Calmodulin phosphorylation and modulation of endothelial nitric oxide synthase catalysis

Abstract

The endothelial NO synthase (eNOS) is regulated by diverse protein kinase pathways, yet eNOS activity ultimately depends on the ubiquitous calcium regulatory protein calmodulin (CaM). In these studies, we establish that CaM itself undergoes phosphorylation in endothelial cells and that CaM phosphorylation attenuates eNOS activation. Using [ 32 P]orthophosphoric acid biosynthetic labeling, we found that CaM is a phosphoprotein in bovine aortic endothelial cells (BAEC) and that the kinase CK2 promotes CaM phosphorylation in BAEC. Phosphorylation of CaM by purified CK2 in vitro reduces the V max of immunopurified eNOS by a factor of 2 but has no effect on the K A for CaM or calcium. Additionally, [ 32 P]orthophosphoric acid biosynthetic labeling of mutant CaM-transfected BAEC revealed that phosphorylation of Ser-81 to alanine mutant CaM (''phosphonull'' S81A mutant) is dramatically reduced relative to WT, whereas phosphorylation of the ''phosphomimetic'' Ser-81 to aspartate (S81D) mutant is unchanged. Further studies using Escherichia coli-expressed and phenylSepharose-purified CaM mutants revealed that the S81A mutation abrogates in vitro CK2-mediated phosphorylation of CaM, whereas phosphorylation of the S81D CaM mutant by CK2 is preserved. Additionally, we found that the phosphomimetic S101D CaM mutant is impaired in its ability to activate eNOS. Taken together, these results suggest that phosphorylation of CaM inhibits eNOS catalysis and proceeds in a hierarchical manner, initially requiring phosphorylation of the CaM Ser-81 residue. We conclude that CaM phosphorylation may represent a unique pathway in the regulation of eNOS signaling and thereby may play a role in modulating NO-dependent vascular responses.T he vascular system uses a network of cell signaling pathways to maintain a delicate homeostatic balance. The endothelial NO synthase (eNOS) is a calmodulin-dependent enzyme that plays a key role in many of these signaling cascades by catalyzing the conversion of L-arginine and oxygen to L-citrulline and the labile gas NO (1). Because NO has fundamental effects on many aspects of vascular physiology (2), it is not surprising that eNOS is tightly regulated by a number of mechanisms. Subcellular localization, acylation, phosphorylation, and direct interaction with other proteins, including calmodulin (CaM), caveolin, and heat shock protein 90, have been shown to modulate eNOS (3). Recently, the phosphorylation of eNOS in endothelial cells has been extensively characterized (4-9); however, the role of phosphorylation pathways in modulating the protein partners of eNOS, such as CaM, is much less well understood.CaM is a ubiquitously expressed, highly conserved acidic protein that mediates a broad range of intracellular calcium-regulated enzymes (10, 11), including eNOS (12). CaM is comprised of 148 aa and has a ''dumbbell'' structure: a long flexible central helix joining two pairs of calcium-binding helix-loop-helix motifs, known as EF hands (13). Upon binding calcium, CaM exposes hydrophobic regions on its s...

Channel-anchored Protein Kinase CK2 and Protein Phosphatase 1 Reciprocally Regulate KCNQ2-containing M-channels via Phosphorylation of Calmodulin

“…Any single aspartate mutations increased KCNQ2 binding to an extent similar to that of CaM(3D). This observation is consistent with a previous report that single aspartate mutation facilitates phosphorylation of other sites in mammalian cells (11). However, co-immunoprecipitation assays using alaninesubstituted mutants identified Thr-80 as the critical residue (Fig.…”

“…For instance, phosphodiesterase exhibits lower binding affinity to phosphorylated CaM without affecting its V max (10). In contrast, the endothelial NO synthetase shows a reduced V max in the presence of phosphorylated CaM without changing its affinity for phosphorylated CaM (11). Regarding the effect of CaM phosphorylation on ion channels, detailed analysis has been described for the SK2 small conductance calcium-activated potassium channels (13,16,17).…”

“…Interestingly, 15-40% of endogenous CaM is known to be phosphorylated (8,9), although the functional role of phosphorylated CaM on KCNQ2 channel remains unknown. Protein kinase CK2 (casein kinase 2) has been identified as key kinase for phosphorylating CaM (10,11). Various functional consequences of phosphorylated CaM have been demonstrated in a number of CaM-dependent proteins (10 -12) including the small conductance calcium-activated potassium channel 2, SK2 channel (13).…”

Characterization of protein kinase CK2 from Trypanosoma brucei

“…However, strong activation towards this substrate was observed, comparable to that seen with wild-type human enzyme by the above authors. Calmodulin is a physiological substrate of CK2 [54], and its CK2 phosphorylation sites (T79, S81, S101) are maintained in T. brucei. CK2 activation by polybasic peptides is not restricted to poly-L-lysine but can be achieved with proteins such as histones [18][19][20], suggesting that this conserved property may be physiologically meaningful.…”

Can glyphosate’s disruption of the gut microbiome and induction of sulfate deficiency explain the epidemic in gout and associated diseases in the industrialized world?

“…Endothelial nitric oxide synthase (eNOS) plays a vital rôle in regulating blood flow, in part through production of nitric oxide (NO) following signaling by calcium uptake and phosphorylation of calmodulin, a regulatory protein [105]. However, eNOS is also capable, especially under hypertensive conditions, of producing superoxide instead of NO in an uncoupled mode [106].…”