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Vascular endothelial growth factor receptor-2 (VEGFR-2) signaling is an obligate requirement for normal development and pathological angiogenesis such as cancer and age-related macular degeneration. Although autophosphorylation of tyrosine 1173 (Y1173) of VEGFR-2 is considered a focal point for its angiogenic signal relay, however, the mechanism of phosphorylation of Y1173, signaling proteins that are recruited to this residue and their role in angiogenesis is not fully understood.
Methodology/Principal Findings
In this study we demonstrate that c-Src kinase directly through its Src homology 2 (SH2) domain and indirectly via c-Cbl binds to phospho-Y1057 of VEGFR-2. Activation of c-Src kinase by a positive feedback mechanism phosphorylates VEGFR-2 at multi-docking site, Y1173. c-Src also catalyzes tyrosine phosphorylation of IQGAP1 and acts as an adaptor to bridge IQGAP1 to VEGFR-2. In turn, IQGAP1 activates b-Raf and mediates proliferation of endothelial cells. Silencing expression of IQGAP1 and b-Raf revealed that their activity is essential for VEGF to stimulate angiogenesis in an in vivo angiogenesis model of chicken chorioallantoic membrane (CAM).
Angiogenesis contributes to the pathology of numerous human diseases ranging from cancer to age-related macular degeneration. Determining molecular mechanism of tyrosine phosphorylation of VEGFR-2 and identification of molecules that are relaying its angiogenic signaling may identify novel targets for therapeutic intervention against angiogenesis-associated diseases. Our study shows that recruitment and activation of c-Src by VEGFR-2 plays a pivotal role in relaying angiogenic signaling of VEGFR-2; it phosphorylates VEGFR-2 at Y1173, facilitates association and activation of IQGAP1 and other signaling proteins to VEGFR-2. IQGAP1-dependent signaling, in part, is critically required for endothelial cell proliferation, a key step in angiogenesis. Thus, Y1057 of VEGFR-2 serves to regulate VEGFR-2 function in a combinatorial manner by supporting both diversity of recruitment of angiogenic signaling proteins to VEGFR-2, and its ability to promote angiogenesis.
Citation: Meyer RD, Sacks DB, Rahimi N (2008) IQGAP1-Dependent Signaling Pathway Regulates Endothelial Cell Proliferation and Angiogenesis. PLoS ONE 3(12): e3848. https://doi.org/10.1371/journal.pone.0003848
Editor: Nick Gay, University of Cambridge, United Kingdom
Received: May 20, 2008; Accepted: November 10, 2008; Published: December 3, 2008
Copyright: © 2008 Meyer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported in part by grants from the National Institutes of Health (NR and DBS) and Massachusetts Lions Foundation grant to the Department of Ophthalmology.
Competing interests: The authors have declared that no competing interests exist.
Activation of vascular endothelial growth factor receptor-2 (VEGFR-2 also called FLK-1/KDR) plays a pivotal role in mediating growth of blood vessels, angiogenesis [1]. VEGF stimulation of VEGFR-2 provokes pleiotropic responses in endothelial cells including endothelial cell growth, survival, differentiation, migration, and tube formation [2]. Autophosphorylation of tyrosine 1173 (Y1173) of VEGFR-2 is a focal point for activation of angiogenic signal relay of VEGFR-2, as it has emerged as a multi-functional docking site involved in the recruitment of multiple signaling proteins including the p85 regulatory subunit of phosphatidylinositol 3-kinase (p85/PI3-K) [3], [4], phospholipase Cγ1 (PLCγ1) [5], [6], Shb [7], and the Shc-related adaptor protein, Sck [8]–[10] and the transmission of a range of biological signals to coordinate endothelial cell function and angiogenesis. The critical and direct role of Y1173 in enabling VEGFR-2 to promote angiogenesis was recently revealed by a gene targeting study where mice homozygous for the mutant VEGFR-2Y1173F knock-in allele died between E8.5 and E9.5 without any organized blood vessels [11] similar to the VEGFR-2 null mice [12]. More recent studies indicate that in addition to Y1173, the kinase domain tyrosine, Y1057 also takes part in VEGFR-2 function and is involved in VEGFR-2-mediated cell proliferation [13]. Y1057 along with Y1173 also is engaged in the recruitment and activation of the ubiquitin E3 ligase, c-Cbl. Phospho-Y1057 contributes to the recruitment of c-Cbl to VEGFR-2 by directly associating with c-Cbl, while phospho-Y1173 indirectly via PLCγ1 participates in its recruitment to VEGFR-2 [14], resulting in the ubiquitination of PLCγ1 and inhibition of angiogenesis in vitro [14].
The recent study indicates that IQGAP1 associates with VEGFR-2 and its activity is required for proliferation of endothelial cells in vitro [26]. It remains, however, unclear how IQGAP1 interacts with VEGFR-2 since it lacks phospho-tyrosine binding domains such as SH2 and PTB and how its activity is regulated by VEGFR-2. IQGAP1 contains multiple protein-interaction domains including calponin homology domain (CHD), poly-proline-binding domain (WW), calmodulin-binding domain (IQ) and rasGTPase-activating protein (GAP)-related domain (GRD) and tyrosine and serine/threonine phosphorylation sites [33] and is involved in multiple cellular functions including calcium/calmodulin signaling, MAPK signaling and regulation of cytoskeletal structure, cell adhesion and cell motility [27], [31], [33].
In this study, we uncovered a surprising correlation between phosphorylation of Y1057 and Y1173 and that phosphorylation of Y1057 plays a multitasking role in mediating VEGFR-2-directed angiogenic signaling events in endothelial cells. Phospho-Y1057 recruits c-Src kinase to VEGFR-2, and in part catalyzes phosphorylation of Y1173 via Src kinases. c-Src also bridges IQGAP1 to VEGFR-2 and directly phosphorylates IQGAP1 and promotes endothelial cell proliferation, a key step in angiogenesis.
Identification of tyrosine 1057 of VEGFR-2 as a binding site for Src kinases
Although stimulation of VEGFR-2 in endothelial cells is known to promote activation of Src kinases [15] and Src activation is linked to endothelial cell permeability, survival and angiogenesis [16], [17], the nature of its association with VEGFR-2, in particular, the putative phospho-tyrosine residue on VEGFR-2 involved in its recruitment is not. Our survey of several primary endothelial cells including, HUVEC, HMVE and BAEC cells showed that c-Src is ubiquitously tyrosine phosphorylated in response to VEGF stimulation (Figure 1A) and associates with VEGFR-2 in a ligand-dependent manner in these primary endothelial cells (Figure 1C). Cell lysates obtained from HEK-293 (human embryonic kidney-293) cells which do not express VEGFR-2 was negative for VEGFR-2 and was used as a negative control (Figure 1C). The cytoplasmic region of VEGFR-2 contains multiple tyrosine phosphorylation sites [18], albeit none of these tyrosine autophosphorylation sites shows obvious sequence similarities to the preferred Src SH2 recognition motif, pYEEI [19]. To analyze association of c-Src with VEGFR-2 we utilized the previously characterized VEGFR-2 chimera [20] herein called CKR which is created by replacing the extracellular domain of VEGFR-2 with that of human Colony stimulating factor-1 receptor (CSF1R) and expressed in PAE (porcine aortic endothelial) cells as an experimental system. In our previous studies we have extensively characterized CKR with VEGFR-2 in a comparative manner. CKR in term of its ability undergo down-regulation, activation, recruitment of signaling proteins and to stimulate cellular responses is identical to VEGFR-2 [3], [14], [20], [21] and data presented in this manuscript. We used this strategy to avoid cross-talk between VEGFR-2 and other VEGF receptors, in particular, VEGFR-1. Our initial observation demonstrated that mutation of tyrosines 799, 820, 925 to phenylalanine (F) and deletion of C-terminus of VEGFR-2 coupled with mutation of tyrosines 1173 and 1212 has no effect on the ability of VEGFR-2 to associate with the SH2 domain of c-Src (Figure 1E). In contrast, mutation of Y1052 to glutamic acid (E) and particularly Y1057, located in the kinase domain, severely reduced the binding of VEGFR-2 with SH2 domain of c-Src (Figure 1G). GST alone does not associate with CKR (Figure S3). Consistent with its reduced binding capability to E1052, the result showed that E1052 only partially reduces the ability of VEGFR-2 to phosphorylate c-Src (Figure 1I). In contrast, the potential of mutant Y1057 (E1057/CKR) to stimulate Src tyrosine phosphorylation was severely compromised and was near to undetectable (Figure 1I). Since both Y1052 and Y1057 are located in the kinase domain we replaced these residues to glutamic acid (E) rather than phenylalanine (F). This type of mutations has been suggested to be useful for characterization of sensitive tyrosine sites on the RTKs (receptor tyrosine kinases) when their mutation to phenylalanine interferes with receptor activation [15]. However, unlike these RTKs, single mutation of Y1052 and Y1057 either to E or F did not interfere with activation of VEGFR-2 in the context of CKR (Figures S1 and S2). Furthermore, to make sure that our observation in CKR also is true in the context of non-chimeric VEGFR-2, we mutated Y1057 to F in the full length VEGFR-2, expressed in PAE cells and similarly analyzed for its ability to bind SH2 domain of c-Src. The result showed that VEGFR-2 associates with SH2-Src in VEGF-dependent manner and mutation of Y1057 inhibited association of VEGFR-2 with c-Src (Figure 1K). Altogether, the data show that Y1057 of VEGFR-2 is a major c-Src binding site on VEGFR-2 and its presence is critically required for VEGFR-2 to activate c-Src.
