Characterization of the Interactions between Calmodulin and Death Receptor 5 in Triple-negative and Estrogen Receptor-positive Breast Cancer Cells AN INTEGRATED EXPERIMENTAL AND COMPUTATIONAL STUDY

Romone M. Fancy; Lingyun Wang; Qinghua Zeng; Hong Wang; Tong Zhou; Donald J. Buchsbaum; Yuhua Song

doi:10.1074/jbc.M116.727727

Characterization of the Interactions between Calmodulin and Death Receptor 5 in Triple-negative and Estrogen Receptor-positive Breast Cancer Cells

AN INTEGRATED EXPERIMENTAL AND COMPUTATIONAL STUDY*

From the Departments of ^‡Biomedical Engineering,
^§Medicine, and
^¶Radiation Oncology, University of Alabama at Birmingham, Birmingham, Alabama 35294

↵2 To whom correspondence should be addressed: Dept. of Biomedical Engineering, The University of Alabama at Birmingham, 803 Shelby Interdisciplinary Biomedical Research Bldg, 1825 University Blvd., Birmingham, AL 35294. Tel.: (205)-996-6939; Fax: (205)-975-4919; E-mail: yhsong{at}uab.edu.

Abstract

Activation of death receptor-5 (DR5) leads to the formation of death inducing signaling complex (DISC) for apoptotic signaling. Targeting DR5 to induce breast cancer apoptosis is a promising strategy to circumvent drug resistance and present a target for breast cancer treatment. Calmodulin (CaM) has been shown to regulate DR5-mediated apoptotic signaling, however, its mechanism remains unknown. In this study, we characterized CaM and DR5 interactions in breast cancer cells with integrated experimental and computational approaches. Results show that CaM directly binds to DR5 in a calcium dependent manner in breast cancer cells. The direct interaction of CaM with DR5 is localized at DR5 death domain. We have predicted and verified the CaM-binding site in DR5 being ³⁵⁴WEPLMRKLGL³⁶³ that is located at the α2 helix and the loop between α2 helix and α3 helix of DR5 DD. The residues of Trp-354, Arg-359, Glu-355, Leu-363, and Glu-367 in DR5 death domain that are important for DR5 recruitment of FADD and caspase-8 for DISC formation to signal apoptosis also play an important role for CaM-DR5 binding. The changed electrostatic potential distribution in the CaM-binding site in DR5 DD by the point mutations of W354A, E355K, R359A, L363N, or E367K in DR5 DD could directly contribute to the experimentally observed decreased CaM-DR5 binding by the point mutations of the key residues in DR5 DD. Results from this study provide a key step for the further investigation of the role of CaM-DR5 binding in DR5-mediated DISC formation for apoptosis in breast cancer cells.

Footnotes

↵1 Supported by Comprehensive Minority Faculty and Student Development Program fellowship from the University of Alabama VP Office for Equity and Diversity.
↵* This work was supported in part by an National Institutes of Health K25 award (5K25 CA140791) (to Y. S.). The authors declare that they have no conflicts of interest with the contents of this article except that Dr. Tong Zhou and Dr. Donald J. Buchsbaum have intellectual property right in TRA-8. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
↵ This article contains supplemental Tables S1 and S2