive North American distributor for Clay Paky.
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Birthday Weekend in Review
16 comments:
- hotpants™ said...
I hope the little one is feeling better. Sounds like you had a fabulous weekend. Bring on the spring weather!
Welcome to SITS. We're happy to have you.
- 23.3.10
- Josh and Cali said...
Happy late birthday! You look stinking cute with E in all of your pictures... Glad you were able to spend some time in the sun with your fam, it doesn't get much better than that!
- 23.3.10
- Kellie said...
st. george was the place to be last weekend! hope you had a good birthday...your little guy is so cute.
- 23.3.10
- Lundell said...
I hope you had a fun Birthday!!! I love St. George...and those black shoes where did you buy them???
- 23.3.10
- Megan said...
What a fun birthday break! Ok and you little guy is so cute--I love his hat! I hope he gets better soon, poor thing. I love all the pictures!
- 23.3.10
- carissa @ lowercase letters said...
your little prince is just charming. he's adorable. love the pictures - super cute! i adore your hair and headband - beauty-full! glad you had a grand bday weekend. oh yes, vacay with babies is a whole lot of work! but of course, totally worth it! :)
- 23.3.10
- Serene is my name, not my life! said...
Love the shoes!
And can I just say how incredibly jealous I am of your gorgeous hair? I want hair like that.
But I was given very fine limp, flat, dead hair. *sniff*
- 23.3.10
- jessica.emily.adams said...
Happy belated birthday, Sar! It was good to see you last week. We need to get together and do lunch (sound like a broken record, don't i?).
- 23.3.10
- The Damsel In DisDress said...
I love your blog! Just found you through MMB. I'm a little scared because I am getting ready to do a sugar cookie post. Oh no!
- 23.3.10
- Meet Virginia said...
So glad you had a great bday, but am so sad that your lil guy got so sick! I hate that! I hope that he gets feeling better super soon!
- 23.3.10
- MommyJ said...
Oh, how I love that baby's face. And seriously. Hope the birthday was awesome.
- 23.3.10
- Cherish said...
Glad you had a great Birthday! I hope Baby E gets better soon. What is your email so that I can add you to my blog? My blog is sooo boring compared to yours though:).
- 23.3.10
- Queenie Jeannie said...
Happy Birthday AND I hope your sweet prince is better now!! ERs are soooo not fun for parents. Hugs!
Welcome to the SITS community!
- 24.3.10
- The McLane Blog Page: said...
Happy belated birthday!!! hope it was great!
- 25.3.10
- Little Lovables said...
you are too cuite in those little bangs and headband, i so wish I could pull that off!
sorry to hear about your little one
- 30.3.10
- Angie said...
Oh my goodness he IS the cutest little boy!! (I can say that cuz mine's going to be 12 this summer) HA! Really your son is adorable!
- 6.4.10


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Video: TechnoServe and McDonald's Partner to Support Coffee Farmers in Guatemala
September 26, 2013
With support from McDonald's, TechnoServe is working to improve the livelihoods of smallholder farmers in Guatemala.
In Guatemala, smallholder farmers like Sandra Lopez face a number of challenges in earning a living from coffee. TechnoServe is working with Sandra and thousands of other farmers to train them in practices that help deliver higher yields – and higher incomes.
This work is happening thanks to generous support from McDonald's. Learn more about efforts by McDonald's to promote coffee sustainability, and watch the video below to see what this work means to Sandra.
Related Blog Posts
Powering Up Renewable Energy in Swaziland
While innovating solutions to timber waste, TechnoServe and its partners unlock an opportunity for a renewable energy power plant with enormous potential for both environmental and social benefits.
The Business Case for Trees
Can simple business solutions make conservation profitable for smallholder farmers?
The Future of Food and Agriculture Lies with Rural Youth
TechnoServe President and CEO William Warshauer talks "Farming, Food and the Future of Work" at the Chicago Council Global Food Security Symposium.