HUVEC, HMVE and BAE cells were stimulated with VEGF-A (100 ηg/ml) for indicated times. Whole cell lysates (WCL) were immunoblotted with an anti-phospho-Src (pY416) antibody (A) or an anti-Src antibody (B). HUVEC, HMVE and HEK-293 cells were either unstimulated (−) or stimulated with VEGF (+) for 10 minutes, lysed, immunoprecipitated with c-Src antibody and immunoblotted with an anti-VEGFR-2 antibody (C). Whole cell lysates (WCL) from the same group was immunoblotted with an anti-VEGFR-2 as a control (D). PAE cells expressing wild type chimeric VEGFR-2 (CKR) and tyrosine mutant CKRs, F799/CKR, F820/CKR, F925/CKR and carboxyl terminus deleted CKR coupled with mutation of Y1173 and Y1212 denoted as ΔCKR/F1173/F1212 was stimulated with CSF-1 for 10 minutes. Cells were lysed, and whole cell lysates were incubated with purified GST-SH2-Src protein. The GST-SH2-Src bound proteins were subjected to Western blot analysis using an anti-VEGFR-2 antibody (E). Whole cell lysates from the same groups was blotted with an anti-VEGFR-2 antibody (F). PAE cells expressing CKR, E1052/CKR, and E1057/CKR were either unstimulated or stimulated with CSF-1 for 10 minutes and cells were lysed and cell lysates was subjected to GST-SH2-Src pull-down assay (G). An immunoblot of whole cell lysates also were probed with an anti-VEGFR-2 antibody (F,H), an anti-phospho-Src (pY416) antibody (I) or an anti-Src antibody (J). PAE cells expressing VEGFR-2 or F1057/VEGFR-2 were prepared and subjected to pull-down analysis as panel E (K) or whole cell lysates was blotted with an anti-VEGFR-2 antibody (L). The results shown in A–L are representative of three separate experiments.
c-Cbl associates with c-Src and participates in the interaction of c-Src with VEGFR-2
A recent study indicates that tyrosine 1057 of VEGFR-2 also serves as a binding site for c-Cbl by directly interacting with its tyrosine kinase binding (TKB) domain [14], raising an interesting possibility as to whether c-Cbl and c-Src competitively bind to Y1057 or c-Cbl acts to bridge c-Src to VEGFR-2 since c-Cbl is known to interact with c-Src via its SH3 domain [22]. To test these two distinct possibilities, we used PAE cells co-expressing CKR either with wild type c-Cbl or Cbl-N, where the TKB domain is deleted [14] and analyzed the ability of GST-SH2 domain of c-Src to interact with VEGFR-2. Over-expression of wild type c-Cbl enhanced association of SH2 domain of c-Src with VEGFR-2, where disabling the binding of c-Cbl with VEGFR-2 had no effect on the binding of SH2-Src to VEGFR-2 (Figure 2A). Quantification of the blots (three independent experiments) showed that indeed interaction of VEGFR-2 with GST-SH2-Src is more than doubled when c-Cbl is over-expressed, where as the deletion of TKB domain almost entirely is inhibited this effect of c-Cbl (Figure 2B). Over-expression of either c-Cbl or c-Cbl-N had no effect on the protein levels of CKR or on its tyrosine phosphorylation (Figure 2C, 2D). Expression of c-Cbl and c-Cbl-N also is shown (Figure 2E). Since up-regulation of c-Cbl by over-expression enhanced association of c-Src with VEGFR-2, we further tested whether silencing expression of c-Cbl by siRNA would reduce interaction of SH2-Src with VEGFR-2. As shown, silencing the expression of c-Cbl in PAE cells reduced the binding of GST-SH2 Src with VEGFR-2 (Figure 2F). Quantification of the blots (three independent experiments) is also shown which further support the hypothesis that endogenous c-Cbl plays a role in bridging c-Src to VEGFR-2 (Figure 2G). The silencing effect of c-Cbl-siRNA on the c-Cbl protein level also is shown (Figure 2H). It showed be noted that c-Cbl is highly expressed in these cells and siRNA only partially reduced its expression (Figure 2H). Altogether, the data show that c-Cbl, in part, facilitates association of Src with VEGFR-2. To firmly establish a direct binding of SH2 domain of c-Src with phospho-Y1057 of VEGFR-2 we synthesized a phosphorylated peptide corresponding to Y1057 and tested its binding ability to GST-SH2 domain of c-Src in an in vitro dot blot assay. The result shows that SH2 domain of Src also directly interacts with phosphorylated Y1057 in a c-Cbl independent manner (Figure 2J), suggesting that the interaction of c-Src with Y1057 of VEGFR-2 is established by a direct binding involving its SH2 domain and an indirect interaction involving c-Cbl. Figure 2K summarizes our observation regarding interaction of c-Src with VEGFR-2 and role of c-Cbl in this process.
Serum-starved PAE cells expressing chimeric VEGFR-2 (CKR) or co-expressing CKR with c-Cbl and CKR with Cbl-N were either unstimulated (−) or stimulated with CSF-1 (+) for 10 minutes. Cells were lysed and subjected to an in vitro pull-down assay using purified GST-SH2-Src protein (A). Quantification of blots of SH2-Src binding to VEGFR-2 of three separate experiments is shown. Each bar represents the mean ±SD of triplicate experiments. *P<0.01 (B). A parallel immunoblot of whole cell lysates (WCL) was probed with an anti-VEGFR-2 antibody (C), with an anti-phosphotyrosine antibody (D) and with an anti-c-Cbl antibody (E). PAE cells co-expressing chimeric VEGFR-2 (CKR) with a control siRNA or Cbl-siRNA were stimulated with CSF-1 for 10 minutes and cells were lysed and prepared for in vitro pull-down assay as panel A (F). A parallel immunoblot of whole cell lysates were probed with an anti-c-Cbl antibody for confirmation of c-Cbl knockdown (H) and anti-Hsp70 antibody as a control for protein loading (I). To detect a direct interaction between the SH2 domain of c-Src and Y1057 of VEGFR-2, the indicated quantities of purified recombinant GST-SH2-Src (top) and GST control (bottom row) were dot blotted as described in the Materials and Methods and detected by an anti-pY1057 VEGFR-2 antibody (J). The graph represents the average binding of SH2-Src to VEGFR-2 in the absence or presence of c-Cbl-siRNA (±SD) of three separate experiments. Quantification of blots of SH2-Src binding to VEGFR-2 of three separate experiments is shown. *P<0.01 (G). All the experiments repeated at least three times. A summary of the proposed interaction of c-Src with VEGFR-2 is shown (K).
