Fig. 3.

The interaction of PGAM5 with BCL-xL is critical for dephosphorylation of BCL-xL. a Photocrosslinking reaction of [³⁵S]Met-labeled BCL-xL proteins, each with the indicated single Lys in the indicated domain replaced by an εANB-Lys, to His₆-tagged PGAM5 protein lacking the N-terminal 90 residues (6H-PGAM5ΔN90). The control reactions omitted the ANB probe, the light (hυ) that activates the ANB probe, or the PGAM5 protein. Cross-linked products were analyzed by SDS-PAGE, alongside with another control reaction containing a Lys-null BCL-xL protein (K0). Radioactive proteins on the gel were detected by phosphor-imaging. The circle indicates the [³⁵S]Met-labeled BCL-xL monomer and arrowheads indicate the BCL-xL/6H-PGAM5ΔN90 photoadducts. The molecular weights (MW) of radioactive protein standards are indicated in kDa on the left side. b The BCL-xL structure, generated from PDB entry 1LXL [39] using the PyMOL program. The NH₂ and COOH termini are labeled. The BH1, 2, 3 and 4 domains are colored in orange, cyan, magenta and green, respectively. The residues that were replaced by εANB-Lys in the photocrosslinking experiment are shown in stick form. The residue S62, which is located in the disordered loop and can be dephosphorylated by PGAM5, is shown in sphere form. c PGAM5/BCL-xL double knockout HeLa cell lines were co-transfected with PGAM5-MYC and FLAG-BCL-xL (WT) or the arginine-6 to aspartic acid (R6D), glycine-138 to alanine (G138A), arginine-139 to aspartic acid (R139D), aspartic acid-189 to asparagine (D189N), or glutamic acid-193 to glutamine (E193Q) mutant as indicated for 24 h. The cells were lysed and immunoprecipitated with anti-FLAG antibody. Co-immunoprecipitated PGAM5-MYC and endogenous BAX and BAK were detected by immunoblotting. d BCL-xL knockout HeLa cells were transfected with FLAG-BCL-xL or the indicated mutants for 24 h, treated with 500 nM vinblastine for 24 h, lysed and analyzed by immunoblotting with the indicated antibodies. e The ratio of p-Ser62 to total WT or mutant FLAG-BCL-xL were calculated from the respective band intensities in the blots in (d). Data are the mean ± SEM of three experiments. *p < 0.05. f HeLa or BCL-xL knockout HeLa cells expressing FLAG-BCL-xL (WT, R6D or R139D) were treated with vinblastine for 48 h, lysed and analyzed by immunoblotting with the indicated antibodies