Figure 3
Cleavage of p65wt-tag protein in Jurkat cells after PMA activation. (a) The RHR consists of two immunoglobulin-like (Ig-like) domains (19–325 amino acid (aa)) connected by a short linker of 5–9 aa. Both domains contact DNA, but only the carboxy-terminal Ig-like domain (191–290 aa) is responsible for the intersubunit dimer formation. The nuclear localization signal (NLS) is located in the carboxy-terminal end (325 aa) of the dimerization domain. The carboxy-terminus of the polypeptide (325–551 aa) contains two transactivation domains, TA1 and TA2 (415–551 aa). The presence of several putative caspase cleavage sites has been indicated with discontinuous arrows for caspase-3-like proteases motifs and with continuous arrows for caspase-6-like proteases motifs. Putative recognition sites for caspase-6 in 91VGKD94 and caspase-3 in 94DCRD97 are indicated. (b) Jurkat cells transiently transfected with pCMV-p65wt-tag expression vector were treated immediately after transfection with PMA and/or the general caspase inhibitor z-VAD-fmk or the caspase inhibitor Ac-DMQD-CHO to inhibit caspase-3 and/or caspase-6. Protein extracts were analyzed 18 hours after transfection by immunoblotting using an antibody against the carboxy-terminus of p65/RelA. (c) Caspase-3 activity was measured in Jurkat cells after treatment with PMA for 18 hours and in the presence of the inhibitors of caspases z-VAD-fmk (100 μM) and Ac-DMQD-CHO (100 μM). Data correspond to the mean of three different experiments and lines on the top of the bars represent the standard deviation. (d) Jurkat cells were transiently transfected with either pCMV-p65wt-tag expression vector (lanes 1 and 2) or each substitution mutant resistant to cleavage by caspase-3 and/or -6 (double amino acid-substitution mutants p65 D94E;D97E-tag (lanes 3 and 4) and p65 V91L;D94E-tag (lanes 9 and 10) were resistant to cleavage by both caspase-3 and caspase-6; single amino acid-substitution mutants p65 D97E-tag (lanes 5 and 6) and p65 V91L-tag (lanes 7 and 8) were resistant to cleavage by caspase-3 and caspase-6, respectively). PMA was added immediately after transfection. After 18 hours of incubation, analysis of protein extracts was performed by immunoblotting using an antibody against the carboxy-terminus of p65/RelA.