Tyrosine 1173 of VEGFR-2 is phosphorylated by Src kinases
Tyrosine 1173 functions as a multitasking residue on VEGFR-2 [3]–[7], [10] and is a bona fide mediator of angiogenic signaling of VEGFR-2 in vivo [11]. Hence we investigated a possible crosstalk between the kinase domain tyrosines (i.e., Y1052 and Y1057) and Y1173 and the potential role of Src kinases in this crosstalk. For this aim, we tested phosphorylation of Y1173 in the background of E1052/CKR and E1057/CKR expressed in PAE cells. The result showed that mutation of 1057 markedly reduced ligand-dependent phosphorylation of Y1173 as detected by an anti-phospho-Y1173 specific antibody (Figure 3A). Mutation of Y1052 only slightly reduced phosphorylation of Y1173, suggesting that Y1057 but not Y1052, is specifically involved in modulation of phosphorylation of Y1173 (Figure 3A). However, mutation of either Y1052 or Y1057 had no major effect on the phosphorylation Y1212 (Figure 3B). Quantification of phosphorylation of Y1173 and Y1212 obtained from three separate independent experiments also is shown (Figure 3D). Since Src kinase binds to Y1057 of VEGFR-2, we tested potential role of Src in the phosphorylation of Y1173. Our analysis showed that co-expression of v-Src with non-chimeric wild-type VEGFR-2 in a transient transfection of HEK-293 cells also increases phosphorylation of Y1173 (Figure 3D), where phosphorylation of Y1052, Y1057 and Y1212 were not effected (Figure 3F–H). The quantification of phosphorylation of Y1173, Y1052, Y1057 and Y1212 also is shown (Figure 3K). Of note, co-expression of v-Src with E1057 mutant VEGFR-2 also rescued the reduced phosphorylation of Y1173 further suggesting that c-Src is involved in catalyzing phosphorylation of Y1173 of VEGFR-2 (data not shown).
Serum-starved PAE cells expressing wild type CKR, E1052/CKR and E1057/CKR were either unstimulated (−) or stimulated with CSF-1 (+) for 10 minutes. Whole cell lysates (WCL) were subjected to Western blot analysis using an anti-phosphoY1173 specific-VEGFR-2 antibody (A), an anti-phosphoY1212 specific-VEGFR-2 antibody (B) or an anti-VEGFR-2 antibody (C). The graph represents the mean phosphorylation of Y1173 and Y1212 of VEGFR-2 (±SD) of three separate experiments. *P<0.01 (D). HEK-293 cells transiently expressing VEGFR-2 alone or co-expressing v-Src were stimulated with VEGF and immunoblotted with anti-pY1173 (E), anti-pY1052 (F), anti-pY1057 (G), anti-pY1212 (H), anti-c-Src (I) and anti-VEGFR-2 (J) antibodies. The blots were also quantified using Image J program (developed by NIH) and represents average tyrosine phosphorylation sites on VEGFR-2 of three separate experiments (±SD) from three separate experiments (K). SYF cells and SYF cells expressing chimeric VEGFR-2 (CKR) were either unstimulated (−) or stimulated with CSF-1 (+) for 10 minutes and cells lysates were prepared and immunoblotted with anti-pY1173 (L), anti-pY1052 (M) and anti-VEGFR-2 (N) antibodies. PAE cells expressing CKR were treated with different concentration of SU6656 for 30 minutes and stimulated with CSF-1 for 10 minutes and cell lysates were prepared as panel A and immunoblotted with anti-pY1173 (O), anti-pY1052 (P) and anti-VEGFR-2 (Q) antibodies. Phosphorylation of Y1173 and Y1052 in response to SU6656 treatment was quantified as panel D and is presented as the mean (±SD) of three separate independent experiments (R). A summary of the proposed model of phosphorylation Y1173 of VEGFR-2 by c-Src and VEGFR-2 itself is shown (S).
To further link Src kinase activity to phosphorylation of Y1173 of VEGFR-2, we tested the phosphorylation of Y1173 of VEGFR-2 in the triple knockout SYF (c-Src, c-Yes and c-Fyn) cells [24]. The data showed that stimulation of SYF cells ectopically expressing chimeric VEGFR-2 (CKR) with ligand increases phosphorylation of Y1052. In contrast, phosphorylation of VEGFR-2 at Y1173 was almost undetectable (Figure 3L and 3M). Only a long exposure of the film detected a weak phosphorylation of Y1173 (data not shown), suggesting that in addition to Src kinases, VEGFR-2 itself also catalyzes phosphorylation of Y1173 of VEGFR-2. Since in the recent years various Src inhibitors were used for therapeutic purposes in cancer and anti-angiogenesis treatments [25], we tested the effect of Src-specific inhibitor, SU6656 to inhibit phosphorylation of VEGFR-2 at Y1173. The result showed that SU6656 selectively inhibits phosphorylation of VEGFR-2 at Y1173 in a dose-dependent manner but had no effect on the phosphorylation of Y1052 (Figure 3O, 3P). The quantification of inhibition of phosphorylation of Y1173 of VEGFR-2 by SU6656 also is shown (Figure 3R). Taken together, the data demonstrate that Src kinases upon activation by VEGFR-2 phosphorylate Y1173 of VEGFR-2 (Figure 3S).
Role of c-Src in VEGFR-2 mediated endothelial cell tube formation and proliferation
Since activation of Src family kinases is linked to angiogenesis [16], [17] we initially analyzed the ability E1052/CKR and E1057/CKR to stimulate endothelial tubulogenesis in vitro. Our analysis showed that mutation of Y1052 had no apparent effect on the ability of CKR to promote tube formation of PAE cells. However, mutation of Y1057 significantly reduced the ability of CKR to stimulate tube formation of PAE cells (Figure 4A). Quantification of tube formation of PAE cells in response to CKR, E1052/CKR and E1057/CKR activation is shown (Figure 4B). To test whether c-Src activation is required for tube formation of PAE cells, we used previously established PAE cells co-expressing CKR with either wild type c-Src or dominantly negative acting kinase inactive Src [15]. The result showed that over-expression of c-Src or dominant negative c-Src in PAE cells do not alter the ability of VEGFR-2 to stimulate tube formation (Figure 4C, 4D), indicating that perhaps c-Src activity is not required for VEGFR-2 to stimulate tube formation of endothelial cells. In contrast, our analysis showed that over-expression of c-Src significantly enhances the ability of VEGFR-2 to stimulate proliferation of PAE cells and over-expression of dominant negative Src (Src kinase-dead) inhibits the VEGFR-2 driven proliferation of PAE cells (Figure 4E). To test the role of c-Src in endothelial cell proliferation further we knocked down c-Src expression in HUVE and HMVE cells by siRNA strategy and tested their VEGF-dependent proliferation. The result showed that silencing the expression of c-Src in endothelial cells significantly inhibits the ability of VEGFR-2 to stimulate proliferation of these primary endothelial cells (Figure 4F, 4G). Interestingly, HMVE cells were more sensitive to silencing of c-Src than HUVE cells since silencing of Src expression almost totally inhibited VEGF-stimulated proliferation (Figure 4G). Taken together, our results identify c-Src as a key component of mitogenic signaling of VEGFR-2 in endothelial cells.
PAE cells expressing CKR, E1052/CKR, and E1057/CKR alone or with c-Src and dominant negative c-Src (DN-Src) were prepared as spheroids and subjected to an in vitro angiogenesis/tubulogenesis assay as described in the Materials and Methods. The pictures shown are representative spheroids from three independent experiments (A, C). Tubulogenesis was also quantified as the mean of ± SD of four sprouting colonies. *P<0.05 (B, D). Equal number of PAE expressing CKR alone or co-expressing CKR with c-Src, and CKR with DN-Src were subjected to proliferation assay as described in the Materials and Methods and expressed as the mean ± SD of triplicate. *P<0.05 (E). HUVE and HMVE cells treated with either a control siRNA or c-Src-siRNA, after 48 hours cells were stimulated with different concentrations of VEGF and subjected to proliferation as Panel C. *P<0.05 (F, G). The Western blots of cells treated with either control siRNA or c-Src-siRNA also are shown (F, G).
Identification of IQGAP1 as a novel Src kinase substrate
IQGAP1 is a scaffold protein and participates in signaling cascades mediated by a diverse group of cell surface receptors [33], including VEGFR-2 [26]. In addition, we recently have identified IQGAP1 as a binding partner of VEGFR-2 by a proteomic approach (our unpublished data), raising the likelihood for the involvement of Y1057 in the recruitment and tyrosine phosphorylation of IQGAP1 by VEGFR-2. Our initial observation showed that IQGAP1 is tyrosine phosphorylated in PAE cells by VEGFR-2 and mutation of Y1057 reduces the ability of VEGFR-2 to stimulate its tyrosine phosphorylation (Figure 5A). IQGAP1 also is tyrosine phosphorylated by other RTKs including, ErbB1/EGFR-1 and PDGFRβ ectopically expressed in PAE cells (our unpublished data), suggesting that IQGAP1 serves as a common substrate for RTKs. In addition, our data show that over-expression of c-Src in PAE cells highly enhanced tyrosine phosphorylation of IQGAP1. In a sharp contrast, over-expression of a dominant negatively acting c-Src inhibited tyrosine phosphorylation of IQGAP1 (Figure 5C).
