TI - PHOSphorylation at Tyr33 is essential for WOX1 to counteract Zfra-mediated apoptosis . AB - PHOSphorylation of WOX1 at Tyr33 is essential for its apoptotic function [8] . In parallel with the above observations ( Figure 9 ) , overexpressed WOX1 did not synergistically increase cell death with Zfra in COS7 cells (Figure 10A) . Also , alteration of Tyr33 to Arg (Y33R) abolished the apoptosis-inducing activity of WOX1 (Figure 10A) . This Y33R mutant could not bind Zfra ( Figure 6 ) , and did not affect Zfra -mediated cell death (Figure 10A) . This relationship was further verified in breast MDA-MB-231 cells (Figure 10B) . We tested the function of SDR domain and a C-terminal tail ( SDR/C ) in WOX1 (Figure 10C) . L929 cells were transfected with cDNA constructs for expressing SDR or SDR/C , followed by culturing for 24 hr in the presence or absence of a synthetic full-length Zfra peptide ( 10 muM ) . Transiently overexpressed SDR/C exerted cell death . Zfra peptide alone had no effect but enhanced cell death caused by SDR/C (Figure 10C) . SDR alone could not cause cell death and Zfra and SDR , in combination , did not increase the cell death (Figure 10C) . Together , these observations indicate a critical role of Tyr33 PHOSphorylation in WOX1 in causing cell death and interacting with Zfra . Also , when Zfra binds to the C-terminal SDR/C region , both Zfra and WOX1 may enhance cell death in a synergistic manner . Protein expression of ECFP-SDR/C and ECFP-SDR is shown . The extent of protein expression was in more than 70% of transfected cells , as determined by fluorescence microscopy . Constructs made for the above experiments are shown ( Figure 10D ) .