Phosphorylation of argininosuccinate synthase by protein kinase A

Corbin, Karen D.; Pendleton, Laura C.; Solomonson, Larry P.; Eichler, Duane C.

doi:10.1016/j.bbrc.2008.10.056

Cited by 6 publications

(7 citation statements)

References 34 publications

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“…The impairment of the peptide-induced production of NO metabolites by both MDLA and L-Name, a specific NOS inhibitor, confirms the activation of AsS and the involvement of NOS in the increase of NO metabolites production by HUVECs. The activation process could be regulated by dynamic AsS phosphorylation, as it has been recently demonstrated (35). However, the involvement of phosphorylation of AsS in the increase of NO production induced by Bj-BPP-10c in HUVECs seems unlikely, since we demonstrated that AsS does not undergo any significant change in phosphorylation degree when HUVECs are treated with the peptide.…”

Section: Discussionmentioning

confidence: 57%

Argininosuccinate Synthetase Is a Functional Target for a Snake Venom Anti-hypertensive Peptide

Guerreiro

Lameu

Oliveira

et al. 2009

Journal of Biological Chemistry

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Bj-BPP-10c is a bioactive proline-rich decapeptide, part of the C-type natriuretic peptide precursor, expressed in the brain and in the venom gland of Bothrops jararaca. We recently showed that Bj-BPP-10c displays a strong, sustained anti-hypertensive effect in spontaneous hypertensive rats (SHR), without causing any effect in normotensive rats, by a pharmacological effect independent of angiotensin-converting enzyme inhibition. Therefore, we hypothesized that another mechanism should be involved in the peptide activity. Here we used affinity chromatography to search for kidney cytosolic proteins with affinity for Bj-BPP-10c and demonstrate that argininosuccinate synthetase (AsS) is the major protein binding to the peptide. More importantly, this interaction activates the catalytic activity of AsS in a dose-dependent manner. AsS is recognized as an important player of the citrulline-NO cycle that represents a potential limiting step in NO synthesis. Accordingly, the functional interaction of Bj-BPP-10c and AsS was evidenced by the following effects promoted by the peptide: (i) increase of NO metabolite production in human umbilical vein endothelial cell culture and of arginine in human embryonic kidney cells and (ii) increase of arginine plasma concentration in SHR. Moreover, ␣-methyl-DL-aspartic acid, a specific AsS inhibitor, significantly reduced the anti-hypertensive activity of Bj-BPP-10c in SHR. Taken together, these results suggest that AsS plays a role in the anti-hypertensive action of Bj-BPP-10c. Therefore, we propose the activation of AsS as a new mechanism for the anti-hypertensive effect of Bj-BPP-10c in SHR and AsS as a novel target for the therapy of hypertension-related diseases.Inhibition of somatic angiotensin-I-converting enzyme (sACE) 3 is a widely used approach in the treatment of hypertension. The first available competitive inhibitors of sACE were the naturally occurring proline-rich oligopeptides from the venom of Bothrops jararaca. Clinical studies using Bj-BPP-9a, teprotide, the most efficient of these snake venom peptides, demonstrated the potential of sACE inhibitors as anti-hypertensive drugs (1). Highly potent inhibitors of sACE, which can be administered orally, have subsequently been developed. The first of these, captopril, was designed employing a theoretical model of the active site of sACE, based on its presumed similarity to the active site of carboxypeptidase A and also with reference to the C terminus of venom proline-rich peptides, which compete with sACE substrates (2). Since captopril reproduced all known pharmacological effects and sACE-inhibiting features of the proline-rich peptides (3), the interest to deepen the investigation of the biological properties of these naturally occurring sACE inhibitors dropped dramatically. However, we recently showed that the Bj-BPP-10c (ϽENWPHPQIPP, where ϽE represents pyroglutamic acid), the most selective inhibitor of the active site at the C-domain of sACE (4), displays a strong and sustained anti-hypertensive effect in spontaneo...

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Section: Discussionmentioning

confidence: 57%

Argininosuccinate Synthetase Is a Functional Target for a Snake Venom Anti-hypertensive Peptide

Guerreiro

Lameu

Oliveira

et al. 2009

Journal of Biological Chemistry

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“…Our in vitro activity assay demonstrates the loss of function when the corresponding G is replaced with an S in the Leishmania enzyme. The specificity of activity of LdASS was supported further by blocking with α-methyl-DL-aspartic acid (MDLA), an ASS specific inhibitor [42], [48]–[49]. MDLA has been shown to be a specific and effective inhibitor of ASS from a variety of organisms [31], [50] and in our study of Leishmania ASS activity.…”