Serum-starved PAE cells expressing CKR (chimeric VEGFR-2) and E1057/CKR were either unstimulated (−) or stimulated with CSF-1 (+) for 10 minutes. Whole cell lysates were subjected to immunoprecipitation with IQGAP1 and Western blot analysis using an anti-phosphotyrosine antibody (A) or anti-IQGAP1 antibody (B). PAE cells co-expressing either wild type c-Src or a dominant negative form of c-Src (KD-Src) were stimulated and subjected to Western blot analysis as panel A using an anti-phosphotyrosine antibody (C). The same membrane striped off the antibody and re-blotted with an anti-IQGAP1 antibody for protein levels (D). SYF cells co-expressing CKR with c-Src or expressing CKR alone were prepared as above and blotted with anti-phosphotyrosine (E), anti-IQGAP1 (F), and anti-VEGFR-2 (G). Cell lysates derived from PAE cells expressing CKR were either precipitated with GST, GST-SH2-Src, (H) or GST-SH3-Src (I) and blotted for IQGAP1 using an anti-IQGAP1 antibody. Human primary dermal vascular endothelial (HMVE) cells were stimulated with VEGF for 10 minutes, cells were lysed and immunoprecipitated with either control antibody (Ctrl.IgG) or c-Src antibody and blotted with anti-IQGAP1 (J). Whole cell lysates was blotted with anti-Src antibody (K). HMVE cells expressing either control siRNA (Ctrl.siRNA) or IQGAP1 siRNA were lysed and blotted for IQGAP1 using an anti-IQGAP1 antibody (L). HMVE cells expressing either control siRNA or IQGAP1 siRNA were subjected to proliferation assay as described in Method section. Error bars represent mean ± SD of triplicate samples. *p<0.001 as compared to proliferation of HMVE cells in response to 100 ηg VEGF-A (M). HMVE cells expressing either control siRNA or IQGAP1-siRNA were unstimulated (−) or stimulated (+) with VEGF for 10 minutes and cells were lysed and immunoprecipitated with an anti-b-Raf antibody and blotted with an anti-phospho-Ser445 antibody (N) or with anti- b-Raf antibody (O). Whole cell lysates was blotted for IQGAP1 (P). Also shown is the graph of the mean phosphorylation of b-Raf where the expression of IQGAP1 is silenced by siRNA. It represents (±SD) of three separate experiments. *P<0.01 (Q). Summary of the proposed VEGFR-2-dependent signal relay in endothelial cell proliferation also is shown (R).
To further establish role of c-Src in phosphorylation of IQGAP1 we show that in SYF knockout cells where Src family kinases (Yes, Fyn and Src) are absent, IQGAP1 is not tyrosine phosphorylated in response to activation of VEGFR-2, where re-introduction of c-Src rescued phosphorylation of IQGAP1 by VEGFR-2 (Figure 5E). Our further studies showed that in an in vitro kinase assay recombinant c-Src protein phosphorylates GST-fusion IQGAP1 (data not shown). Taken together, the data support the hypothesis that IQGAP1 directly associates with c-Src and undergoes c-Src-dependent tyrosine phosphorylation. Hence to further establish as to whether c-Src interacts with IQGAP1 we tested the ability of SH2 and SH3 domains of c-Src to associate with IQGAP1 in an in vitro pull-down assay. The result showed that SH2 domain of c-Src interacts with IQGAP1 independent of its tyrosine phosphorylation since SH2 domain of c-Src forms a complex with IQGAP1 prior to stimulation of cells with ligand (Figure 5H). Additional analysis using PAE cells null for chimeric VEGFR-2 revealed that indeed association of SH2 domain of Src with IQGAP1 is independent of VEGFR-2 (data not shown). In contrast to the SH2 domain, the SH3 domain of c-Src failed to interact with IQGAP1 with or without stimulation of VEGFR-2 (Figure 5I). The data suggest that SH2 domain mediates the interaction of c-Src with IQGAP1 by a novel mechanism that is independent of tyrosine phosphorylation of IQGAP1.
IQGAP1 signaling pathway is required for VEGF-induced angiogenesis
To establish the biological importance of IQGAP1 in VEGFR-2 signaling, we initially examined its role in proliferation of endothelial cells. To this end, we employed siRNA-mediated knockdown strategy and analyzed proliferation of endothelial cells in response to VEGF stimulation. IQGAP1 siRNA significantly reduced expression of IQGAP1 in HMVE cells (Figure 5L). We further analyzed these cells for their ability to undergo proliferation in response to VEGF. Silencing the expression of IQGAP1 in primary endothelial cells, but not control siRNA, significantly inhibited VEGF-dependent cell proliferation (Figure 5M), suggesting that VEGFR-2/c-Src orchestrated tyrosine phosphorylation of IQGAP1 serves an important role in endothelial cell proliferation. Our recent study indicates that IQGAP1 is capable of modulating activity of b-Raf [27], to test whether IQGAP1 activity is also required for b-Raf phosphorylation in endothelial cells in response to VEGFR-2 activation we tested phosphorylation of b-Raf where expression of IQGAP1 is knocked down by siRNA. The data show that stimulation of endothelial cells with VEGF promotes phosphorylation of b-Raf at Ser 455 and silencing expression of IQGAP1 notably reduced its phosphorylation (Figure 5N). Quantification of phosphorylation of Ser 455 of b-Raf based on the three independent experiments also is shown (Figure 5Q). The reduced phosphorylation of b-Raf in IQGAP1 siRNA treated cells was not due to the differential amount of protein since relatively equal amount of b-Raf protein is present in each group (Figure 5O). The expression of IQGAP1 in cells treated with control siRNA or IQGAP1 siRNA also is shown (Figure 5P). In short, the data indicate that VEGFR-2-dependent endothelial cell proliferation, in part, is established by Src, IQGAP1and b-Raf axis (Figure 5R).
To further address the physiological importance of c-Src, IQGAP1and b-Raf in angiogenesis in an in vivo setting, we used chicken chorioallantoic membrane (CAM) angiogenesis assay where their expression were knocked down by siRNA strategy. The result showed that targeting IQGAP1, c-Src and b-Raf individually by siRNA suppresses the ability of VEGF to stimulate angiogenesis (Figure 6A), where control siRNA had no negative effect on angiogenesis (Figure 6A). Furthermore, quantification of CAM assay showed that VEGF treatment of CAM induced robust angiogenic response where IQGAP1-siRNA, c-Src-siRNA and b-Raf-siRNA each decreased VEGF-induced angiogenesis (Figure 6B). As noted the siRNA-mediated inhibition of VEGF-induced angiogenesis was near to baseline angiogenesis (Figure 6B) underscoring the importance of this pathway for VEGF-induced angiogenesis. The ability of these specific siRNAs to knockdown expression of c-Src, IQGAP1 and b-Raf also are analyzed (Figure 6C, 6E, 6G). The same membranes were re-probed for PLCγ1 as a loading control (Figure 6D, 6F, 6H). In sum, our data demonstrate that IQGAP1, c-Src and b-Raf pathway plays a critical role both in vitro and in vivo in VEGF-mediated angiogenesis.
9-day old chicken chorioallantoic membrane (CAM) (n = 10 CAM for each experimental group) were prepared as described in Materials and Method section and exposed to filter paper disks saturated with siRNAs of either IQGAP1, Src, b-Raf or scrambled control siRNA for 24 hours and then incubated in the presence or in absence VEGF for an additional 72 hours. Pictures were taken by Leica Camera under microscope and a representative pictures from each group are shown (A). Blood vessels were quantified by Leica imaging software system. Each bar represents the mean ±SD of 10 CAM (n = 10). *P<0.05 versus control siRNA plus VEGF (B). CAM tissue was prepared and subjected to Western blot analysis using an anti-IQGAP1 antibody (C), anti-c-Src antibody (E), and anti-b-Raf antibody (G). The same membranes were re-blotted with an anti-PLCγ1 antibody as a control for protein loading (D, F, H).