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GMCT's community advisory committee (CAC) plays an important role, providing our trust with insights from a diverse range of cultural, religious and community groups.
The CAC is involved in the development of community engagement projects and activities which support open conversations with the community around our products and services, and on the broader issues around death and dying. The committee also provides a channel for GMCT to communicate information or seek responses from the community. The CAC advocates on behalf of GMCT to local individuals and groups.
Committee members pose with GMCT staff during their August 2016 meeting, held at Quang Duc Buddhist Monastery in Fawkner.
It is a legislative requirement for Class A cemetery trusts to form and engage a community advisory committee. Guidelines for Class A cemetery trust CACs are available here.
This committee meets six times per year to advise GMCT of the community's feeling about specific matters and issues at particular cemeteries.
The CAC comprises trust members and community members who represent various cultural, religious and interest groups local to GMCT cemeteries in the north, west and east of Melbourne.
Expressions of interest are open for new members and we hope to continue to grow the membership in 2017.
Please see the links to the expression of interest and CAC application forms.
Please contact firstname.lastname@example.com for more information or to submit an application.
Meg Jenkins
Meg Jenkins has a longstanding relationship with the community and not-for-profit organisations. For over 30 years, Meg has been passionate about volunteering and community development - particularly through her involvement with Williamstown Cemetery. She has a rich experience in the coordination and management of various community programs, committees and associations.
Meg holds a particular interest in the rituals, monuments and customs that individuals and groups use to honour and remember their relatives and ancestors.
Venerable Thich Nguyen Tang
Venerable Tang is a practising monk and community leader. Arriving in Australia from Vietnam in 1998, Venerable Tang quickly became involved in bridging the gaps between new and established Australians and contributing to Vietnamese-to-English translations of key Buddhist texts.
He currently volunteers as a Buddhist chaplain at a range of Melbourne hospitals and has held a number of roles as part of community committees. Venerable Tang also leads weekly retreats and services as the spiritual leader of Quang Duc Buddhist Monastery.
Janet Borg
Janet Borg has worked and consulted extensively in the environmental and sustainability sector. She has held a number of board and committee roles across various organisations dedicated to social responsibility and improving business practice.
Janet has currently turned towards community engagement with her harp practice Soothing Your Soul. Janet plays her harp in hospital, palliative care and other settings as a form of music therapy.
Vivienne Jackson
Vivienne Jackson is a career social worker and community development practitioner who has worked across a range of diverse local and state government organisations.
Vivienne's most recent experience has been supporting the needs and aspirations of residents with disabilities within the City of Whittlesea. She brings a passion for community advocacy and social inclusiveness to her role with the CAC.
Allocation of Orthometals funding
GMCT works with international company Orthometals to recycle metals following cremator processes. Funds raised through this recycling are used to support charities and aligned support organisations identified by the CAC.
Partnership with the Australian Centre for Grief and Bereavement (ACGB)
The partnership with ACGB has provided funds via Orthometals to support:
Dying To Know Day 2016
CAC members attended and contributed to discussion about death and dying during GMCT's Dying to Know Day event, which provided community members with an opportunity to take a tour of a mausoleum and learn about the cremation process from our crematorium supervisor.
GMCT focus areas
The CAC is consulted on pricing and engagement planning across GMCT cemeteries, including the new developments at Keilor Cemetery, Northern Memorial Park and Lilydale Memorial Park.
GMCT's community engagement plan 2016-18 is currently under development.


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This is for certified Engineers only here in Qatar.


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Connecting Families to Senior Living
Talk to a local advisor for FREE
Talk to a local Advisor about this community
Contact this community at:
(866) 333-2174
497 South Lake St
Facility License: 0012700
Caretypes Provided: Residential Care Home
Pricing is available for the following rooms:
*Prices quoted are monthly rental charges and are provided by the communities themselves. Actual prices may differ due to one-time fees, timing and care services required. Speak with your Senior Living Advisor to learn more.
Our apologies. Pricing is currently unavailable for this particular community.
Our family owned 2-8 bed Assisted Living homes are located in Montello, tranquility with a touch of history. One story buildings to make it easier for people with mobility issues. Our only mission is to make your loved one feel comfortable, and at home. We provide a very safe environment for your loved one: Door alarms on 24 hours a day. Monthly checks on Smoke, Heat, and Carbon Monoxide Detectors. Annual professional services on Heat Detectors, Smoke Detectors, Sprinkler Systems, Fire Extinguishers, & Heating/Cooling Systems. Staff trained quarterly in Emergency situations ex: Fire, Flooding, Wandering Persons, Tornado. All rooms are private. Private Bathrooms are available - but limited. We are State Certified to provide services for: Alzheimers/Dementia, Physically Disabled, Developmentally Disabled, Advanced Age, and Hospice Services.
Schedule an appointment with an Advisor
out of reviews
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Call Now (866) 333-2174
Get the latest tips, news, and advice on aging and caregiving.
A Place for Mom is the largest assisted living referral service. We are paid by our participating communities, therefore our service is offered at no charge to families.
Copyright ©2017 A Place for Mom, Inc. All rights reserved.


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High 235mm x Wide 132mm x Deep 65mm / Pocket Depth 30mm
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This double A6 portrait brochure holder is an ideal way of distributing literature to customers and to the public. Made out of clear acrylic, this A6 literature holder is a robust retail display unit. The brochure holder has a large flat base to stand stable on a countertop. With its two large pockets, the A6 literature holder can be easily accessed whether full or empty. This A6 portrait brochure holder is very lightweight and can be moved around or repositioned with ease. Due to its transparent design, customers can view the front literature on display from various angles.
Personalise the front of the A6 brochure holder with a logo sticker, brand image or contact details. There are other literature holders available in our range to compliment this A6 brochure holder, such as a banner stand or other wall mounted literature holders. This double A6 portrait brochure holder would look perfect in an exhibition hall to inform people on product specifications or in a perfume shop to distribute adverts about celebrities latest fragrances.
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Displaysense Ltd. Rye Street, Bishop's Stortford, Hertfordshire, CM23 2HG, UK. Copyright © 1979-2016 Displaysense


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Bus Katowice Katowicy • Silesian Voivodeship » Perigueux Périgueux
3 coach and bus timetables from Katowice to Perigueux • 4 bus companies • euroTicket, Eurolines Switzerland, Eurolines France and Busfor offer • Cheap online tickets starting from 143 EUR / 154 USD
Fares starting from 143 EUR / 154 USD
Here at rebrutto.com you can buy your bus tickets from Katowice to Perigueux.
When you go to tickets offer you can also verify your fare in booking system, to which you will be redirected after clicking BUY button. Fill the forms as if you would book your ticket. Set the number and type of tickets, look for available discounts and deals. Cancel your booking without consequences whenever you want, as well just before the payment.
Katowice to Perigueux coach and bus online tickets
Get your bus ticket from: Busfor, Eurolines France, Eurolines Polska, Eurolines Switzerland, euroTicket or Intercars Basque on Katowice - Perigueux route.
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View more bus services from Katowice to France at Katowice to France tickets. For your return travel options, visit our offer at Perigueux to Katowice or Perigueux to other cities in Poland.
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