title: The cAMP-specific phosphodiesterase PDE4A5 is cleaved downstream of its SH3 interaction domain by caspase-3. Consequences for altered intracellular distribution.
abstract: The unique N-terminal region of the cAMP-specific phosphodiesterase PDE4A5, which confers an ability to bind to certain protein SH3 domains, is cleaved during apoptosis in both Rat-1 fibroblasts and PC12 cells. Cleavage was abolished by the caspase-3-selective inhibitor, z-DEVD-CHO but not the caspase-1 selective inhibitor, z-YVAD-CHO. Caspase-3 treatment of PDE4A5, expressed either transiently in COS cells or generated in vitro by coupled transcription translation, generated a similar cleavage product of 100 kDa compared with the native 110-kDa PDE4A5. This product could be detected immunochemically with an antibody raised to a C-terminal PDE4A5 peptide but not an antibody raised to the N terminus of PDE4A5, indicating that caspase-3 caused N-terminal cleavage of PDE4A5. Deletion of the putative caspase-3 cleavage site, (69)DAVD(72), in PDE4A5, or generation of either the D72A or the D69A mutants, ablated the ability of caspase-3 to cause cleavage. The N-terminal truncate PDE4A5-DeltaP3 was engineered to mimic the caspase-cleaved product of PDE4A5. This showed altered catalytic activity and, unlike PDE4A5, was unable to interact with the SH3 domain of the tyrosyl kinase, LYN. Although both PDE4A5 and PDE4A5-DeltaP3 were localized at cell cortical regions (ruffles), the distinct perinuclear association noted for both PDE4A5 and LYN was not seen for PDE4A5-DeltaP3. Staurosporine-induced apoptosis caused a marked redistribution of PDE4A5 but not PDE4A8 in stably transfected Rat-1 cells. The PDE4-selective inhibitor, rolipram together with the adenylyl cyclase activator forskolin, caused a synergistic increase in the apoptosis of Rat-1 cells. Overexpression of PDE4A5 in Rat-1 cells protected against 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 remove the SH3 binding domain that is unique to this isoform within the PDE4A family and to alter its intracellular targeting.
authors.affiliation: Molecular Pharmacology Group, Division of Biochemistry & Molecular Biology, Davidson Building, Institute of Biomedical & Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
authors.forename: E
authors.initials: E
authors.lastname: Huston
authors.forename: M
authors.initials: M
authors.lastname: Beard
authors.forename: F
authors.initials: F
authors.lastname: McCallum
authors.forename: N J
authors.initials: NJ
authors.lastname: Pyne
authors.forename: P
authors.initials: P
authors.lastname: Vandenabeele
authors.forename: G
authors.initials: G
authors.lastname: Scotland
authors.forename: M D
authors.initials: MD
authors.lastname: Houslay
mesh_terms: D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D020134:Catalytic Domain; D002454:Cell Differentiation; D002460:Cell Line; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D016716:PC12 Cells; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D014162:Transfection; D018909:src Homology Domains
chemical_list: D015853:Cysteine Proteinase Inhibitors; D011994:Recombinant Proteins; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D019311:Staurosporine