The cAMP-specific phosphodiesterase PDE4A5 is cleaved downstream of its SH3 interaction domain by caspase-3 - Consequences for altered intracellular distribution

The unique N-terminal region of the cAMP-specific phosphodiesterase PDE4A5, which confers an ability to bind to certain protein SH3 domains, is cleave d during apoptosis in both Rat-1 fibroblasts and PC12 cells. Cleavage was a bolished by the caspase-3-selective inhibitor, z-DEVD-CHO but not the caspa se-1 selective inhibitor, z-YVAD-CHO, Caspase-3 treatment of PDE4A5, expres sed either transiently in COS cells or generated in vitro by coupled transc ription translation, generated a similar cleavage product of 100 kDa compar ed with the native 110-kDa PDE4A5. This product could be detected immunoche mically with an antibody raised to a C-terminal PDE4A5 peptide but not an a ntibody raised to the N terminus of PDE4A5, indicating that caspase-3 cause d N-terminal cleavage of PDE4A5. Deletion of the putative caspase-3 cleavag e site, (69)DAVD(72), in PDE4A5, or generation of either the D72A or the D6 9A mutants, ablated the ability of caspase-3 to cause cleavage. The N-termi nal truncate PDE4A5-Delta P3 was engineered to mimic the caspase-cleaved pr oduct of PDE4A5. This showed altered catalytic activity and, unlike PDE4A5, was unable to interact with the SH3 domain of the tyrosyl kinase, LYN, Alt hough both PDE4A5 and PDE4A5-Delta P3 mere localized at cell cortical regio ns (ruffles), the distinct perinuclear association noted for both PDE4A5 an d LYN mas not seen for PDE4A5-Delta P3. Staurosporine-induced apoptosis cau sed a marked redistribution of PDE4A5 but not PDE4A8 in stably transfected Rat-1 cells. The PDE4-selective inhibitor, rolipram together with the adeny lyl cyclase activator forskolin, caused a synergistic increase in the apopt osis of Rat-1 cells. Overexpression of PDE4A5 in Rat-1 cells protected agai nst staurosporine-induced apoptosis in contrast to overexpression of PDE4A8 , which potentiated apoptosis, PDE4A5 may be the sole PDE4 family member to provide a substrate for caspase-3 cleavage and this action serves to remov e the SH3 binding domain that is unique to this isoform within the PDE4A fa mily and to alter its intracellular targeting.