In this report, we have identified a novel angiogenic pathway emanating from phospho-Y1057 of VEGFR-2. Phosphorylation of Y1057 plays a combinatorial role in VEGFR-2 signaling; it recruits c-Src kinase to VEGFR-2, regulates phosphorylation of multi-docking Y1173 site, mediates VEGFR-2-dependent proliferation of endothelial cells and angiogenesis through IQGAP1 and b-Raf. One of the most interesting aspects of this study is the discovery of an unexpected role of Src family kinases in the phosphorylation of Y1173 of VEGFR-2. Y1173 of VEGFR-2 is considered to be a bona fide mediator of angiogenic signaling of VEGFR-2 in vivo since its mutation abrogates its ability to stimulate angiogenesis during mouse embryonic development [6], [11] and Y1173 functions as a multi-docking residue on VEGFR-2 that recruits p85/PI3kinase, PLCγ1, Shb, and Sck [3]–[7], [10]. Thus, phosphorylation of Y1173 by Src kinases represents one pathway by which the intensity and extent of Y1173-dependent signaling of VEGFR-2 is controlled. Conversely, inhibition of Src kinases could skew the binding of these signaling proteins to VEGFR-2 and their subsequent activation by VEGFR-2.
A recent study indicates that c-Cbl directly interacts with Y1057 of VEGFR-2 [14]. Interestingly, not only does c-Src bind directly to Y1057, but c-Cbl provides an additional mechanism for c-Src to interact with VEGFR-2 by serving as an "adaptor" which in turn may modulate its activation by VEGFR-2. Activation of c-Src is linked to vascular permeability [16], endothelial cell survival and angiogenesis [16], [17]. Our findings show that although c-Src activity is dispensable for VEGFR-2-dependent tube formation of endothelial cells, its activity is highly critical for VEGF-stimulated endothelial cell proliferation and angiogenesis in CAM model of angiogenesis. The mechanism by which c-Src contributes to VEGFR-2-dependent cell proliferation is linked, in part to its ability to phosphorylate Y1173, which is known to regulate VEGFR-2-mediated endothelial cell proliferation [5]–[7]. c-Src also associates with IQGAP1 in endothelial cells and catalyzes tyrosine phosphorylation of IQGAP1 which acts as a downstream target of VEGFR-2 and c-Src to stimulate endothelial cell proliferation. A recent work has demonstrated that c-Src regulates activation of Raf-1 (also known as c-Raf) in endothelial cells [17]. c-Src also associates directly with another Raf family protein, b-Raf, leading to activation of MAPK pathway in a Ras-independent manner [27], [33]. Our demonstration that IQGAP1 is activated by VEGFR-2 further supports earlier studies that shows VEGFR-2 activates MAPK pathway in Ras-independent manner [31].
In conclusion, in this study we have identified and defined a molecular switch by which VEGFR-2 regulates endothelial cell proliferation and stimulates angiogenesis by identifying Y1057 of VEGFR-2 that undergoes phosphorylation and orchestrates recruitment and activation of c-Src and modulates endothelial cell proliferation via IQGAP1-dependent signaling pathway.
Materials and Methods
Reagents, antibodies, siRNAs and vectors
Recombinant human VEGF-A and colony stimulating factor-1 (CSF-1) were purchased from R&D Systems (Minneapolis, MN). Mouse monoclonal anti-phosphotyrosine antibody 4G10 and anti-IQGAP1 antibody were purchased from Upstate Biotechnology (Lake Placid, NY). Rabbit anti-phospho-VEGFR-2 (pY1052) and rabbit polyclonal anti-phospho-VEGFR-2 (pY1057) were made in collaboration with Upstate Biotechnology (Lake Placid, NY). Rabbit polyclonal anti-VEGFR-2 sera were raised against either a glutathione S-transferase-VEGFR-2 kinase insert domain fusion protein (1410) or a GST-VEGFR-2 carboxyl-terminus fusion protein (1412) [18], [21]. Rabbit monoclonal anti-phospho-VEGFR-2 (pY1173) was purchased from Cell Signaling Technology (Beverly, MA). Rabbit polyclonal anti-PLCγ1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The oligonucleotide inserted into pSUPER.retro.puro vector used to produce c-Cbl siRNA was described elsewhere [14], and other oligonucleotide siRNA used in this study are described in supplementary Data S1.
Cell culture, cell lines and retroviral infection
Porcine aortic endothelial (PAE) cells were grown in 10% FBS cells and lack endogenous expression of VEGFR-2 [20]. Primary human dermal microvascular endothelial cells (HMVE cells) were purchased from (Cell Applications, Inc. San Diego) and human umbilical vein endothelial (HUVE) cells, bovine vascular endothelial cells (BAE) cells and human embryonic kidney cells (HEK-293) cells were purchased from ATCC and were grown in 10% FBS plus growth factor supplements and penicillin/ streptomycin. SYF cells are triple knockout (Src, Yes and Fyn) fibroblast cells and they were used as described [15]. Creation of the VEGFR-2 chimera (CKR), in which the extracellular domain of VEGFR-2 is replaced with that of the human CSF-1R, has been previously described [18]. The oligonucleotide siRNA for c-Src was 5′-AAACTCCCCTTG-CTCATGTAC-3′. The oligonucleotide siRNA used for IQGAP1was cloned into pSUPER.retro.puro vector as previously described [27]. Avian siRNAs generated for c-Src, IQGAP1 and b-Raf are the following; siRNAs of avian c-Src were; CAUUGCCAAGGUCAGCGAU UU and AUGACGCCAC AGCGCAGGC UU, siRNA for avian b-raf were; UUGGCUGGGACACUGACAU UU and AGGAUAGGAUCUGGAUCAU UU; siRNA for avian IQGAP1 was; ACUCUGCAAGCCUUACA GAUU. Creation of mutant VEGFR-2s and other related methods are described in supplementary Data S1.
In vitro angiogenesis/tubulogenesis assay
Angiogenesis assay was performed essentially as described [14], [28]. Briefly, PAE cells were suspended in DMEM containing 1% fetal bovine serum and 0.24% (w/v) carboxymethylcellulose (4000 centipoise) in non-adherent round-bottom 96-well plates. After 24 h, all cells formed one single spheroid per well. Spheroids were cultured for additional 48 h before using them in the in vitro angiogenesis assay in the manner previously described [14], [28]. Sprouting and tubulogenesis was observed under an inverted phase-contrast microscope and pictures were taken using a Leica digitial camera system. For statistical analysis purposes four sprouting colonies were used for each experimental condition and their sprouting was quantified using image J program (NIH).
Immunoprecipitation, immunoblotting and in vitro pull-down assay
Cells were prepared for immunoprecipitation as described [14]. Briefly, cells were grown in 10-cm culture dishes until 80–90% confluent and after serum starvation, cells were left either resting or stimulated with appropriate ligands as indicated in figure legends. Cells were lysed and normalized whole cell lysates were subject to immunoprecipitation by incubating with appropriate antibodies. Immunocomplexes were captured by incubating with either Protein-A Sepharose or Protein-G-Agarose beads and immunoprecipitated protein were subjected to immunoblotting analysis. Additional experimental details are provided in Supplementary Data S1. In some occasions the blots were scanned and they were subsequently quantified using Image J program (NIH).
Statistical Analysis
For statistical purposes the blots of three independent experiments were scanned and blots were quantified using NIH Image J program. Comparison of the different parameters for the blots was determined by repeated measures analysis of variance (ANOVA). Significant differences were assigned using Kruskal-Wallis post hoc test. The criterion for significance for all tests was set at p < 0.05. Specific software was used to assist in the data analysis (GraphPadPrism v4.0b, GraphPad Software, San Diego, USA).
GST-Pull down assay
In vitro GST fusion protein binding experiments were performed as described [30]. To this end, equal numbers of endothelial cells were grown to 90% confluency prior to serum starvation for overnight. Unstimulated or ligand-stimulated cells were lysed in ice-cold lysis buffer supplemented with 2 mM Na3VO4 and a protease inhibitor cocktail. Equal amounts of the appropriate immobilized GST fusion proteins were incubated with normalized whole cell lysates by rocking for 3 h at 4°C. The beads were washed four times in the presence of protease inhibitors and Na3VO4, and proteins were eluted and analyzed in Western blot analysis using appropriate antibody.
Dot blot assay
Purified recombinant GST, GST-SH2-c-Src and GST-SH3-c-Src were eluted from glutathione-Sepharose beads and subjected to dot blot. Further information regarding this assay is provided in Supplementary Data S1.
