Abstract
Overexpression of the cellular FLICE-like inhibitory protein (cFLIP) has been reported in a number of tumor types. As an inactive procaspase-8 homologue, cFLIP is recruited to the intracellular assembly known as the Death Inducing Signaling Complex (DISC) where it inhibits apoptosis, leading to cancer cell proliferation. Here we characterize the molecular details of the interaction between cFLIPL and calmodulin, a ubiquitous calcium sensing protein. By expressing the individual domains of cFLIPL, we demonstrate that the interaction with calmodulin is mediated by the N-terminal death effector domain (DED1) of cFLIPL. Additionally, we mapped the interaction to a specific region of the C-terminus of DED1, referred to as DED1 R4. By designing DED1/DED2 chimeric constructs in which the homologous R4 regions of the two domains were swapped, calmodulin binding properties were transferred to DED2 and removed from DED1. Furthermore, we show that the isolated DED1 R4 peptide binds to calmodulin and solve the structure of the peptide-protein complex using NMR and computational refinement. Finally, we demonstrate an interaction between cFLIPL and calmodulin in cancer cell lysates. In summary, our data implicate calmodulin as a potential player in DISC-mediated apoptosis and provide evidence for a specific interaction with the DED1 of cFLIPL.
Highlights
IntroductionCFLIP (cellular FLICE-like inhibitory protein) is a key anti-apoptotic protein over-expressed in multiple types of tumor cells [1, 2]
CFLIP is a key anti-apoptotic protein over-expressed in multiple types of tumor cells [1, 2]
Following reports of calmodulin acting as an antagonist of the TRAIL pathway, we queried the possibility of a specific interaction between calmodulin and the major Death Inducing Signaling Complex (DISC) anti-apoptotic regulator, cellular FLICE-like inhibitory protein (cFLIP)
Summary
CFLIP (cellular FLICE-like inhibitory protein) is a key anti-apoptotic protein over-expressed in multiple types of tumor cells [1, 2]. CFLIP inhibits extracellular receptor-mediated (or extrinsic) apoptosis, which in tumor cells enables a mechanism for cell survival and uncontrolled proliferation [3]. Cancer cells displaying high levels of cFLIP expression appear to become resistant to chemotherapeutic agents [3,4,5,6,7,8,9]. One aim is to induce cell death in tumor cells, but in many cases these cells display resistance to receptor-mediated apoptosis and cFLIP appears to be involved in this. DED-Mediated Interaction of cFLIP and Calmodulin nigms.nih.gov/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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