Caspases are associated with apoptosis but have been reported to play key roles in protein processing under non-apoptotic conditions. Upon phosphorylation, STAT (signal transducer and activator of transcription) proteins regulate transcription and affect multiple cellular processes. We reported that Stat3 expression was reduced following caspase activation and biochemical analysis, conducted under cell-free conditions, revealed that caspases directly cleave this protein. We now report studies to (i) map the location of caspase cleavage sites in Stat3, and (ii) evaluate the impact of Stat3 proteolysis on transactivation. In whole cells, staurosporin (STS, 12h, 1uM) exposure resulted in Stat3 cleavage and the generation of several fragments detected with C- or N-terminal Stat3 antibodies with apparent molecular weights >40kD. Both Stat3 cleavage and fragment formation were blocked by the caspase inhibitor zVAD-FMK (40uM). Next, a series of c-Myc tagged Stat3 truncation mutants (corresponding to amino acids 1-130, 1-320, 1-688, 588-770 and 1-770) were expressed in HEK293T cells. Transfected cells then were exposed to 1uM STS (12h) and fragment cleavage patterns analyzed by Western-blot. This revealed that, with the exception of the small N-terminal (1-130) mutant, all Stat3 fragments were cleaved following STS exposure, a finding consistent with the identification of multiple caspase recognition sites in the sequence of Stat3. These findings were supported by cell-free experiments where exposure of purified full-length Stat3 to a mixture of caspases 1-10 (37ox90 mins) resulted in Stat3 disappearance that also corresponded with the formation of a fragment in the 50kD range. Experiments to sequence and identify fragment(s) generated under cell-free conditions are planned. Analysis of the functional consequence of Stat3 proteolysis employed Stat3 driven luciferase reporter assays. Initial studies examined the effect of STS (+/-zVAD) on reporter activity and revealed that STS significantly reduced luciferase activity, an effect partially blocked by zVAD and correlated with STS-induced reductions in pY705-Stat3 expression. Parallel experiments assessed the impact of Stat3 fragment expression on reporter activity in HEK293T cells under non-apoptotic conditions and revealed that the expression of all Stat3 fragments examined, except one corresponding to amino acids 1-688, slightly increased (<150%) reporter activity. In contrast, expressing the 1-688 fragment increased reporter activity ∼5-fold. These findings reveal that Stat3 contains multiple caspase cleavage sites and that caspase-mediated Stat3 proteolysis can modulate Stat3 signaling under apoptotic and non-apoptotic conditions (supported by CA-102128 and Rhode Island Hospital).

[Proc Amer Assoc Cancer Res, Volume 47, 2006]