Cell proliferation assay
Cell proliferation was evaluated by direct cell counting. Endothelial cells were seeded at a density of 2×104 cells/well in 24-well plates and cultured overnight; the cells were then incubated in serum-free medium for 12 hours. Cells were stimulated with recombinant human CSF-1 or VEGF-A at different concentrations as indicated in figure legends, and after 48 hours they were washed with PBS, harvested by mild trypsinization, and counted with a hematocytometer. Experiments were performed in triplicate and 3 separate experiments were performed and where appropriate, values were presented as means of ± SD.
Chicken chorioallantoic membrane (CAM) angiogenesis assay
Pathogen free grade fertilized chick embryos (purchased from SPAFAS, Preston, CT) were incubated for 10 days (37°C with 70% humidity). A hole was made with a drill over the air sac at the end of the egg as described [16]. A small window was cut in the egg shell over the dropped CAM, in order to place growth factor and siRNA on the CAM [16], [17]. Filter disks were soaked in cortisone acetate, VEGF (200 ηg), siRNA and plus transfection agent and the disk filters were applied to the CAM of 9 day chick embryos and incubated at 37°C for 72 hours. 10 CAM were used per each group. Pictures were taken by Leica Camera under microscope from three different area randomly and these areas were further used to quantify blood vessels using Leica imaging software system. Statistical Analysis: Data represent mean ±SD of 10 CAM which includes three different fields from each CAM was used unless otherwise stated. Statistical significance was calculated by ANOVA soft ware program (prism v4.0b), considering p<0.05 as statistically significant. Tissue preparation for Western blotting: CAM tissue was also harvested for Western blotting. For this purpose CAM tissue was snap frozen in liquid nitrogen and samples were homogenized in a modified RIPA buffer. Five CAMs from each experimental group were homogenized in 1 ml of the RIPA buffer. Equal amounts of protein were subjected to Western blot analysis using desired antibody as described in the figure legends.
Supporting Information
Figure S1.
Effect of mutation of tyrosines 1052 and 1057 to phenylalanine: Equal number of serum-starved PAE cells expressing wild type CKR, F1052/CKR, F1057/CKR and F1052/F1057/CKR were either unstimulated (−) or stimulated with CSF-1 (+) for 10 minutes. Total cell lysates were subjected to Western blot analysis using an anti-phosphoY1052 specific-VEGFR-2 antibody (A), an anti-phosphoY1057 specific anti-VEGFR-2 antibody (B), an anti-phosphotyrosine antibody (C), and an anti-VEGFR-2 antibody (D). Equal number of serum-starved PAE cells expressing wild type CKR, F1052/CKR, F1057/CKR and F1052/F1057/CKR without stimulation were lysed and immunoprecipitated with an anti-VEGFR-2 antibody. The immunoprecipitated proteins were subjected to an in vitro kinase assay with increasing concentrations of ATP as indicated and described in Materials and Methods and immunoblotted with an anti-phosphotyrosine antibody (E). The same membranes were re-blotted with an anti-VEGFR-2 antibody for proteins levels (F).
Figure S2.
Effect of mutation of Tyrosines 1052 and 1057 to glutamic acid. Partial amino acid sequence of mouse VEGFR-2 containing Y1052 and Y1057 and the schematic presentation of VEGFR-2 are shown (A). Equal number of serum-starved PAE cells expressing wild type CKR, E1052/CKR, E1057/CKR and E1052/E1057/CKR were either unstimulated (−) or stimulated with CSF-1 (+) for 10 minutes. Total cell lysates were subjected to Western blot analysis using an anti-phosphoY1052 specific-VEGFR-2 antibody (B), an anti-phosphoY1057 specific anti-VEGFR-2 antibody (C), an anti-phosphotyrosine antibody (D), and an anti-VEGFR-2 antibody (E). Equal number of serum-starved PAE cells expressing wild type CKR, E1052/CKR, E1057/CKR and E1052/E1057/CKR without stimulation were lysed and immunoprecipitated with an anti-VEGFR-2 antibody. The immunoprecipitated proteins were subjected to an in vitro kinase assay with increasing concentrations of ATP as indicated and described in Materials and Methods and immunoblotted with an anti-phosphotyrosine antibody (F). The same membranes were re-blotted with an anti-VEGFR-2 antibody for proteins levels (G). The image analysis program was used to quantify the data. The tyrosine phosphorylation values were normalized over total protein (H). Arbitrary unit (AU).
Figure S3.
GST-Src but not GST binds to VEGFR-2: Serum-starved PAE cells expressing CKR were either unstimulated (−) or stimulated (+) with CSF-1 for 10 minutes, lysed, and whole cell lysates were incubated with purified GST alone or GST-SH2-Src protein. The GST-SH2-Src bound proteins were subjected to Western blot analysis using an anti-VEGFR-2 antibody (A). Whole cell lysates were blotted with anti-VEGFR-2 antibody as a control (B).
Author Contributions
Conceived and designed the experiments: RDM NR. Performed the experiments: RDM NR. Analyzed the data: RDM NR. Contributed reagents/materials/analysis tools: DBS. Wrote the paper: NR.
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text:
Jeremy Carroll wrote: > > I had deliberately decided not to offer any reasons, since, as far as I can > see, such discussion is out of order - it is about topics that have never > been raised as issues. Yes, well I've been disappointed that we often spend far more time discussing process than actual technical issues. > > However, since you ask - > > I hope that semantic web users will be able to migrate from RDF thru RDFS > thru OWL Lite thru OWL DL thru OWL Full - possibly stopping off where their > needs are met; possibly skipping a stage if they have specific needs that > are only met at higher levels. This is an interesting (and not unexpected) position, which highlights a fundamental problem with our arrangement of languages. There is the (mis)conception that RDFS is simpler than OWL Lite which is simpler than OWL DL which is simpler than OWL Full, yet 'simpler' exactly how? Cognitively? For implementation? In terms of "contraints on the language" might we say that RDFS is the least constrained, followed by OWL Full, then OWL DL and then OWL Lite? I can see why Peter might make certain decisions based upon this latter view, whereas you have certain objections based upon a different (former?) view. We all must agree that the semantic layering is a compromise and one that has left certain questions unanswered -- can we implement a complete OWL full reasoner? etc. So how we organize the 4 languages in a line depends on our differing priorities. These are issues we need to plainly discuss. > > Simply dropping things from the RDFS and RDF vocabulary, apparantly on an > editorial whim, does not involve any sort of "case". > > So I can't live with OWL Lite prohibiting rdfs:seeAlso and rdfs:isDefinedBy > because of the additional migration cost for users moving from RDFS to OWL > Lite. I offer no other defense of these properties - I am not sure that > there is much of one (I agree with your "wishy washy"). Yes, well, to speak plainly, I expect that if you we forbidden to use the terms "rdfs:seeAlso" and "rdfs:isDefinedBy" under penalty of death, that you would be able to design a long and productive life for yourself. Let's not use the term "can't live with" too lightly, eh? > > In summary, it is important to me that the migration path works, which means > that RDF and RDFS features should not be arbitrarily dropped from OWL Lite. Interesting position -- I really (really) wonder what the role of 'vanilla' RDFS will be. Instead I'd hope the migration path might be: OWL Lite -> OWL DL -> OWL Full. Given OWL, what does RDFS add to the picture -- is it easier to implement? is it more powerful? is it really easier to learn (given real world ontological or modelling problems)? JonathanReceived on Thursday, 23 January 2003 22:27:56 UTC
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subset: commoncrawl


text:
Michigan-Air Force: No. 3 Wolverines Upset in The NCAA First RoundMarch 27, 2009
No. 15 ranked Air Force men's ice hockey team shut out No. 3 University of Michigan in the NCAA East Regional semifinal on Friday, 2-0, in Bridgeport, Conn.
Despite out-shooting the Falcons by a 43-13 margin, Michigan was shut out for the fifth time this season and the first time in 71 games of NCAA Tournament play.
The Wolverines failed to convert seven power-play opportunities en route to the loss and had more shots on the man-advantage (16) than Air Force accumulated in the entire game (13).
Michigan went up 5-on-3 for 1:19 early in the opening period after Air Force received penalties at 3:32 and 4:13 for contact to the head and crosschecking, but Michigan could not muster a shot past Air Force net minder, Andrew Volkening, with their two-man advantage.
The Falcons, on the power play, earned three straight man-advantages, capitalizing on the last to take a 1-0 lead at 15:18 of the first period.
