Annals of Oncology Advance Access originally published online on May 13, 2008
Annals of Oncology 2008 19(7):1356-1357; doi:10.1093/annonc/mdn293
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letters to the editor |
Insulin receptor substrate protein 53 (IRSp53) as a binding partner of antimetastasis molecule NESH, a member of Abelson interactor protein family
Involvement of NESH in cancer invasion and metastasis is now very important and interesting. In order to clarify the molecular mechanisms, we identified insulin receptor substrate protein 53 (IRSp53) as a partner of NESH utilizing a conventional yeast two-hybrid screen with a proline-rich domain of NESH as bait. The specific interaction was confirmed by coimmunoprecipitation assay and colocalization analysis in mammalian cells. The carboxyl-terminal SH3 domain of IRSp53 is responsible for the interaction with the proline-rich domain of NESH. NESH may play a critical role in regulating the actin cytoskeleton required for membrane ruffling induced by the IRSp53 pathway.
We observed that NESH expression caused a marked reduction in motility and exhibited significant reduction in tumor metastatic potential in vivo [1]. NESH expression is also frequently lost in invasive cancer cell lines despite its ubiquitous expression in normal tissues. We, however, also found that use of imatinib to treat NESH-expressed cancer cells greatly enhances its invasive tumor growth in vivo [2]. An unknown antimetastasis pathway in which NESH is involved might be regulated by abelson tyrosine kinase [3]. To identify proteins interacting with the NESH, we used a two-hybrid system to screen a human complementary DNA library with a NESH protein as bait. More than four positive preys were identified, and subsequent sequence analysis revealed one of the preys to be IRSp53.
To determine whether IRSp53 and NESH might interact, we first determined the association by glutathione S-transferase (GST) pull-down assays. We found that the SH3 domain of IRSp53 was associated with the proline-rich region of NESH in NIH3T3 cells but not with control GST protein (data not shown). We examined the biochemical in vivo interactions between NESH and IRSp53 (Figure 1A). NIH3T3 cells were transfected with expression plasmids for NESH. The cell lysates were subjected to immunoprecipitation using either anti-NESH or anti-IRSp53 antibodies. As shown in Figure 1A, NESH, but not control, was coprecipitated with IRSp53. Reciprocally, IRSp53 was coprecipitated with NESH but not with the mock antibody. We next carried out double immunofluorescent staining for IRSp53 and NESH in 3Y1 cells. Analysis with confocal microscopy clearly indicated that NESH and IRSp53 colocalized in 3Y1 cells (Figure 1B). We also investigated the subcellular localization of IRSp53 in NIH3T3 and human umbilical vein endothelial cells, and the IRSp53 staining again overlapped with that of NESH (data not shown). These results indicated an interaction between IRSp53 and NESH in vivo. The two-hybrid assay revealed that the SH3 region of IRSp53 was necessary for this interaction since the truncated protein of IRSp53 did not interact with NESH (data not shown). Several studies have indicated the involvement of IRSp53 in growth factor-mediated membrane ruffling. Therefore, we examined the effects of NESH expression on platelet-derived growth factor (PDGF)-induced membrane ruffling. Treating the cells with 5 ng/ml of PDGF for 10 min induced rapid membrane ruffling that was not present in untreated cells (Figure 1C, 1–2). However, when the cells were transiently transfected with the NESH expression plasmid, PDGF-mediated membrane ruffling was undetectable (Figure 1C, 3–5, and D).
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Tumor metastasis is a dynamic multistep process involving increased migration to the distant site. A variety of molecules function and interact with each other in order to metastasize. NESH as well as IRSp53 is localized to the edge of the lamellipodial protrusions, in which the migration requires polymerization of actin. IRSp53 can associate with Rac as small guanosine triphosphatases of the Rho family through its amino-terminal domain and plays a pivotal role in lamellipodia formation in motile cells [4]. Binding of activated Rac and WAVE2 via IRSp53 induces actin filament assembly, reorganization and membrane ruffling in NIH3T3 cells. The manner of regulation of this activity, however, remains largely unknown. Here, we show that overexpression of NESH potently blocks PDGF-stimulated membrane ruffling in mammalian cells. NESH binds IRSp53-SH3 domain which is identical with binding site of WAVE2 [5], which might compete with WAVE2 for binding to IRSp53.
funding
Grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology in Japan and Nara Women's University Intramural Grant for Project Research.
1 Department of Environmental Health, Nara Women's University, Nara 630-8506
2 Department of Molecular Pathogenesis
3 Second Department of Internal Medicine, Nagoya University School of Medicine, Nagoya 466-8550
4 Department of Hematology/Oncology, Teikyo University School of Medicine, Tokyo, Japan
* (E-mail: smatsuda{at}cc.nara-wu.ac.jp)
Acknowledgements
The authors declare that they have no competing financial interests.
Footnotes
These authors contributed equally to this work. 
References
1. Ichigotani Y, Yokozaki S, Fukuda Y, et al. Forced expression of NESH suppresses motility and metastatic dissemination of malignant cells. Cancer Res (2002) 62:2215–2219.
2. Matsuda S, Ichigotani Y, Okumura N, et al. NESH protein expression switches to the adverse effect of imatinib mesylate. Mol Oncol (2008) doi: 10.1016/j.molonc.2008.03.003.
3. Terauchi K, Shimada J, Uekawa N, et al. Cancer-associated loss of TARSH gene expression in human primary lung cancer. J Cancer Res Clin Oncol (2006) 132:28–34.[CrossRef][Web of Science][Medline]
4. Disanza A, Mantoani S, Hertzog M, et al. Regulation of cell shape by Cdc42 is mediated by the synergic actin-bundling activity of the Eps8-IRSp53 complex. Nat Cell Biol (2006) 8:1337–1347.[CrossRef][Web of Science][Medline]
5. Oda A, Miki H, Wada I, et al. WAVE/Scars in platelets. Blood (2005) 105:3141–3148.
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