CCR Translation for the Article from Vascular Endothelial Growth Factor Receptor-1 Is Synthetic Lethal to Aberrant β-Catenin Activation in Colon Cancer
Abstract Purpose: The Wnt/β-catenin (β-cat) signaling cascade is a key regulator of development, and dysregulation of Wnt/β-cat contributes to selected cancers, such as colorectal, breast, and hepatocellular carcinoma, through abnormal activation of Wnt target genes. To identify novel modulators of the Wnt/β-cat pathway that may emerge as therapeutic targets, we did an unbiased high-throughput RNA interference screen. Experimental Design: A synthetic oligonucleotide small interfering RNA library targeting 691 known and predicted human kinases was screened in Wnt3a-stimulated human cells in a live cell luciferase assay for modulation of Wnt/β-cat–dependent transcription. Follow-up studies of a selected high-confidence “hit” were conducted. Results: A robust quartile-based statistical analysis and secondary screen yielded several kinases worthy of further investigation, including Cdc2L1, Lmtk3, Pank2, ErbB3, and, of note, vascular endothelial growth factor receptor (VEGFR)1/Flt1, a receptor tyrosine kinase (TK) with putative weak kinase activity conventionally believed to be a negative regulator of angiogenesis. A series of loss-of-function, genetic null, and VEGFR TK inhibitor assays further revealed that VEGFR1 is a positive regulator of Wnt signaling that functions in a glycogen synthase kinase-3β (GSK3β)–independent manner as a potential synthetic lethal target in Wnt/β-cat–addicted colon carcinoma cells. Conclusions: This unanticipated non-endothelial link between VEGFR1 TK activity and Wnt/β-cat signaling may refine our understanding of aberrant Wnt signaling in colon carcinoma and points to new combinatorial therapeutics targeted to the tumor cell compartment, rather than angiogenesis, in the context of colon cancer. (Clin Cancer Res 2009;15(24):7529–37)
Color me yellow: Poly(ADP-ribose) polymerases (PARPs) play a major role in cellular survival and maintenance of energy stores after genotoxic insult. The colorimetric PARP substrate ADP-ribose-pNP can be used to monitor PARP activity. By monitoring the production of p-nitrophenolate, the kinetic parameters of PARP-1, tankyrase, and PARP-4 could be evaluated. ADP=adenosine diphosphate, pNP=p-nitrophenoxy.
Gelb strahlt: Poly(ADP-Ribose)-Polymerasen (PARPs) spielen eine zentrale Rolle für das Überleben der Zellen und die Aufrechterhaltung von Energiespeichern nach einem gentoxischen Angriff. Das kolorimetrische PARP-Substrat ADP-Ribose-pNP kann zur Verfolgung der PARP-Aktivität genutzt werden. Anhand der Produktion von p-Nitrophenolat sind die Kinetikparameter von PARP-1, Tankyrase und PARP-4 ermittelbar. ADP = Adenosindiphosphat, pNP = p-Nitrophenoxy. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2001/2007/z603988_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
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Abstract Fragment complementation systems based on various luciferase enzymes have allowed for a more complete exploration of the protein interactome within the context of the cellular environment over biologically-relevant timescales. However, this strategy has been plagued by lingering questions regarding the reversibility of the systems that involve luciferase complementation fragments. To further address the need for tools to study the protein interactome, we have developed a novel set of multicolored heteroprotein fragment complementation systems based on various firefly and click beetle luciferases that utilize the same substrate, D-luciferin. Selected heteroprotein fragment complementation systems enabled simultaneous dual-color quantification of multiple protein interactions within live cells in realtime through use of spectral deconvolution analysis. Importantly, we have rigorously characterized the reversibility that is inherent in these systems. Reversibility was noted by the reproduction of association and dissociation constants when probing FK506 blockade of rapamycin-induced mTOR/FKBP interactions. The mean apparent Kd value upon rapamycin induction was 0.25 ± 0.05 nM, irrespective of the composition of the paired luciferases, spectral characteristics, total photon flux, or fold-inducibility. Competitive inhibition by FK506 with all the different pairs, when tested in the presence of 10 nM rapamycin, yielded a mean apparent Ki value of 1.6 ± 0.9 nM, consistent with conventional protein assays. Additionally, using split click-beetle green luciferase fused to the C-termini of EGFR, a more temporally dynamic system, we were able to monitor reversible protein conformational changes associated with the cytoplasmic domains of EGFR upon ligand-induced activation. Addition of EGF resulted in a biphasic response, characterized by an immediate drop in luciferase activity within the first minute after EGF addition followed by a rapid recovery of signal. The initial signal loss correlated with asymmetric movement of the cytosolic C-termini of EGFR dimers and adoption of a constrained conformation after phosphorylation. The subsequent recovery in luciferase activity corresponded to additional conformational changes that placed the relaxed C-terminal tails once again in close proximity. Addition of the MAPK inhibitor U0126 (10 µM) resulted in a blunted recovery of click-beetle green luciferase activity, again demonstrating the requirement for MAPK-mediated phosphorylation to induce post-activation rearrangements of the cytoplamic domain of the receptor. These dual-color reversible protein interaction reporters should enable dynamic analysis of a variety of protein networks in living cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-137.
We compare the folding transition state (TS) of ubiquitin previously identified by using ψ analysis to that determined by using φ analysis. Both methods attempt to identify interactions and their relative populations at the rate-limiting step for folding. The TS ensemble derived from ψ analysis has an extensive native-like chain topology, with a four-stranded β-sheet network and a portion of the major helix. According to φ analysis, however, the TS is much smaller and more polarized, with only a local helix/hairpin motif. We find that structured regions can have φ values far from unity, the canonical value for such sites, because of structural relaxation of the TS. Consequently, these sites may be incorrectly interpreted as contributing little to the structure of the TS. These results stress the need for caution when interpreting and drawing conclusions from φ analysis alone and highlight the need for more specific tools for examining the structure and energetics of the TS ensemble.
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