After receiving the puck from Jeff Hajner, Derrick Burnett skated to the middle of the right circle where he took a wrist shot that flew into the top left corner of the net for the go-ahead tally.
The score came on Air Force's third shot of the game.
At the first intermission, the Wolverines were out shooting the Falcons by a 16-3 margin.
AHA Player of the Year and Hobey Baker finalist, Jacques Lamoureux, put Air Force up by a pair at 7:02 in the second period.
Matt Fairchild made a cross-ice pass to Lamoureux just outside the crease and slid it past goaltender Bryan Hogan's left side for the two-goal lead.
Neither team scored for the remainder of the frame.
At the second intermission, Michigan had 27 shots to Air Force's eight. The Wolverines were 0-for-6 on the power play.
At 2:08 in the third period, UMich gained another one man-advantage when Greg Flynn was whistled for interference. Michigan had three shots in the power play; none of which found a way between the pipes.
Michigan pulled Hogan from the net with 2:36 remaining in the contest, but could not generate the offense it needed for the comeback.
"Well everything happened that needed to happen for us tonight, said Air Force head coach Frank Serratore. "We had several bounces go our way; we found a way to get a lead. We were fortunate to get some bounces, we had an incredible game in goal from Volkening and we were fortunate to win a huge game."
"This was a disappointing loss for our team," said Michigan head coach Red Berenson. "We expected a tough game and got one. This was not an upset. This was a very tough Air Force team. They held on to the lead. Congratulations to them. Give their team a lot of credit. Their goalie played a tremendous game. We couldn't ask anymore of our team tonight."
The Wolverines finish the season 29-12.
The game marked Michigan's national-best 19th consecutive NCAA Tournament appearance. In the 19-year run, Michigan has collected at least one win 15 times.
Britney Milazzo is a Contributor for Bleacher Report.


text:
Ibis Belfast Queens Quarter
Ibis Belfast Queens Quarter Rates and Deals
Ibis Belfast Queens Quarter Reviews
537 reviews
Cheap and Comfortable Hotel in Belfast Nov 01, 2012
The hotel location is very convenient, 5 minutes walk from Victoria train station, with plenty of cafes and restaurants around it. City centre and shopping areas is within 15 minutes walk from the hotel. 2 blocks away is Ulster Museum and Queens University of Belfast and Belfast Botanic Garden.
The hotel site at old building next to the church with clear signboard outside the building and it is easy to find. The receptionist is friendly and efficient and my room is ready. The room itself is small and basic with modern furniture, television, desk, air condition, kettle for tea/coffee, hair dryer, and en-suite bathroom and free internet in the room.
The bed is very comfortable and the room itself very quite eventhough I have the room facing the street.
The hotel has one small café in the lobby but I didn't try it. Overall, enjoy my stay in Belfast.
5 / 5 TravBuddies found this review helpful/trustworthy
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Ibis Belfast Queens Quarter Overview
Located just outside of the city center and adjacent to Queen's University is the Hotel Ibis Queens Quarter Belfast. It is one of the more conveniently placed hotels in Belfast and is within walking distance of the city's primary shopping, dining, entertainment, and cultural areas. It is only minutes from Ulster Hall, where many concerts take place, and St. George's Market, which is the largest in the city. It is also close to Odyssey Center, Windsor Park, Victoria Square and the lovely City Hall too. The Hotel Ibis Queens Quarter Belfast is located at 75 University Street, and is 15.6 miles from the Belfast International Airport.
The 56 bedrooms in the Hotel Ibis Queens Quarter Belfast are all supplied with personal safes, climate controls, satellite TV, beverage stations, high-speed Internet access, and luxurious bathrooms with complimentary toiletries.
The Hotel Ibis Queens Quarter Belfast provides guests with a nice collection of amenities, including a business service center, in-house restaurant, lounge with bar service, parking on the premises, free daily breakfast, currency exchange, and 24-hour front desk.
Ibis Belfast Queens Quarter Amenities


text:
Duxbury building projects win Town Meeting support
Duxbury's Annual Town Meeting has approved spending on three major building projects, including a new police station, an expanded fire station, and new high and middle schools estimated to run well over $100 million.
All three projects, however, require voters to pass Proposition 2 1/2 overrides at the March 26 town election that would allow the town's debt for the work to be excluded from the annual tax cap.
Total construction costs for the projects are expected to increase the annual tax bill for the median-value tax property — $6,000 on a $481,100 property — by almost $900, according to the town's fiscal advisory committee.
The $6.25 million plan to build a new police station is expected to add $85 to the median tax bill. The $3.7 million approved for an expanded fire station would add $58. Full construction costs for the school project will add an estimated $733.
While clearly the most ambitious project, the schools require just partial funding this year, since the town plans to borrow only $2.9 million for design costs. The total construction cost is estimated to be $130 million, but the state school building program has pledged to reimburse 43.5 percent to the town.
Town Meeting gave its backing Saturday, after a grass-roots group that supports the new schools, Duxbury Cares, held 18 public forums over the last two months to spread information about the project, said chairwoman Karen Wong.
"Our goal was to get information out,'' Wong said. "The more people believed in the project, the more people joined our operation. It took on a life of its own.''
School officials are planning to replace the town's aging high and middle schools, which serve 1,800 students, with two new schools built on the current middle school site. The plan, backed by the state's School Building Authority, calls for the schools to be built close enough together to share some facilities. The plan would also save energy and use less land.
State officials have urged the town to make use of state-approved school designs to save money on architectural costs and fast-track the project to take advantage of a favorable construction market. By taking part in the Model School Program, the town receives a higher rate of reimbursement.
Going into Saturday's Town Meeting, in which all registered local voters are entitled to participate, the school project appeared to be controversial because of its cost in combination with other needed projects.
But school building supporters swelled Town Meeting attendance to 1,300 people, and the proposal to fund design costs passed overwhelmingly by a voice vote.
The new police station project was also approved by a voice vote.
"The police station is a dinosaur and needs to be replaced. It's well recognized by the residents,'' said Town Manager Richard MacDonald on Monday.
The plan approved by Town Meeting calls for building a 15,200-square-foot building on town property on Mayflower Street next to the transfer station.
The new building would replace an undersized and dilapidated station that has a basement that floods, leaky roofs, problems with electronics, a lack of space, and other problems.
For the fire station project, Town Meeting approved $3.7 million in construction costs for a renovation and major addition to the Tremont Street station after approving $300,000 last year for engineering and architectural plans.
If the override is approved at the polls, construction could begin soon.
"The fire station looks fine from the outside, but it needs work,'' MacDonald said.
While both projects received strong support at Town Meeting last weekend, earlier improvement projects for both stations failed at the polls two years ago.
School project backers struck a note of caution as well, despite the strong showing.
"Town Meeting is just the first hurdle,'' Wong said. "The ballot box has been even more challenging.''
Local taxpayers will get something of a break next year on health insurance costs, a budget-buster in many communities, which were held to a 1 percent annual increase.
The town's ability to manage its health care costs helped hold down the overall $55 million budget to a 1.7 percent increase over the current year, which includes a $1 million increase in spending on schools, MacDonald said.
Robert Knox can be reached at firstname.lastname@example.com.


text:
Peyton Manning tossed three interceptions in the first quarter on Monday night and the Atlanta Falcons took advantage of it. The Denver Broncos were not able to recover from these early turnovers and lost in a close 27-21 game.
I truly believe that Manning cost the Broncos the game this time. The three interceptions looked like they all could have been partly or fully Manning's fault. A couple of them looked like the Falcons' mixed coverage was confusing and on one, it looked like the Falcons were confused and happened to be in the right place at the right time. Whatever the reasons are for the interceptions, it was clear that Manning wasn't protecting the football as usual.
I know that Manning would agree and he would take full responsibility for the loss. Here is what he had to say about the turnovers:
"I don't make any excuses for it," Manning said. "Three poor decisions. Three throws into coverage. Just have to be able to eliminate those. I put our team in too far of a hole. I'm sure when I see the film I'll see someone open short underneath on the check down."
The Falcons took advantage early and started with a 20-0 lead. The Broncos did, however, slow down the Falcons' passing attack by holding Julio Jones to 15 yards of total offense. QB Matt Ryan had a good, but not overwhelming, game by passing for 219 yards and 2 touchdowns.
The key here was that the Falcons didn't commit any turnovers. The Broncos had four turnovers and even then brought the game within one possession, but it was too late.
