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

Elaine Huston, Matthew Beard, Fraser McCallum, Nigel J. Pyne, Peter Vandenabeele, Grant Scotland, Miles D. Houslay

Research output: Contribution to journalArticle

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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.
Original languageEnglish
Pages (from-to)28063-28074
Number of pages12
JournalJournal of Biological Chemistry
Volume275
Issue number36
DOIs
Publication statusPublished - 26 May 2000

Fingerprint

src Homology Domains
Phosphoric Diester Hydrolases
Caspase 3
Apoptosis
Rats
Staurosporine
Phosphodiesterase 4 Inhibitors
Rolipram
Caspase 1
Aptitude
Antibodies
COS Cells
PC12 Cells
Colforsin
Transcription
Fibroblasts
Caspases
Adenylyl Cyclases
Catalyst activity
Protein Isoforms

Keywords

  • 3',5'-cyclic-AMP phosphodiesterases
  • amino acid sequence
  • animals
  • binding sites
  • cos cells
  • caspase 3
  • caspases
  • catalytic domain
  • cell differentiation
  • cell line
  • cyclic nucleotide phosphodiesterases, type 4
  • cysteine proteinase inhibitors
  • kinetics
  • molecular sequence data
  • PC12 cells
  • rats
  • recombinant proteins
  • staurosporine
  • transfection
  • src homology domains

Cite this

Huston, Elaine ; Beard, Matthew ; McCallum, Fraser ; Pyne, Nigel J. ; Vandenabeele, Peter ; Scotland, Grant ; Houslay, Miles D. / The cAMP-specific phosphodiesterase PDE4A5 is cleaved downstream of its SH3 interaction domain by caspase-3 : consequences for altered intracellular distribution. In: Journal of Biological Chemistry. 2000 ; Vol. 275, No. 36. pp. 28063-28074.
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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.",
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The cAMP-specific phosphodiesterase PDE4A5 is cleaved downstream of its SH3 interaction domain by caspase-3 : consequences for altered intracellular distribution. / Huston, Elaine; Beard, Matthew; McCallum, Fraser; Pyne, Nigel J.; Vandenabeele, Peter; Scotland, Grant; Houslay, Miles D.

In: Journal of Biological Chemistry, Vol. 275, No. 36, 26.05.2000, p. 28063-28074.

Research output: Contribution to journalArticle

TY - JOUR

T1 - The cAMP-specific phosphodiesterase PDE4A5 is cleaved downstream of its SH3 interaction domain by caspase-3

T2 - consequences for altered intracellular distribution

AU - Huston, Elaine

AU - Beard, Matthew

AU - McCallum, Fraser

AU - Pyne, Nigel J.

AU - Vandenabeele, Peter

AU - Scotland, Grant

AU - Houslay, Miles D.

PY - 2000/5/26

Y1 - 2000/5/26

N2 - 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.

AB - 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.

KW - 3',5'-cyclic-AMP phosphodiesterases

KW - amino acid sequence

KW - animals

KW - binding sites

KW - cos cells

KW - caspase 3

KW - caspases

KW - catalytic domain

KW - cell differentiation

KW - cell line

KW - cyclic nucleotide phosphodiesterases, type 4

KW - cysteine proteinase inhibitors

KW - kinetics

KW - molecular sequence data

KW - PC12 cells

KW - rats

KW - recombinant proteins

KW - staurosporine

KW - transfection

KW - src homology domains

U2 - 10.1074/jbc.M906144199

DO - 10.1074/jbc.M906144199

M3 - Article

C2 - 10829034

VL - 275

SP - 28063

EP - 28074

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 36

ER -