in-vitro-methylation-assay-to-study-protein-arginine-methylation

<em>In vitro </em>Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

The Soft Agar Colony Formation Assay

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft ...

University of Illinois at Chicago

Infection of SARS-associated coronavirus (SARS-CoV) induced a strong anti-nucleocapsid (anti-N) antibody response. However, the pathophysiological significance of the anti-N antibodies in SARS pathogenesis is largely unknown. To profile the anti-N antibodies, a phage-displayed scFv library was prepared from mice immunized with heat-inactivated SARS-CoV-infected Vero E6 cell lysate. Specific anti-N scFvs were isolated by panning against a recombinant nucleocapsid protein and reactivity was confirmed with phage-ELISA. Sequence analysis indicated that two of the isolated anti-N scFv clones were identical and displayed a high homology with an scFv specific for interleukin 11 (IL-11), an anti-inflammatory cytokine derived from bone marrow stroma cells. In a neutralization assay, IL-11-induced STAT 3 phosphorylation in rat intestinal epithelial IEC-18 cells was completely suppressed by the anti-N scFv clone L9N01.

Cross-reactivity of antibody against SARS-coronavirus nucleocapsid protein with IL-11.

In the present study, a phage-displayed random peptide library was used to identify surrogate peptide ligands for orphan GPCR mas. Sequence analysis of the isolated phage clones indicated a selective enrichment of some peptide sequences. Moreover, multiple alignments of the isolated phage clones gave two conserved peptide motifs from which we synthesized peptide MBP7 for further evaluation. Characterization of the representative phage clones and the synthetic peptide MBP7 by immunocytochemistry revealed a strong punctate cell surface staining in CHO cells expressing mas-GFP fusion protein. The isolated phage clones and synthetic peptide MBP7 induced mas internalization in a stable CHO cell clone (MC0M80) over-expressing mas. In addition, MBP7-stimulated phospholipase C activity and intracellular calcium mobilization in these same cells. In summary, we have demonstrated a systematic approach to derive surrogate peptide ligands for orphan GPCRs. With this technique, we have identified two conserved peptide motifs which allow us to identify potential protein partners for mas, and have generated a peptide agonist MBP7 which will be invaluable for functional characterization of the mas oncogene.

Identification and characterization of surrogate peptide ligand for orphan G protein-coupled receptor mas using phage-displayed peptide library.

In Drosophila, activation of Jun N-terminal Kinase (JNK) mediated by Frizzled and Dishevelled leads to signaling linked to planar cell polarity. A biochemical delineation of WNT-JNK planar cell polarity was sought in mammalian cells, making use of totipotent mouse F9 teratocarcinoma cells that respond to WNT3a via Frizzled-1. The canonical WNT-beta-catenin signaling pathway requires both G alpha o and G alpha q heterotrimeric G-proteins, whereas we show that WNT-JNK signaling requires only G alpha o protein. G alpha o propagates the signal downstream through all three Dishevelled isoforms, as determined by epistasis experiments using the Dishevelled antagonist Dapper1 (DACT1). Suppression of either Dishevelled-1 or Dishevelled-3, but not Dishevelled-2, abolishes WNT3a activation of JNK. Activation of the small GTPases RhoA, Rac1 and Cdc42 operates downstream of Dishevelled, linking to the MEKK 1/MEKK 4-dependent cascade, and on to JNK activation. Chemical inhibitors of JNK (SP600125), but not p38 (SB203580), block WNT3a activation of JNK, whereas both the inhibitors attenuate the WNT3a-beta-catenin pathway. These data reveal both common and unique signaling elements in WNT3a-sensitive pathways, highlighting crosstalk from WNT3a-JNK to WNT3a-beta-catenin signaling.

G alpha o mediates WNT-JNK signaling through dishevelled 1 and 3, RhoA family members, and MEKK 1 and 4 in mammalian cells.

The Wnt-beta-catenin canonical signaling pathway is crucial for normal embryonic development, and aberrant expression of components of this pathway results in oncogenesis. Upon scanning for the mitogen-activated protein kinase (MAPK) pathways that might intersect with the canonical Wnt-beta-catenin signaling pathway in response to Wnt3a, we observed a strong activation of p38 MAPK in mouse F9 teratocarcinoma cells. Wnt3a-induced p38 MAPK activation was sensitive to siRNAs against Galpha(q) or Galpha(s), but not against either Galpha(o) or Galpha(11). Activation of p38 MAPK is critical for canonical Wnt-beta-catenin signaling. Chemical inhibitors of p38 MAPK (SB203580 or SB239063) and expression of a dominant negative-version of p38 MAPK attenuate Wnt3a-induced accumulation of beta-catenin, Lef/Tcf-sensitive gene activation, and primitive endoderm formation. Furthermore, epistasis experiments pinpoint p38 MAPK as operating downstream of Dishevelleds. We also demonstrate that chemical inhibition of p38 MAPK restores Wnt3a-attenuated GSK3beta kinase activity. We demonstrate the involvement of G-proteins and Dishevelleds in Wnt3a-induced p38 MAPK activation, highlighting a critical role for p38 MAPK in canonical Wnt-beta-catenin signaling.

p38 mitogen-activated protein kinase regulates canonical Wnt-beta-catenin signaling by inactivation of GSK3beta.

