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. 2012 Aug 21;31(20):3961–3975. doi: 10.1038/emboj.2012.236

Figure 1.

Figure 1

Identification of a phosphorylation-sensitive protein complex consisting of AATF and MRLC3. (A) An oriented (pSer/pThr) phosphopeptide library, biased towards the basophilic phosphorylation motif of Chk1/2 and MK2, was immobilized on streptavidin beads. The phospho ϕXRXXpT and non-phosphorylated ϕXRXXT peptide libraries were screened for interaction against in vitro translated, 35S-Met-labelled proteins. (B) Identification of MRLC3 as a non-phospho binder occurred in pool 16B11 and through progressive subdivision to a single clone. (C) Yeast two-hybrid screening revealed AATF as an interactor of MRLC3. We further characterized this interaction through co-immunoprecipitation (co-IP), performed in the presence or absence of 1 μM okadaic acid (OA). FLAG.MRLC3 was immunoprecipitated from HEK293T cells co-expressing V5.AATF. FLAG.GFP served as a control. Lane 3 shows an interaction of FLAG.MRLC3 with V5.AATF, which was abolished by OA-mediated Ser/Thr phosphatase inhibition 1 h prior to lysis (lane 4). (D) The MRLC3:AATF complex is sensitive to UV-C-induced DNA damage. FLAG.MRLC3 and V5.AATF-expressing HEK293T cells were UV-C irradiated (20 J/m2) 30 min prior to lysis and IP with anti-FLAG beads. FLAG.GFP served as a negative control. While V5.AATF co-precipitated with FLAG.MRLC3 in the absence of UV-C, the interaction was abrogated in the presence of DNA damage. (E) Reversal of the co-IP experiment is shown in (D). Anti-FLAG IP reveals AATF.FLAG:V5.MRLC3 complexes that display strong sensitivity to UV-C-induced DNA damage. FLAG.GFP served as a negative control. (F) Endogenous AATF:MRLC complexes display UV-C sensitivity. AATF was immunoprecipitated from HCT116 cells that were mock-treated or exposed to UV-C (20 J/m2) 30 min prior to lysis and IP. GFP IP served as a negative control (lanes 1 and 2). While substantial amounts of MRLC co-immunoprecipitated with AATF (lane 3), this interaction was abolished by UV-C-induced DNA damage (lane 4). (G) Endogenous AATF:MRLC3 complexes are sensitive to the topoisomerase-II inhibitor doxorubicin. AATF was immunoprecipitated from HCT116 cells that were mock-treated or exposed to doxorubicin (1 μM) 1 h prior to IP. GFP antibody served as a negative control (lanes 1 and 2). Doxorubicin (lane 4) disrupted the interaction between AATF and MRLC (lane 3). Figure source data can be found with the Supplementary data.