Deformed Epidermal Autoregulatory Factor-1 (DEAF1) Interacts with the Ku70 Subunit of the DNA-Dependent Protein Kinase Complex

<div><p>Deformed Epidermal Autoregulatory Factor 1 (DEAF1) is a transcription factor linked to suicide, cancer, autoimmune disorders and neural tube defects. To better understand the role of DEAF1 in protein interaction networks, a GST-DEAF1 fusion protein was used to isolate interacting proteins in mammalian cell lysates, and the XRCC6 (Ku70) and the XRCC5 (Ku80) subunits of DNA dependent protein kinase (DNA-PK) complex were identified by mass spectrometry, and the DNA-PK catalytic subunit was identified by immunoblotting. Interaction of DEAF1 with Ku70 and Ku80 was confirmed to occur within cells by co-immunoprecipitation of epitope-tagged proteins, and was mediated through interaction with the Ku70 subunit. Using <em>in vitro</em> GST-pulldowns, interaction between DEAF1 and the Ku70 subunit was mapped to the DEAF1 DNA binding domain and the C-terminal Bax-binding region of Ku70. In transfected cells, DEAF1 and Ku70 colocalized to the nucleus, but Ku70 could not relocalize a mutant cytoplasmic form of DEAF1 to the nucleus. Using an <em>in vitro</em> kinase assay, DEAF1 was phosphorylated by DNA-PK in a DNA-independent manner. Electrophoretic mobility shift assays showed that DEAF1 or Ku70/Ku80 did not interfere with the DNA binding of each other, but DNA containing DEAF1 binding sites inhibited the DEAF1-Ku70 interaction. The data demonstrates that DEAF1 can interact with the DNA-PK complex through interactions of its DNA binding domain with the carboxy-terminal region of Ku70 that contains the Bax binding domain, and that DEAF1 is a potential substrate for DNA-PK.</p> </div

DEAF1 interacts with the DNA-PK complex.

<p>Recombinant GST and GST-DEAF1 fusion proteins were isolated from bacterial extracts, bound to glutathione-Sepharose beads, and incubated overnight in the presence (+) or absence (−, buffer only) of PC-3 cell lysates. Interacting proteins were eluted, separated by SDS-PAGE, and stained with Coomassie blue (<b>A</b>) or analyzed by Western blot with an antibody to DNA-PKcs (<b>B</b>). The 70 kDa and 80 kDa proteins (arrows) were determined by mass spectrometry to be Ku70 and Ku80. (<b>C</b>) CV1 cells were transfected with expression plasmids for DEAF1-FLAG and/or HA-tagged Ku proteins. Cell lysates were immunoprecipitated (IP) with anti-FLAG coupled beads followed by Western blot analysis with anti-HA antibody. (<b>D</b>) Reversing the epitopes described in (C), lysates of cells transfected with DEAF1 and FLAG-tagged or HA-tagged Ku proteins were immunoprecipitated with anti-FLAG coupled beads followed by Western blot analysis with anti-DEAF1 antibody. Levels of the proteins in 1.0% of the cell lysates (inputs) used in the immunoprecipitation reactions in C and D were assessed using the antibodies indicated. The results are representative of two independent experiments.</p

Ku70 is unable to relocalize a cytoplasmic form of DEAF1 to the nucleus.

<p>CV-1 cells were transfected either alone or in combinations with DEAF1-HA, HA-Ku70, GFP-DEAF1, or GFP-DEAF1nls (mutated nuclear localization signal). Cellular localization of HA-tagged proteins were determined by indirect immunofluorescence using anti-HA antibodies and secondary antibodies conjugated to CY3 and GFP-tagged proteins were determined by intrinsic GFP fluorescence.</p

DEAF1 interacts with Ku70.

<p>GST pull-downs assays were performed by incubation of <i>in vitro</i> translated, [³⁵S]methionine-labeled Ku70, Ku80, or DEAF1 with the indicated GST fusion proteins and GST. 10% of the <i>in vitro</i> translated proteins used in the pull-downs are shown on the left of each panel. The results are representative of two independent experiments.</p

The DEAF1 DNA binding domain interacts with Ku70.

<p>(<b>A</b>) DEAF1 proteins with C-terminal and/or N-terminal deletions were radiolabeled with [³⁵S]methionine by <i>in vitro</i> translation and used in GST pull-downs. 10% of the [³⁵S]-labeled proteins (10% input) used in the pull-downs are shown to the left of the pull-down results obtained with GST-Ku70 and GST. Schematic representations of all the DEAF1 translated proteins tested are shown in the left panel. Results are summarized as a positive interaction (+) or no interaction (−). Ku80 interaction with GST-Ku70 was used as a positive control. (<b>B</b>) A schematic summary of the DEAF1 and Ku70 proteins and the interaction domains deduced from these experiments. Also shown are the Bax interaction domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033404#pone.0033404-Cohen1" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033404#pone.0033404-Subramanian1" target="_blank">[24]</a>, the MYND (zinc binding) domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033404#pone.0033404-Lutterbach1" target="_blank">[25]</a>, and DBD (DNA binding domain).</p

DNA-PK phosphorylates DEAF1, and sequence specific DNA competes with Ku70 for binding to DEAF1.

<p>(<b>A</b>) DNA-PK and [γ-³²P]ATP were incubated with GST or GST-DEAF1 bound to glutathione-sepharose beads in the absence (−) or presence (+) of dsDNA (salmon sperm DNA sheared to ∼300 bp). Bound proteins were washed, eluted, and separated by SDS-PAGE; and the phosphorylated proteins were detected by autoradiography. Protein levels used in the reactions were assessed in separate SDS-PAGE gels stained with Coomassie blue. (<b>B</b>) Electrophoretic mobility shift assays were performed using the indicated FLAG proteins purified from HEK 293T/17 cells and either ³²P-labeled dsDNA probes N52-69 (DNA ligand of DEAF1) or Mut 2 (mutated DNA ligand that DEAF1 does not bind). The purities of the proteins used in the assays are shown in Coomassie blue stained SDS-PAGE gels on the left. (<b>C and D</b>) GST pull-down experiments using GST-Ku70 (550–609) or GST with <i>in vitro</i> translated, [³⁵S]methionine-labeled DEAF1 (167–565) were performed in the absence (−) or presence (+) of 15 µg of DEAF1 promoter DNA (<i>C</i>), or increasing amounts of N52-69 DNA (0.04 mg–0.6 mg) or 0.6 mg of Mut 2 DNA (<i>D</i>). 10% of the <i>in vitro</i> translated [³⁵S]methionine-labeled proteins used in each pull-down experiment are shown on the left. The results are representative of two independent experiments.</p

DEAF1 interacts with the C-terminal end of Ku70.

<p>(<b>A and B</b>) GST-Ku70 fusion proteins with N-terminal and/or C-terminal deletions of Ku70 were used in pull-downs with <i>in vitro</i> translated [³⁵S]methionine-labeled DEAF1 (167–565) shown in (A) or DEAF1 (155–326) shown in (B). The results are representative of two independent experiments. (<b>C</b>) GST tags were reversed relative to (A) and (B) and GST-DEAF1 was used to pull-down <i>in vitro</i> translated Ku70 peptides (right panel). A schematic representation of all the Ku70 translated proteins tested is shown in the left panel. Results are summarized as a positive interaction (+) or no interaction (−).</p

GABA A receptor subunit Γ2 and Δ subtypes confer unique kinetic properties on recombinant GABA A receptor currents in mouse fibroblasts

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65434/1/j.1469-7793.1999.027af.x.pd