Wnt/beta-catenin canonical pathway is critical for normal embryonic development; mutations and aberrant expression of specific components of this pathway can be oncogenic. Mitogen-activated protein kinase (MAPK) pathways, prominent in intracellular signaling, have been shown to have unique and provocative roles that impact the Wnt/beta-catenin signaling. We discuss recent insights that implicate the three major pathways of the MAPK network, i.e., mediated by p38, c-Jun N-terminal (JNK) kinase and Extra-cellular-Regulated Kinases (ERK) and their downstream signaling elements in Wnt/beta-catenin signaling. Novel "crosstalk" among MAPK and Wnt/beta-catenin canonical signaling pathways is essential. A fuller understanding of how such signaling is integrated during development is a high-value target for future research.

Mitogen-activated protein kinases and Wnt/beta-catenin signaling: Molecular conversations among signaling pathways.

Canonical Wnt/beta-catenin signaling is crucial during embryonic development. Upon Wnt stimulation, Dishevelled proteins relay the signal from upstream Frizzled receptors to downstream effectors. By using affinity purification followed by ion-trap mass spectrometry we identified K-homology splicing regulator protein (KSRP) as a novel Dishevelled-interacting protein. We show that KSRP negatively regulates Wnt/beta-catenin signaling at the level of post-transcriptional CTNNB1 (beta-catenin) mRNA stability. Thus, Dishevelled-KSRP complex operates in Wnt regulation of beta-catenin, functioning post-transcriptionally upon CTNNB1 mRNA stability.

Dishevelled-KSRP complex regulates Wnt signaling through post-transcriptional stabilization of beta-catenin mRNA.

Wnt/β-catenin signaling is essential for normal mammalian development. Wnt3a activates the Wnt/β-catenin pathway through stabilization of β-catenin; a process in which the phosphoprotein Dishevelled figures prominently. Protein arginine methylation in signaling complexes containing Dishevelled was investigated. Mass spectrometry of a prominent arginine-methylated, Dishevelled-associated protein identified the Ras GTPase activating protein-binding protein 1 G3BP1. Stimulation of totipotent mouse embryonic F9 cells with Wnt3a provoked increased methylation of G3BP1. We show that G3BP1 is a novel Ctnnb1 mRNA binding protein. Methylation of G3BP1 constitutes a molecular switch that regulates Ctnnb1 mRNA in response to Wnt3a. Thus, the protein arginine methylation that targets G3BP1 acts as a novel regulator of Ctnnb1 mRNA.

Arginine methylation of G3BP1 in response to Wnt3a regulates β-catenin mRNA.

Wnt signaling is initiated upon binding of Wnt proteins to Frizzled proteins and their co-receptors LRP5 and 6. The signal is then propagated to several downstream effectors, mediated by the phosphoprotein scaffold, dishevelled. We report a novel role for arginine methylation in regulating Wnt3a-stimulated LRP6 phosphorylation. G3BP2, a dishevelled-associated protein, is methylated in response to Wnt3a. The Wnt3a-induced LRP6 phosphorylation is attenuated by G3BP2 knockdown, chemical inhibition of methyl transferase activity or expression of methylation-deficient mutants of G3BP2. Arginine methylation of G3BP2 appears to be a Wnt3a-sensitive 'switch' regulating LRP6 phosphorylation and canonical Wnt-β-catenin signaling.

Wnt3a-stimulated LRP6 phosphorylation is dependent upon arginine methylation of G3BP2.

Dishevelled, a phosphoprotein scaffold, is a central component in all the Wnt-sensitive signaling pathways. In the present study, we report that Dishevelled is post-translationally modified, both in vitro and in vivo, via arginine methylation. We also show protein arginine methyl transferases 1 and 7 as the key enzymes catalyzing Dishevelled methylation. Interestingly, Wnt3a stimulation of F9 teratocarcinoma cells results in reduced Dishevelled methylation. Similarly, the methylation-deficient mutant of Dishevelled, R271K, displayed spontaneous membrane localization and robust activation of Wnt signaling; suggesting that differential methylation of Dishevelled plays an important role in Wnt signaling. Thus arginine methylation is shown to be an important switch in regulation of Dishevelled function and Wnt signaling.

Dishevelled3 is a novel arginine methyl transferase substrate.

Targeted therapies for cancer bring the hope of specific treatment, providing high efficacy and in some cases lower toxicity than conventional treatment. Although targeted therapeutics have helped immensely in the treatment of several cancers, like chronic myelogenous leukemia, colon cancer, and breast cancer, the benefit of these agents in the treatment of lung cancer remains limited, in part due to the development of drug resistance. In this review, we discuss the mechanisms of drug resistance and the current strategies used to treat lung cancer. A better understanding of these drug-resistance mechanisms could potentially benefit from the development of a more robust personalized medicine approach for the treatment of lung cancer.

Non-small-cell lung cancer: molecular targeted therapy and personalized medicine - drug resistance, mechanisms, and strategies.

