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Grap-2, a novel RET binding protein, is involved in RET mitogenic signaling
Leopold Ludwig1, Heidi Kessler, Cuong Hoang-Vu
1Department of Internal Medicine I, University of Ulm, 89081 Ulm, Germany.
Abstract:
Signal transduction of the RET receptor tyrosine kinase is involved in developmental processes as well as in neoplastic transformation. Activation of RET initiates receptor autophosphorylation on specific tyrosines that act as docking sites for downstream signaling molecules. Using the cytoplasmatic part of RET as bait in a yeast two-hybrid screen, we identified a novel SH2 and SH3 domain containing adaptor protein previously termed Grap-2/Grf40/GrpL/GRID and its murine homologue as Gads/Mona, respectively. This protein, predominantly expressed in cells of hematopoietic origin, is involved in signaling downstream of the T-cell receptor and the receptor for monocyte colony-stimulating factor. Here, we show that Grap-2 is also expressed in neuroendocrine tumors and cell lines known to bear mutated forms of RET. Endogenously expressed RET and Grap-2 coimmunoprecipitate from lysates of a medullary thyroid carcinoma cell line. Grap-2 directly associates with RET in pull-down experiments using in vitro translated proteins. Overexpression of Grap-2 inhibits RET-induced NF-kappaB activation, and cotransfection of Grap-2 significantly reduces focus formation induced by oncogenic RET in NIH 3T3 cells. Taken together, these results suggest that besides being involved in tyrosine kinase signaling in hematopoietic cells, Grap-2 plays a tissue-specific role as an inhibitor of RET mitogenic signaling.
Insights
Grap-2, a novel adaptor protein, inhibits RET receptor tyrosine kinase signaling in neuroendocrine tumors. This finding suggests a tissue-specific inhibitory role for Grap-2 in RET-driven cancer development.
Area of Science:
- Molecular Biology
- Cell Biology
- Oncology
Background:
- The RET receptor tyrosine kinase is crucial for development and implicated in cancer.
- RET activation involves autophosphorylation and docking of signaling molecules.
- Adaptor proteins mediate downstream signaling pathways.
Purpose of the Study:
- To identify novel proteins interacting with RET.
- To investigate the role of Grap-2 in RET signaling.
- To explore Grap-2's function in neuroendocrine tumors.
Main Methods:
- Yeast two-hybrid screening to identify interacting proteins.
- Coimmunoprecipitation and pull-down assays to confirm protein interactions.
- Functional assays including NF-kappaB activation and focus formation assays.
Main Results:
- Grap-2, an SH2 and SH3 domain-containing adaptor protein, was identified as a RET-interacting protein.
- Grap-2 is expressed in neuroendocrine tumors and coimmunoprecipitates with RET.
- Grap-2 overexpression inhibits RET-induced NF-kappaB activation and focus formation.
Conclusions:
- Grap-2 interacts with RET and inhibits its mitogenic signaling in a tissue-specific manner.
- Grap-2 may act as a tumor suppressor in RET-driven neuroendocrine cancers.
- This study reveals a novel inhibitory role for Grap-2 in tyrosine kinase signaling.