Binding of Regulatory Subunits of Cyclic AMP-Dependent Protein Kinase to Cyclic CMP Agarose

Hammerschmidt, Andreas; Chatterji, Bijon; Zeiser, Johannes; Schröder, Arne; Genieser, Hans‐Gottfried; Pich, Andreas; Kaever, Volkhard; Schwede, Frank; Wolter, Sabine; Seifert, Roland

doi:10.1371/journal.pone.0039848

Cited by 21 publications

(12 citation statements)

References 25 publications

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“…However, the much more logical hypothesis is that hitherto unrecognized specific cUMP effector proteins exist. Such new cUMP effector proteins could be identified with cUMP agarose binding techniques coupled with mass spectrometry detection [28]. We are currently performing such studies but cannot yet report a candidate cUMP-binding protein at this point of time.…”

Section: Discussionmentioning

confidence: 99%

ExoY from Pseudomonas aeruginosa is a nucleotidyl cyclase with preference for cGMP and cUMP formation

Beckert

Wolter

Hartwig

et al. 2014

Biochemical and Biophysical Research Communications

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In addition to the well known second messengers cAMP and cGMP, mammalian cells contain the cyclic pyrimidine nucleotides cCMP and cUMP. Soluble guanylyl cyclase and soluble adenylyl cyclase produce all four cNMPs. Several bacterial toxins exploit mammalian cyclic nucleotide signaling. The type III secretion protein ExoY from Pseudomonas aeruginosa induces severe lung damage and effectively produces cGMP. Here, we show that transfection of mammalian cells with ExoY or infection with ExoY-expressing P. aeruginosa not only massively increases cGMP but also cUMP levels. In contrast, the structurally related CyaA from Bordetella pertussis and edema factor from Bacillus anthracis exhibit a striking preference for cAMP increases. Thus, ExoY is a nucleotidyl cyclase with preference for cGMP and cUMP production. The differential effects of bacterial toxins on cNMP levels suggest that cUMP plays a distinct second messenger role.

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Section: Discussionmentioning

confidence: 99%

ExoY from Pseudomonas aeruginosa is a nucleotidyl cyclase with preference for cGMP and cUMP formation

Beckert

Wolter

Hartwig

et al. 2014

Biochemical and Biophysical Research Communications

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“…The large cUMP increases in the lung following infection with P. aeruginosa suggest that this cNMP plays a specific role in signal transduction and in the pathophysiology of lung function. Therefore, current research efforts are directed towards the identification of specific cUMP-binding proteins using the cNMP agarose affinity technique [5]. Since there are striking differences in degradation of cUMP and cGMP in the lung, we assume that the two cNMPs exhibit different functions.…”

Section: Resultsmentioning

confidence: 99%

Pseudomonas aeruginosaExoY, a cyclic GMP- and cyclic UMP-generating nucleotidyl cyclase

Seifert

Hartwig

Wolter

et al. 2013

BMC Pharmacol Toxicol

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“…2, 3, and 4 and Table 1). As alternative approach, cCMP agarose affinity binding studies may be used to identify specific cCMP-binding proteins (Hammerschmidt et al 2012). …”

Section: Anɵ-p-crebmentioning

confidence: 99%

N4-monobutyryl-cCMP activates PKA RIα and PKA RIIα more potently and with higher efficacy than PKG Iα in vitro but not in vivo

Wolter

Dove

Golombek

et al. 2014

Naunyn-Schmiedeberg's Arch Pharmacol

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There is increasing evidence for a role of cytidine 3',5'-cyclic monophosphate (cCMP) as second messenger. In a recent study, we showed that cCMP activates both purified guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase Iα (PKG Iα) and adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) isoenzymes with the regulatory subunits RIα and RIIα. Moreover, the membrane-permeant cCMP analog dibutyryl (DB)-cCMP induces effective vasodilation and inhibition of platelet aggregation via PKG Iα, but not via PKA. These data prompted us to conduct a systematic analysis of the effects of cyclic nucleotide (cNMP) analogs on purified PKG Iα and PKA RIα and RIIα We also studied the effect of DB-cCMP on PKA-dependent phosphorylation of the transcription factor cAMP response-binding protein (CREB) in S49 wild-type lymphoma cells and S49 kin(-) cells, devoid of the catalytic subunit of PKA. The major cellular metabolite of the prodrug DB-cCMP, N(4)-monobutyryl (4-MB)-cCMP, was a partial and low-potency activator of purified PKG Iα and a full and moderate-potency activator of PKA RIα and RIIα. Sp-cCMPS and Sp-cAMPS activated PKA RIα and RIIα with much higher potency and efficacy than PKG Iα. Molecular modeling suggested that the cytidine ring interacts with PKG Iα mainly via hydrophobic interactions, while the butyryl group projects away from the kinase. In contrast to DB-cAMP, DB-cCMP did not induce PKA-dependent phosphorylation in intact cells. Taken together, our data show that N(4)-monobutyryl-cCMP (4-MB-cCMP) activates PKA RIα and PKA RIIα more potently and with higher efficacy than PKG Iα in vitro but not in vivo. cNMP phosphorothioates constitute a starting point for the development of PKA activators with high selectivity relative to PKG.

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Binding of Regulatory Subunits of Cyclic AMP-Dependent Protein Kinase to Cyclic CMP Agarose

Cited by 21 publications

References 25 publications

ExoY from Pseudomonas aeruginosa is a nucleotidyl cyclase with preference for cGMP and cUMP formation

ExoY from Pseudomonas aeruginosa is a nucleotidyl cyclase with preference for cGMP and cUMP formation

Pseudomonas aeruginosaExoY, a cyclic GMP- and cyclic UMP-generating nucleotidyl cyclase

N4-monobutyryl-cCMP activates PKA RIα and PKA RIIα more potently and with higher efficacy than PKG Iα in vitro but not in vivo

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