In non-small cell lung cancer cell lines, activation of β-catenin independent signaling, via Wnt7a/Frizzled9 signaling, leads to reversal of cellular transformation, reduced anchorage-independent growth and induction of epithelial differentiation. miRNA expression profiling on a human lung adenocarcinoma cell line (A549) identified hsa-miR29b as an important downstream target of Wnt7a/Frizzled9 signaling. We show herein that hsa-miR29b expression is lost in non-small cell lung cancer (NSCLC) cell lines and stimulation of β-catenin independent signaling, via Wnt7a expression, in NSCLC cell lines results in increased expression of hsa-miR29b. Surprisingly, we also identify specific regulation of hsa-miR29b by Wnt7a but not by Wnt3, a ligand for β-catenin-dependent signaling. Interestingly, knockdown of hsa-miR29b was enough to abrogate the tumor suppressive effects of Wnt7a/Frizzled9 signaling in NSCLC cells, suggesting that hsa-miR29b is an important mediator of β-catenin independent signaling. Finally, we show for the first time that hsa-miR29b plays an important role as a tumor suppressor in lung cancer by targeting murine double mutant 2 (MDM2), revealing novel nodes for Wnt7a/Frizzled9-mediated regulation of NSCLC cell proliferation.

hsa-miR29b, a critical downstream target of non-canonical Wnt signaling, plays an anti-proliferative role in non-small cell lung cancer cells via targeting MDM2 expression.

G-protein-coupled receptors (GPCR) are the largest family of cell surface molecules that play important role/s in a number of biological and pathological processes including cancers. Earlier studies have highlighted the importance of Wnt7a signaling via its cognate receptor Frizzled9, a GPCR, in inhibition of cell proliferation, anchorage-independent growth, and reversal of transformed phenotype in non small cell lung cancer primarily through activation of the tumor suppressor, PPARγ. However, the G-protein effectors that couple to this important tumor suppressor pathway have not been identified, and are of potential therapeutic interest. In this study, by using two independent Wnt7a/Frizzled9-specific read-outs, we identify Gα16 as a novel downstream effector of Wnt7a/Frizzled9 signaling. Interestingly, Gα16 expression is severely down-regulated, both at the messenger RNA levels and protein levels, in many non small cell lung cancer cell lines. Additionally, through gene-specific knock-downs and expression of GTPase-deficient forms (Q212L) of Gα16, we also establish Gα16 as a novel regulator of non small cell lung cancer cell proliferation and anchorage-independent cell growth. Taken together, our data not only establish the importance of Gα16 as a critical downstream effector of the non-canonical Wnt signaling pathway but also as a potential therapeutic target for the treatment of non small cell lung cancer.

Heterotrimeric G-protein, Gα16, is a critical downstream effector of non-canonical Wnt signaling and a potent inhibitor of transformed cell growth in non small cell lung cancer.

ArticleTotal : 2	Year
Publication title Cited by 8	2014
Publication title Cited by 525	2014

Article	Year
Cross-reactivity of antibody against SARS-coronavirus nucleocapsid protein with IL-11. Biochemical and biophysical research communications\| PubMed ID: 16263078	2005
Identification and characterization of surrogate peptide ligand for orphan G protein-coupled receptor mas using phage-displayed peptide library. Biochemical pharmacology\| PubMed ID: 16336942	2006
G alpha o mediates WNT-JNK signaling through dishevelled 1 and 3, RhoA family members, and MEKK 1 and 4 in mammalian cells. Journal of cell science\| PubMed ID: 18187455	2008
p38 mitogen-activated protein kinase regulates canonical Wnt-beta-catenin signaling by inactivation of GSK3beta. Journal of cell science\| PubMed ID: 18946023	2008
Mitogen-activated protein kinases and Wnt/beta-catenin signaling: Molecular conversations among signaling pathways. Communicative & integrative biology\| PubMed ID: 19513264	2009
Dishevelled-KSRP complex regulates Wnt signaling through post-transcriptional stabilization of beta-catenin mRNA. Journal of cell science\| PubMed ID: 20332102	2010
Arginine methylation of G3BP1 in response to Wnt3a regulates β-catenin mRNA. Journal of cell science\| PubMed ID: 21652632	2011
Wnt3a-stimulated LRP6 phosphorylation is dependent upon arginine methylation of G3BP2. Journal of cell science\| PubMed ID: 22357953	2012
Dishevelled3 is a novel arginine methyl transferase substrate. Scientific reports\| PubMed ID: 23150776	2012
Non-small-cell lung cancer: molecular targeted therapy and personalized medicine - drug resistance, mechanisms, and strategies. Pharmacogenomics and personalized medicine\| PubMed ID: 23690695	2013
hsa-miR29b, a critical downstream target of non-canonical Wnt signaling, plays an anti-proliferative role in non-small cell lung cancer cells via targeting MDM2 expression. Biology open\| PubMed ID: 23862015	2013
Heterotrimeric G-protein, Gα16, is a critical downstream effector of non-canonical Wnt signaling and a potent inhibitor of transformed cell growth in non small cell lung cancer. PloS one\| PubMed ID: 24204697	2013

Rama Kamesh Bikkavilli

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