Figure 10

Phosphorylation of WOX1 at Tyr33 is essential for its apoptotic function and for counteracting Zfra-mediated cell death. (A) Transiently overexpressed EGFP-Zfra and EGFP-WOX1 induced death of COS7 fibroblasts (n = 8, mean ± standard deviation). Zfra counteracted with WOX1 in causing cell death. Alteration of Tyr33 to Arg33 (Y33R) abolished the apoptosis-inducing activity of WOX1, and this mutant did not block Zfra-mediated cell death. Expression of EGFP-tagged Zfra, WOX1 and WOX1(Y33R) is shown in fluorescent micrographs (400× magnification). WOX1 is shown in the perinuclear area, whereas the Y33R mutant tends to aggregate in the nuclei. Zfra is expressed ubiquitously in cells. (B) Similar experiments were carried out in breast MDA-MB-231 cells. Again, Zfra and WOX1 did not increase cell death in a synergistic manner (n = 8, mean ± standard deviation). The Y33R mutant lost its apoptotic function and had no apparent effect on Zfra-mediated cell death. (C) L929 cells were transfected with the SDR domain plus a C-terminal tail (SDR/C) or the SDR domain alone (tagged with ECFP at the N-terminus). These cells were cultured for 24 hr in the presence or absence of a synthetic full-length Zfra peptide (10 μM), and the extent of cell death was determined by crystal violet staining. Zfra enhanced cell death caused by SDR/C (n = 8, mean ± standard deviation). SDR alone could not cause cell death, and Zfra did not increase the cell death. Protein expression of ECFP-SDR/C and ECFP-SDR is shown. (D) Constructs made for the above experiments are shown.