Interaction of caspase-3 with the cyclic GMP binding cyclic GMP specific phosphodiesterase (PDE5a1)

Mhairi J Frame, Rothwelle Tate, David R Adams, Keith M Morgan, M D Houslay, Peter Vandenabeele, Nigel J Pyne

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Here, we show that recombinant bovine PDE5A1 is proteolysed by recombinant caspase-3 in in vitro and transfected Cos-7 cells. In addition, the treatment of PDE5A1-transfected Cos-7 and PC12 cells with staurosporine, an apoptotic agent that activates endogenous caspase-3, also induced proteolysis and inactivation of PDE5A1. These findings suggest that there is specificity in the interaction between caspase-3 and PDE5A1 that requires application of an apoptotic stimulus. The potential proteolysis of the [778]DQGD[781] site in PDE5A1 by caspase-3 might affect cGMP's hydrolyzing activity as this is within the boundary of the active site. We therefore created a truncated D781 mutant corresponding exactly to the potential cleavage product. This mutant was expressed equally well compared with the wild-type enzyme in transfected Cos-7 cells and was inactive. Inactivity of the truncated mutant was not due to potential misfolding of the enzyme as it eluted from gel filtration chromatography in the same fraction as the wild-type enzyme. Homology model comparison with the catalytic domain of PDE4B2 was used to probe a functional role for the region in PDE5A1 that might be cleaved by caspase-3. From this, we can predict that a caspase-3-mediated cleavage of the [778]DQGD[781] motif would result in removal of the C-terminal tail containing Q807 and F810, which are potentially important amino acids required for substrate binding.
LanguageEnglish
Pages962-970
Number of pages9
JournalEuropean Journal of Biochemistry
Volume270
Issue number5
DOIs
Publication statusPublished - Mar 2003

Fingerprint

Cyclic GMP
Phosphoric Diester Hydrolases
Caspase 3
Proteolysis
Catalytic Domain
Enzymes
Staurosporine
PC12 Cells
Chromatography
Gel Chromatography
Gels
Amino Acids
Substrates

Keywords

  • 3',5'-Cyclic-GMP Phosphodiesterases
  • animals
  • apoptosis
  • blotting, western
  • cos cells
  • caspase 3
  • caspases
  • cattle
  • cyclic nucleotide phosphodiesterases, type 5
  • DNA, complementary
  • enzyme inhibitors
  • hydrolysis
  • models, molecular
  • PC12 cells
  • protein binding
  • rats
  • staurosporine

Cite this

Frame, Mhairi J ; Tate, Rothwelle ; Adams, David R ; Morgan, Keith M ; Houslay, M D ; Vandenabeele, Peter ; Pyne, Nigel J. / Interaction of caspase-3 with the cyclic GMP binding cyclic GMP specific phosphodiesterase (PDE5a1). In: European Journal of Biochemistry . 2003 ; Vol. 270, No. 5. pp. 962-970.
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Interaction of caspase-3 with the cyclic GMP binding cyclic GMP specific phosphodiesterase (PDE5a1). / Frame, Mhairi J; Tate, Rothwelle; Adams, David R; Morgan, Keith M; Houslay, M D; Vandenabeele, Peter; Pyne, Nigel J.

In: European Journal of Biochemistry , Vol. 270, No. 5, 03.2003, p. 962-970.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Interaction of caspase-3 with the cyclic GMP binding cyclic GMP specific phosphodiesterase (PDE5a1)

AU - Frame, Mhairi J

AU - Tate, Rothwelle

AU - Adams, David R

AU - Morgan, Keith M

AU - Houslay, M D

AU - Vandenabeele, Peter

AU - Pyne, Nigel J

PY - 2003/3

Y1 - 2003/3

N2 - Here, we show that recombinant bovine PDE5A1 is proteolysed by recombinant caspase-3 in in vitro and transfected Cos-7 cells. In addition, the treatment of PDE5A1-transfected Cos-7 and PC12 cells with staurosporine, an apoptotic agent that activates endogenous caspase-3, also induced proteolysis and inactivation of PDE5A1. These findings suggest that there is specificity in the interaction between caspase-3 and PDE5A1 that requires application of an apoptotic stimulus. The potential proteolysis of the [778]DQGD[781] site in PDE5A1 by caspase-3 might affect cGMP's hydrolyzing activity as this is within the boundary of the active site. We therefore created a truncated D781 mutant corresponding exactly to the potential cleavage product. This mutant was expressed equally well compared with the wild-type enzyme in transfected Cos-7 cells and was inactive. Inactivity of the truncated mutant was not due to potential misfolding of the enzyme as it eluted from gel filtration chromatography in the same fraction as the wild-type enzyme. Homology model comparison with the catalytic domain of PDE4B2 was used to probe a functional role for the region in PDE5A1 that might be cleaved by caspase-3. From this, we can predict that a caspase-3-mediated cleavage of the [778]DQGD[781] motif would result in removal of the C-terminal tail containing Q807 and F810, which are potentially important amino acids required for substrate binding.

AB - Here, we show that recombinant bovine PDE5A1 is proteolysed by recombinant caspase-3 in in vitro and transfected Cos-7 cells. In addition, the treatment of PDE5A1-transfected Cos-7 and PC12 cells with staurosporine, an apoptotic agent that activates endogenous caspase-3, also induced proteolysis and inactivation of PDE5A1. These findings suggest that there is specificity in the interaction between caspase-3 and PDE5A1 that requires application of an apoptotic stimulus. The potential proteolysis of the [778]DQGD[781] site in PDE5A1 by caspase-3 might affect cGMP's hydrolyzing activity as this is within the boundary of the active site. We therefore created a truncated D781 mutant corresponding exactly to the potential cleavage product. This mutant was expressed equally well compared with the wild-type enzyme in transfected Cos-7 cells and was inactive. Inactivity of the truncated mutant was not due to potential misfolding of the enzyme as it eluted from gel filtration chromatography in the same fraction as the wild-type enzyme. Homology model comparison with the catalytic domain of PDE4B2 was used to probe a functional role for the region in PDE5A1 that might be cleaved by caspase-3. From this, we can predict that a caspase-3-mediated cleavage of the [778]DQGD[781] motif would result in removal of the C-terminal tail containing Q807 and F810, which are potentially important amino acids required for substrate binding.

KW - 3',5'-Cyclic-GMP Phosphodiesterases

KW - animals

KW - apoptosis

KW - blotting, western

KW - cos cells

KW - caspase 3

KW - caspases

KW - cattle

KW - cyclic nucleotide phosphodiesterases, type 5

KW - DNA, complementary

KW - enzyme inhibitors

KW - hydrolysis

KW - models, molecular

KW - PC12 cells

KW - protein binding

KW - rats

KW - staurosporine

U2 - 10.1046/j.1432-1033.2003.03464.x

DO - 10.1046/j.1432-1033.2003.03464.x

M3 - Article

VL - 270

SP - 962

EP - 970

JO - European Journal of Biochemistry

T2 - European Journal of Biochemistry

JF - European Journal of Biochemistry

SN - 0014-2956

IS - 5

ER -