Cyclic AMP-independent Activation of Protein Kinase A by Vasoactive Peptides

Protein kinase A (PKA) is an important effector enzyme commonly activated by cAMP. The present study focuses on our finding that the vasoactive peptide endothelin-1 (ET1), whose signaling is not coupled to cAMP production, stimulates PKA in two independent cellular models. Using an in vivo assay for PKA activity, we found that ET1 stimulated PKA in HeLa cells overexpressing ET1 receptors and in aortic smooth muscle cells expressing endogenous levels of ET1 receptors. In these cell models, ET1 did not stimulate cAMP production, indicating a novel mechanism for PKA activation. The ET1-induced activation of PKA was found to be dependent on the degradation of inhibitor of κB, which was previously reported to bind and inhibit PKA. ET1 potently stimulated the nuclear factor-κB pathway, and this effect was inhibited by overexpression of the inhibitor of κB dominant negative mutant (IκBαm) and by treatment with the proteasome inhibitor MG-132. Importantly, IκBαm and MG-132 had similar inhibitory effects on ET1-induced activation of PKA without affecting Gs-mediated activation of PKA or ET1-induced phosphorylation of mitogen-activated protein kinase. Finally, another vasoactive peptide, angiotensin II, also stimulated PKA in a cAMP-independent manner in aortic smooth muscle cells. These findings suggest that cAMP-independent activation of PKA might be a general response to vasoactive peptides. Protein kinase A (PKA) is an important effector enzyme commonly activated by cAMP. The present study focuses on our finding that the vasoactive peptide endothelin-1 (ET1), whose signaling is not coupled to cAMP production, stimulates PKA in two independent cellular models. Using an in vivo assay for PKA activity, we found that ET1 stimulated PKA in HeLa cells overexpressing ET1 receptors and in aortic smooth muscle cells expressing endogenous levels of ET1 receptors. In these cell models, ET1 did not stimulate cAMP production, indicating a novel mechanism for PKA activation. The ET1-induced activation of PKA was found to be dependent on the degradation of inhibitor of κB, which was previously reported to bind and inhibit PKA. ET1 potently stimulated the nuclear factor-κB pathway, and this effect was inhibited by overexpression of the inhibitor of κB dominant negative mutant (IκBαm) and by treatment with the proteasome inhibitor MG-132. Importantly, IκBαm and MG-132 had similar inhibitory effects on ET1-induced activation of PKA without affecting Gs-mediated activation of PKA or ET1-induced phosphorylation of mitogen-activated protein kinase. Finally, another vasoactive peptide, angiotensin II, also stimulated PKA in a cAMP-independent manner in aortic smooth muscle cells. These findings suggest that cAMP-independent activation of PKA might be a general response to vasoactive peptides. endothelin-1 inhibitor of κB isoproterenol lipopolysaccharide nuclear factor κB protein kinase A rat aortic smooth muscle cells vasodilator-stimulated phosphoprotein mitogen-activated protein Dulbecco's modified Eagle's medium fetal bovine serum angiotensin II Endothelin-1 (ET1)1 is a vasoactive peptide implicated in embryonic development and in pathophysiology of cardiovascular, renal, and respiratory systems (1Parris R.J. Webb D.J. Vasc. Med. 1997; 2: 31-43Crossref PubMed Scopus (28) Google Scholar,2Ortega Mateo A. de Artinano A.A. Pharmacol. Res. 1997; 36: 339-351Crossref PubMed Scopus (106) Google Scholar). Two types of ET1 receptors, namely ETA and ETB, have been cloned and identified as typical G protein-coupled receptors (3Adachi M. Yang Y.Y. Furuichi Y. Miyamoto C. Biochem. Biophys. Res. Commun. 1991; 180: 1265-1272Crossref PubMed Scopus (94) Google Scholar, 4Sakamoto A. Yanagisawa M. Sakurai T. Takuwa Y. Yanagisawa H. Masaki T. Biochem. Biophys. Res. Commun. 1991; 178: 656-663Crossref PubMed Scopus (247) Google Scholar). ETA receptors are coupled to Gq/11, G12/13, and Giheterotrimeric G proteins, leading to stimulation of phospholipase C, small GTPase RhoA, and inhibition of adenylyl cyclase, respectively (5Vogelsang M. Broede-Sitz A. Schafer E. Zerkowski H.R. Brodde O.E. J. Cardiovasc. Pharmacol. 1994; 23: 344-347Crossref PubMed Google Scholar, 6Takagi Y. Ninomiya H. Sakamoto A. Miwa S. Masaki T. J. Biol. Chem. 1995; 270: 10072-10078Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 7Wu-Wong J.R. Opgenorth T.J. J. Cardiovasc. Pharmacol. 1998; 31 Suppl. 1: 185-191Crossref Scopus (21) Google Scholar, 8Mao J. Yuan H. Xie W. Simon M.I. Wu D. J. Biol. Chem. 1998; 273: 27118-27123Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). The coupling of ET1 receptors to Gs is controversial. A modest cAMP response to ET1 was reported by some investigators (9Aramori I. Nakanishi S. J. Biol. Chem. 1992; 267: 12468-12474Abstract Full Text PDF PubMed Google Scholar, 10El-Mowafy A.M. White R.E. Biochem. Biophys. Res. Commun. 