N-terminal and C-terminal domains of calmodulin mediate FADD and TRADD interaction

FADD (Fas–associated death domain) and TRADD (Tumor Necrosis Factor Receptor 1-associated death domain) proteins are important regulators of cell fate in mammalian cells. They are both involved in death receptors mediated signaling pathways and have been linked to the Toll-like receptor family and innate immunity. Here we identify and characterize by database search analysis, mutagenesis and calmodulin (CaM) pull-down assays a calcium-dependent CaM binding site in the α-helices 1–2 of TRADD death domain. We also show that oxidation of CaM methionines drastically reduces CaM affinity for FADD and TRADD suggesting that oxidation might regulate CaM-FADD and CaM-TRADD interactions. Finally, using Met-to-Leu CaM mutants and binding assays we show that both the N- and C-terminal domains of CaM are important for binding

FADD–calmodulin interaction: A novel player in cell cycle regulation

AbstractAnalyses of knockout and mutant transgenic mice as well as in vitro studies demonstrated a complex role of FADD in the regulation of cell fate. FADD is involved in death receptor induced apoptosis, cell cycle progression and cell proliferation. In a search for mechanisms that might regulate FADD functions, we identified, upon the screening of a lambda-phage cDNA library, calmodulin (CaM) as a novel FADD interacting protein. CaM is a key mediator of signals by the secondary messenger calcium and it is an essential regulator of cell cycle progression and cell survival. Here, we describe the identification and characterization of two calcium dependent CaM binding sites in the alpha helices 8–9 and 10–11 of FADD. Phosphorylation of human FADD at the C-terminal serine 194, by casein kinase I alpha (CKIα), has been shown to regulate FADD-dependent non-apoptotic activities. Remarkably, we showed that both FADD and CaM are CKIα substrates and that in synchronized HeLa cells, FADD, CaM and CKIα co-localize at the mitotic spindle in metaphase and anaphase. Moreover, complementation experiments in Jurkat FADD−/− T cells indicated that: a) cells expressing FADD mutants in the CaM binding sites are protected from Taxol-induced G2/M cell cycle arrest; b) FADD/CaM interaction is not required for Fas receptor-mediated apoptosis although Fas and CaM might compete for binding to FADD. We suggest that the interplay of FADD, CaM and CKIα may have an important role in the regulation of cell fate

Methiones oxidation impairs CaM interaction with FADD and TRADD.

<p><b>A</b>: CaM oxidation analysis. Coomassie blue stained 15% SDS-PAGE loaded with the indicated <i>Xenopus laevis</i> His-CaM proteins. <b>B</b>. Schematic representation of GST-FADD and GST-TRADD proteins used in pull-down assays. The DED, DD and the S194 phosphorylation site of human FADD are indicated. <b>C</b>: GST pull-down assays. The indicated GST proteins bound to gluathione-sepharose beads were incubated with recombinant His-CaM proteins, as indicated; 50% of the eluted proteins were subjected to SDS-PAGE (12%) and western blot analysis. I indicates the input of the CaM proteins used in binding assays. The data shown are representative of at least three independent experiments. </p

Binding assays of Met-to-Leu CaM mutants.

<p>Binding assays of Met-to-Leu CaM mutants.</p

Met-to-Leu CaM mutants.

<p>Methionine residues located in the N-teminal (Met 36, 51, 71, 72), C-terminal (Met 109, 124, 144, 145) and linker region (Met 76) of CaM are listed. Mutant description reflects the relative position of Met-to-Leu substitutions as schematically indicated; 9L all nine methionine mutated, 8L 109M and 8L 124M eight methionine mutated with the exception respectively of residues 109M or 124M.</p><p>Met-to-Leu CaM mutants.</p

Characterization of a CaM binding site in TRADD.DD.

<p><b>A</b>: schematic representation of the GST-TRADD mutants. The N-terminal domain (N) and the Death Domain (DD) of human TRADD are indicated. <b>B</b> and <b>C</b>: western blot with GST specific antibody of GST-TRADD mutants (top panels, I stands for inputs) and CaM pull-down assays (bottom panels, E stands for eluates). The GST fusion proteins indicated were pull-down with CaM-sepharose beads in binding buffer with 2 mM Ca²⁺ (<b>B, C</b>) or EGTA (<b>B</b>). <b>D</b>: CaM blot overlay assay. <i>E coli</i> BL21 lysates expressing N-TRADD (lane 1) or untransformed (Ctrl) (lane 2) were used as negative controls. Top panel a) shows the ponceau stained filter and bottom panel b) the western blot probed with biotin-conjugated His-CaM. <b>E</b>: GST-TRADD pull-down assays. The GST-TRADD proteins indicated, bound to glutathione-sepharose beads, were incubated with His-CaM in binding buffer with 2 mM Ca²⁺ or EGTA. Top panel a) shows the ponceau stained filter and bottom panel b) the western blot probed with CaM specific antibody. I indicates the input of His-CaM. <b>F</b>: GST-TRADD pull-down assays. Bound proteins were analyzed by 12% SDS-PAGE and autoradiography. Panel a) shows the coomassie stained gel where I indicates the input of ³⁵S-FADD. Panel b) shows the corresponding autoradiogram. <b>G</b>: Immunoprecipitation (IP) assay. Total lysates of Hek 293T cells expressing HA-FADD and Flag-TRADD proteins were probed with Flag or HA monoclonal antibodies. Cell lysates were IP with Flag-resin, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116251#sec002" target="_blank">material and methods</a>. The data shown are representative of at least three independent experiments.</p

Ca²⁺-dependent binding of CaM to TRADD.

<p><b>A</b>: CaM target database analysis. Amino acid sequences of the predicted CaM binding sites in human TRADD are shown along with the corresponding probability scores. <b>B</b>: autoradiography of CaM pull-down assay. (I) shows input of ³⁵S-TRADD (∼ 33 kDa) incubated with CaM sepharose (CaM) or control sepharose (Ctrl) beads in Ca²⁺ or EGTA binding buffer. <b>C</b>: autoradiography of pull-down assay of ³⁵S-CaM with GST or GST-TRADD. Panel a) shows a 12% SDS-PAGE stained with coomassie and panel b) the corresponding autoradiogram for ³⁵S-CaM. <b>D</b>: western blot of His-CaM pull-down assays. His-CaM or His-Ctrl (control, cyclophilin) bound to Ni-NTA agarose beads were incubated with cell lysates, as indicated. TL indicates total cell lysates. Positions of the molecular weight standards are indicated. The data shown are representative of at least three independent experiments.</p

Effect of Met-to-Leu substitutions in CaM on FADD and TRADD binding.

<p><b>A</b>: CaM mutants and SDS-PAGE analysis. Coomassie stained gel (a) and western blot analysis (b) of CaM mutants, identified by a numerical code detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116251#pone.0116251.t001" target="_blank">Table 1</a>. <b>B</b>: GST pull-down assays. Native (N) or oxidized (Ox) CaM mutants were incubated with the indicated GST proteins bound to glutathione-sepharose beads. 50% of the eluted proteins were analyzed by 12% SDS-PAGE and blotted to nitrocellulose stained with ponceau (a) and processed for western blot (b). I indicates the input of recombinant CaM proteins (250 ng). CaM mutants are identified by a numerical code as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116251#pone.0116251.t001" target="_blank">Table 1</a>. <b>A</b> and <b>B</b>: the black vertical lines in all panels indicate that non-adjacent lanes from the same gel or blots are shown. The data shown are representative of three independent experiments.</p