Abstract
Calmodulin-dependent activation of endothelial nitric-oxide synthase is generally considered to follow a transient increase in intracellular calcium levels. However, a number of physiological stimuli (e.g. endothelial shear-stress, insulin) are known to activate endothelial nitric oxide (eNOS) via a non-classical, "calcium-independent" pathway. Recent findings demonstrate that such stimuli elicit the phosphorylation of a C-terminal residue in eNOS (Ser(1179) in the bovine isoform), rendering eNOS active at resting levels of intracellular calcium. However, the mechanistic basis for this mode of eNOS activation remains unknown. Protein modeling led us to consider that the C terminus of eNOS may fulfill an autoinhibitory function that can be disrupted by phosphorylation of serine 1179. To test this possibility we contrasted the phenotype of wild type bovine eNOS with that of a mutant lacking C-terminal residues 1179-1205 (CDelta27 eNOS). Despite no observed difference in calmodulin affinity, CDelta27 eNOS exhibited a 5-fold reduction in EC(50) for calcium and a 2-4-fold increase in maximal catalytic activities. In these phenotypic properties, CDelta27 accurately mimics phospho-Ser(1179) wild type eNOS. We conclude that the C terminus imposes a significant barrier to the activation of eNOS by calmodulin binding and that this barrier can be functionally disabled by Ser(1179) phosphorylation-elicited enzyme activation.
Highlights
Nitric-oxide synthases (NOSs)1 comprise a family of three mammalian gene products that each possess an N-terminal heme-containing oxygenase domain and a C-terminal flavincontaining reductase domain, bridged by a canonical calmodulin (CaM)-binding polypeptide [1]
Subsequent cloning of all three NOS isoforms revealed a conserved bidomain structure consisting of an N-terminal oxygenase domain and a C-terminal reductase domain, bridged by a 25–35-amino acid CaM-binding domain
From the cloned sequences it was apparent that the reductase domain of all the NOS isoforms bears striking homology to mammalian cytochrome P-450 reductase (CPR) [34, 35]
Summary
Nitric-oxide synthases (NOSs) comprise a family of three mammalian gene products that each possess an N-terminal heme-containing oxygenase domain and a C-terminal flavincontaining reductase domain, bridged by a canonical calmodulin (CaM)-binding polypeptide [1]. We previously demonstrated that the FMN-binding subdomain of Ca2ϩ-dependent isoforms of nitric-oxide synthase (cNOS) contains a 45-amino acid insertion peptide that functions as an autoinhibitory control element (ACE) [4]. Binding of CaM to cNOS was postulated to displace the ACE by virtue of domain overlap, thereby eliciting enzyme activation Support for this prediction was subsequently provided by demonstrations that deletion of the entire ACE from eNOS [5, 6] or nNOS [7, 8] results in a marked reduction in the Ca2ϩ concentration required for activation of NO synthesis. Recognition sites on cNOSs that interact with the ACE to modulate activity have not been elucidated That such sites exist within the reductase domain is suggested by the finding that Ca2ϩ/CaM-dependent control of electron flux remains intact in isolated eNOS- and nNOS-derived reductase domains [9, 10]. The peptide extension of NOSs is found in no other FAD-containing flavoprotein that otherwise bears NOS-homology
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