Frame, Mhairi J and Tate, Rothwelle and Adams, David R and Morgan, Keith M and Houslay, M D and Vandenabeele, Peter and Pyne, Nigel J (2003) Interaction of caspase-3 with the cyclic GMP binding cyclic GMP specific phosphodiesterase (PDE5a1). European Journal of Biochemistry, 270 (5). pp. 962-970. ISSN 0014-2956Full text not available in this repository. (Request a copy from the Strathclyde author)
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 DQGD 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 DQGD motif would result in removal of the C-terminal tail containing Q807 and F810, which are potentially important amino acids required for substrate binding.
|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, Pharmacy and materia medica, Biochemistry|
|Subjects:||Medicine > Pharmacy and materia medica|
|Department:||Faculty of Science > Strathclyde Institute of Pharmacy and Biomedical Sciences|
|Depositing user:||Pure Administrator|
|Date Deposited:||15 Nov 2011 14:53|
|Last modified:||04 May 2016 20:24|