Section: Discussionmentioning

confidence: 70%

Leishmania donovani Argininosuccinate Synthase Is an Active Enzyme Associated with Parasite Pathogenesis

et al. 2012

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BackgroundGene expression analysis in Leishmania donovani (Ld) identified an orthologue of the urea cycle enzyme, argininosuccinate synthase (LdASS), that was more abundantly expressed in amastigotes than in promastigotes. In order to characterize in detail this newly identified protein in Leishmania, we determined its enzymatic activity, subcellular localization in the parasite and affect on virulence in vivo.Methodology/Principal FindingsTwo parasite cell lines either over expressing wild type LdASS or a mutant form (G128S) associated with severe cases of citrullinemia in humans were developed. In addition we also produced bacterially expressed recombinant forms of the same proteins. Our results demonstrated that LdASS has argininosuccinate synthase enzymatic activity that is abolished using an ASS specific inhibitor (MDLA: methyl-D-L-Aspartic acid). However, the mutant form of the protein is inactive. We demonstrate that though LdASS has a glycosomal targeting signal that binds the targeting apparatus in vitro, only a small proportion of the total cellular ASS is localized in a vesicle, as indicated by protection from protease digestion of the crude organelle fraction. The majority of LdASS was found to be in the cytosolic fraction that may include large cytosolic complexes as indicated by the punctate distribution in IFA. Surprisingly, comparison to known glycosomal proteins by IFA revealed that LdASS was located in a structure different from the known glycosomal vesicles. Significantly, parasites expressing a mutant form of LdASS associated with a loss of in vitro activity had reduced virulence in vivo in BALB/c mice as demonstrated by a significant reduction in the parasite load in spleen and liver.Conclusion/SignificanceOur study suggests that LdASS is an active enzyme, with unique localization and essential for parasite survival and growth in the mammalian host. Based on these observations LdASS could be further explored as a potential drug target.

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“…Indeed, recent work using mass spectrometry analysis showed that AS is phosphorylated at site Ser-352 (human sequence) (9); however, the biological significance of this phosphorylation was not defined. In addition, Corbin et al (8) showed that phosphorylation of AS was altered in response to VEGF treatment, yet the phosphorylation site was unknown. In this study, we demonstrated that AS Ser-328 phosphorylation supports the calcium-dependent stimulation of NO production in vascular endothelial cells and is mediated by PKC␣.…”

Section: Discussionmentioning

confidence: 99%

“…Despite the fact that relatively recent studies have shown that AS is a phospho-protein (8,9), no physiologically relevant phosphorylation sites have been reported. In 2008, an automated phospho-proteome analysis revealed that AS is phosphorylated at serine 352 in HeLa cells, but the physiologic significance was not established (9).…”

Section: Endothelial Nitric-oxide Synthase (Enos) Utilizes L-argininementioning

confidence: 99%

“…In particular, both VEGF and insulin stimulation of NO production were chosen for two reasons. First, there is evidence that AS phosphorylation changed in response to VEGF treatment, which was mediated via PKA signaling (8). Second, both VEGF and insulin are known to stimulate endothelial NO production by promoting phosphorylation of Ser-1179 of eNOS (13,14).…”

Section: Identification Of the Argininosuccinate Synthase Ser-328mentioning

confidence: 99%

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Protein Kinase Cα Phosphorylates a Novel Argininosuccinate Synthase Site at Serine 328 during Calcium-dependent Stimulation of Endothelial Nitric-oxide Synthase in Vascular Endothelial Cells

Haines

Corbin

Pendleton

et al. 2012

Journal of Biological Chemistry

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Background: Argininosuccinate synthase (AS) is critical for endothelial nitric oxide production, yet little is known about its regulation. Results: AS Ser-328 phosphorylation increased with calcium stimulation and decreased with PKC␣ interference. Conclusion: PKC␣ phosphorylates AS at Ser-328 under calcium-dependent stimulatory conditions to support nitric oxide production. Significance: Knowledge of how AS is regulated is essential in understanding nitric oxide homeostasis.

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Phosphorylation of argininosuccinate synthase by protein kinase A

Cited by 6 publications

References 34 publications

Argininosuccinate Synthetase Is a Functional Target for a Snake Venom Anti-hypertensive Peptide

Argininosuccinate Synthetase Is a Functional Target for a Snake Venom Anti-hypertensive Peptide

Leishmania donovani Argininosuccinate Synthase Is an Active Enzyme Associated with Parasite Pathogenesis

Protein Kinase Cα Phosphorylates a Novel Argininosuccinate Synthase Site at Serine 328 during Calcium-dependent Stimulation of Endothelial Nitric-oxide Synthase in Vascular Endothelial Cells

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