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

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.Keywords: apoptosis; caspases; cyclic GMP; phosphodiesterase; proteases.Members of the phosphodiesterase (PDE) family catalyze the hydrolysis of cyclic nucleotides to inactive 5¢ nucleotides. Therefore, they terminate the action of agents, such as b-adrenergic agonists and nitric oxide, which use cAMP and cGMP as Ôsecond-messengersÕ, respectively, to initiate cellular responses.There are at least 11 members of the PDE family (PDE1-11) that are encoded by different genes. These isoforms have different specificities for cAMP and cGMP, are regulated by several different protein kinases, e.g. protein kinase A, protein kinase B (Akt pro-oncogene), extracellular signalregulated kinase (ERK) and CAM kinase, and allosteric molecules (e.g. cyclic nucleotides, Ca 2+ ) and display distinct tissue distribution [1-3]. PDE5A1 is a major cGMP-binding protein expressed in lung [4] where it is believed to have a key role in regulating nitric oxide signaling. There are at least two isoforms (termed PDE5A1 and 2) [4]. The enzyme has a highaffinity for cGMP at both noncatalytic (GAF domains) and catalytic sites, is a dimeric protein with a subunit molecular mass of 93-98 kDa [5]. The enzyme is phosphorylated at S92 and activated by both protein kinase A and protein kinase G [6][7]. Here we explore the possibility that PDE5A1 may be regulated by caspase-3 as sequence inspection shows that the bovine enzyme contains five putative caspase consensus sites: DHWD(26-29), DEGD(134-137), DEKD(289-292), DCSD(365-368) and DQGD(778-781) (Fig. 1). Of these sites, only two show strong consensus for caspase-3: DHWD(26-29) and DQGD(77...

SOD and inhaled nitric oxide normalize phosphodiesterase 5 expression and activity in neonatal lambs with persistent pulmonary hypertension

“…There are five consensus caspase-3 sites in the PDE5 enzyme including one at the cGMP-hydrolytic site. Others have previously demonstrated that caspase-3 can cause decreased PDE5 activity by proteolytic degradation of PDE5 (16,17). Consistent with this hypothesis, we found a significant increase in caspase-3 activity in lung tissue from 1DSB lambs relative to fetal controls and a nonsignificant trend toward increased caspase-3 activity in PPHN fetal lambs relative to fetal controls (Fig.…”

“…Since PDE5 mRNA is unchanged in both groups, it seems likely that the decrease in PDE5 protein is a result of increased proteolytic degradation. Caspase-3 is the only enzyme that has previously been described to regulate PDE5 degradation (16,17). Consistent with this hypothesis, we observed increased caspase-3 activity in the 1DSB lambs and a trend toward increased caspase-3 activity in PPHN fetuses relative to the control fetuses (Fig.…”

Testosterone/Estradiol Ratio Regulates NO-Induced Bladder Relaxation and Responsiveness to PDE5 Inhibitors

“…However, PDE5 gene and protein expression in bladder, along with its immonodistribution, is neither gender specific nor androgen dependent. The apparent paradox of a gender‐related reduction of activity of PDE5 in the presence of similar amount of expression can be explained by at least four different mechanisms: (i) phosphorylation of PDE5—it results in a gain of function of 50–70% of the in vitro cGMP catalytic activity [35]; (ii) binding of cGMP to the cGMP pocket in PDE5—such binding stimulates the catalytic activity of approximately 10‐fold and it can occur even in the absence of PDE5 phosphorylation [36]; (iii) activity of protein phosphatase 1 (PP1)—deactivation and dephosphorylation of PDE5 have been shown to be carried out by PP1 [37]; and (iv) cleavage of PDE5 by caspase‐3—hydrolyzing the 98‐kDa PDE5 protein into an 82‐kDa fragment [38]. However, it should be recognized that in the female bladder, less cGMP degradation is occurring and therefore a higher amount of cGMP should be available to allosterically increase PDE5 activity [39,40], therefore activating more than inhibiting the enzyme activity.…”

Expression, Distribution and Regulation of Phosphodiesterase 5

“…The bovine PDE5A1 has been shown to be a substrate for caspase-3, which hydrolyzed the 98-kd PDE5A1 protein into an 82-kd fragment [64]. This in vitro reaction was specific for caspase-3 because caspase-2, -12 and -14 did not significantly cleave the recombinant PDE5A1.…”