I will guarantee you one thing: Manning will work even harder and make sure that something like this doesn't happen again. This could easily be a great thing for the Broncos because they will realize that they still have to do things as close to perfect as possible to win. There will be times at this stage of Manning's career where the rest of the team has to pick him up during his outings that are not up to par.
You can comment on this article below. Your opinions and thoughts will be greatly appreciated.
Follow me on Twitter @Broncos_Media
Like me on Facebook Mark Stringer – RantSports.com
Add me to your circle on Google + Mark Stringer


text:
Massachusetts proposes new congressional district map
Congressional (Proposed) District Statistics
Mass House Legislative Statistics
Mass Senate Legislative Statistics
BOSTON (AP) – A new congressional map would lump two members of the state's all-Democratic delegation into a single district, creating one new district without an incumbent.
The proposed map, unveiled Monday by the Legislature's Redistricting Committee, would also create the state's first true "minority-majority" district, in which minority groups compromise a majority of the voting age population.
The new districts reflect the loss of one of the state's 10 seats in the House, due to population shifts measured by the most recent U.S. census. The map must still be approved by the full Legislature and signed by Gov. Deval Patrick.
The plan would place U.S. Reps. Stephen Lynch of South Boston and William Keating of Quincy into a new district by pulling Quincy into the district currently represented by Lynch.
Keating, a freshman, whose family owns a home on Cape Cod, would have the option of running in a newly-formed district that includes the Cape – which Keating currently represents – and parts of southeastern Massachusetts.
Keating could not immediately be reached for comment.
The proposed map makes major changes in western Massachusetts by essentially carving up the current 1st District, now represented by Rep. John Olver, who recently announced that he would retire at the end of his term.
Berkshire County would be folded into a new district with Springfield, currently represented by Rep. Richard Neal.
Rep. James McGovern of Worcester would reside in a district that would stretch north to pick up portions of Franklin and Hampshire counties, and south to the Rhode Island state line.
Advocates for minority voters cheered the committee's creation of the redrawn Boston-area district now represented by Rep. Michael Capuano of Somerville.
Carving out a true minority-majority district was a state goal of the redistricting panel.
"I'm pleased with the fact that we have been able to increase the number of minorities both racially and ethnically in this congressional district, and do it by essentially reflecting the movement of people of color into Boston and around Boston," said Rep. Byron Rushing, D-Boston, a member of the committee.
Massachusetts saw its Latino and Asian American population each jump 46 percent over the last 10 years. The state's black population – mainly driven by immigrants from Haiti – saw a rise of 26 percent, while the white population fell by 1.9 percent, according to date from the 2010 census.
The newly-created district, where no incumbent currently resides, would include Cape Cod, Plymouth, New Bedford and part of Fall River.
Rep. Barney Frank, whose district currently stretches from the affluent Boston suburbs of Newton and Brookline to southeastern Massachusetts, would lose New Bedford but retain part of Fall River, which is split in the new map.
The proposed map also makes a major change in the Merrimack Valley region by moving Lawrence into the district now represented by Rep. Niki Tsongas of Lowell.


text: GAVILAN COLLEGE, CA - As of Fall Semester, 2011, Gavilan College is a smoke-and-tobacco-free learning environment. Smoking and use of tobacco products and non-FDA approved nicotine delivery systems (such as "e-cigs") are prohibited on all areas of campus, except for designated smoking areas. FREE smoking-cessation counseling and FDA-approved nicotine replacement therapy can be obtained through the office of Student Health, through a partnership with Breathe California.The new policy was approved by the Gavilan College Board of Trustees at their June meeting, following years of discussion, surveys, and planning by students and student groups to restrict smoking on campus. Student Health nurse Alice Dufresne-Reyes, has been a champion for the new policy. "This moves our campus to significantly better health," she said pointing out that second-hand smoke is particularly dangerous for people with asthma and other breathing difficulties.Approximately three years ago, the Associated Student Body raised the concern of smoking on campus, through an appeal to the Health, Safety, Facilities and Grounds Committee. This led to the creation of a Smoking / Tobacco Cessation Task Force with strong student involvement, which met monthly to study the issue, create the policy, and advocate throughout the campus community.In September 2010, Gavilan College was identified by the Santa Clara County Department of Public Health (SCCPHD) as one of the five schools of higher learning in Santa Clara County that would receive additional training, and health education materials supporting a smoking cessation policy. Gavilan College was the only community college identified in this proposal. ( The remaining community colleges in Santa Clara County already had created smoking control policies.) The California Youth Advocacy Network received grant funding from SCCPHD to provide training and materials for the task force members, and attended the monthly meetings.Smoking is now prohibited in any campus location that is not specifically designated as a smoking area. Five designated smoking areas have been created in the campus parking lots. Each is marked with a sign, blocked off to traffic, and contains a fire-safe container for cigarette butts. The lots with smoking areas are shown on Campus Maps.###


text:
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Why not just:
alias findstring="find . -type f -iname "*$1*"
I don't know, you tell me ..
Your command didn't work on my box, no output.
Its just using the name of the file pattern matching that find does, rather than piping it out to use grep for the same purpose. I would assume its more system efficient.
It won't output anything, if there is no file to match.
[[email protected] ~/dev]$ ll cgfxShader.so
-r-xr-xr-x 1 david.ward sysadmins 394578 May 20 14:09 cgfxShader.so
[[email protected] ~/dev]$ find . -type f -iname "*cgfxsh*"
It's not for filenames, it's for searching for strings inside files.
some implementations of grep won't output the filename if it's searching a single file, and sometimes xargs passes single filenames to grep. So, either add -n2 to the xargs call or -H to the grep call.
!!Critical Error!!
functions and shell scripts can use "$1" - aliases should *NOT*
to expose the bug, you would have to set a static positional
parameter when you invoke bash...
bash -s argv1
alias ekko='echo $1'
ekko test
Also, you need to use the `-print0 | -0` construct -
the completely correct version would be:
alias findstring='find . -type f -print0 | xargs -0 grep -H'
Oh yeah.
Ok, so if its to grep files, then why not something like:
grep -r "$1" ./*
@DaveQB: yes, your last command does the same thing, but there's still a good purpose for having this alias.
On some older implementations of grep, the -r (recursive) option is not implemented (Older HP-UX for example).
Upvoted cause I found myself in need for this command a couple times
Fair enough
asmoore82: Thanks alot for your correction, I also discovered when playing some more with aliases that you could not use $1 and what I had done was only because I had the perception that it would work for aliases and so it did until I tried with 2 parameters and found out I was wrong all along.
Will you please explain the `-print0 | -0` construct ?
And what about the bash command thing you talked about earlier in your comment, does it enable using $1 like in scripts?
the -print0 and -0 combo between find|xargs says that all filenames (that is, strings) passed out of find are null-terminated (via -print0) and xargs expects the input strings to be null terminated (via -0). This covers the case when filenames have spaces. You could also use the multi-argument -exec option of find so I think the command would be:
find . -type f -exec grep -H {} "search string" \;
Although instead of an alias you could us a bash function which does support arguments, although you have to be a little careful about quoting. I have a command in my bashrc for grepping many files:
function grepfiles () { grep -lr "$1" $2; }
use the --include option to restrict the search to .c files or whatever. In bash 4.0 and zsh (and possibly in other shells) there is the ** globbing syntax which means "at any depth" so potentially you could accomplish what you are trying to do with:
function findstring () { grep -H "$1" **/"$2" }
find . -type f -exec grep -H "searchy bandit" {} \;
I think you should put the search string after grep, your way doesn't work for me, it thinks your search argument is the filename.
If I use your grepfiles function like:
grepfiles STRING2FIND *
It only returns one result, not all containing "STRING2FIND"
Which --include option are you talking about? I don't see --include in grep --help ..
[[email protected] test]$ function findstring () { grep -H "$1" **/"$2" }
This happens when I try to assign the function, have to terminate with CTRL+C
I now tried with bash 4.0 and readline 6.0 and this is what happens:
[[email protected] ~]$ findstring stylesheet *.php
grep: **/something.php: No such file or directory
[[email protected] ~]$
in .bashrc
function findstring() { grep -H "$1" **/"$2"; }
If I try with
function findstring() { grep -H "$1" "$2"; }
It finishes instantly and doesn't find anything.
If I try with
function findstring () { grep -H "$1" **/"$2" }
bash: /home/dezza/.bashrc: line 12: syntax error: unexpected end of file
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