1998; 251: 494-500Crossref PubMed Scopus (18) Google Scholar, 11Rebsamen M.C. Church D.J. Morabito D. Vallotton M.B. Lang U. Am. J. Physiol. 1997; 273: E922-E931PubMed Google Scholar), whereas no response or inhibition of cAMP levels was shown by others (5Vogelsang M. Broede-Sitz A. Schafer E. Zerkowski H.R. Brodde O.E. J. Cardiovasc. Pharmacol. 1994; 23: 344-347Crossref PubMed Google Scholar, 7Wu-Wong J.R. Opgenorth T.J. J. Cardiovasc. Pharmacol. 1998; 31 Suppl. 1: 185-191Crossref Scopus (21) Google Scholar, 12Takuwa N. Takuwa Y. Yanagisawa M. Yamashita K. Masaki T. J. Biol. Chem. 1989; 264: 7856-7861Abstract Full Text PDF PubMed Google Scholar, 13Lin W.W. Chuang D.M. Mol. Pharmacol. 1993; 44: 158-165PubMed Google Scholar, 14James A.F. Xie L.H. Fujitani Y. Hayashi S. Horie M. Nature. 1994; 370: 297-300Crossref PubMed Scopus (80) Google Scholar, 15Zhu Y. Yang H.T. Endoh M. Am. J. Physiol. 1997; 273: H119-H127PubMed Google Scholar). Moreover, there was no convincing evidence that the main target of cAMP, the protein kinase A (PKA), could be activated by ET1. The PKA holoenzyme is a tetrameric complex consisting of two catalytic subunits (PKAc) bound to a homodimer of two regulatory subunits (PKAr). The established mechanism of PKA activation in response to various hormones involves stimulatory G proteins, Gs, which activate adenylyl cyclase resulting in production of cAMP. Binding of cAMP to PKAr leads to a release and activation of PKAc (16Taylor S.S. Buechler J.A. Yonemoto W. Ortega Mateo A. de Artinano A.A. Annu. Rev. Biochem. 1990; 59: 971-1005Crossref PubMed Scopus (949) Google Scholar, 17Francis S.H. Corbin J.D. Crit. Rev. Clin. Lab. Sci. 1999; 36: 275-328Crossref PubMed Scopus (260) Google Scholar). Recently, a novel mechanism for PKA activation by lipopolysaccharide (LPS) has been described that is related to the nuclear factor-κB (NFκB) pathway (18Zhong H. SuYang H. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1997; 89: 413-424Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar). NFκB is a transcription factor that is commonly activated during immune and inflammatory responses (19Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5515) Google Scholar, 20Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4550) Google Scholar). Under basal conditions, NFκB exists in an inactive state bound to its natural inhibitor IκB. Activation of NFκB occurs as a result of agonist-induced phosphorylation and degradation of IκB followed by a release of free NFκB. Apparently, a certain pool of PKAc also exists in a complex with IκB (18Zhong H. SuYang H. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1997; 89: 413-424Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar). Under basal conditions, IκB retains PKAc in the inactive state, presumably by masking its ATP binding site. LPS-induced phosphorylation and degradation of IκB results in a release and activation of PKAc (18Zhong H. SuYang H. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1997; 89: 413-424Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar). However, except for bacterially derived LPS, there was no evidence that other physiological agonists are able to activate PKA by this mechanism. The present study demonstrates for the first time that ET1 stimulates PKA activity by a cAMP-independent mechanism involving degradation of IκB. Moreover, our data suggest that this is most likely a general phenomenon common for vasoactive peptides. The cDNA for ETA receptor was kindly provided by Dr. Masashi Yanagisawa (University of Texas, South Western Medical Center, Dallas, TX). The cDNA for FLAG-tagged vasodilator-stimulated phosphoprotein (VASP) was a gift from Dr. Michael Uhler (University of Michigan, Ann Arbor, MI). The cDNA for the dominant negative mutant of PKA (δR1α) was a gift from Dr. Stanley McKnight (University of Washington, Seattle, WA). The cDNA for the phosphorylation-deficient S32A,S36A mutant of mouse IκBα (IκBαm) was a gift from Dr. Inder Verma (The Salk Institute, La Jolla, CA). The phosphorylation-deficient S19A,S23A mutant of mouse IκBβ (IκBβm) was generated by polymerase chain reaction, and its identity was confirmed by sequencing. The NFκB-driven luciferase reporter plasmid was described previously (21Xie P. Browning D.D. Hay N. Mackman N. Ye R.D. J. Biol. Chem. 2000; 275: 24907-24914Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Endothelin-1, isoproterenol, tumor necrosis factor α, and MG-132 were from Calbiochem. Angiotensin II was from Peninsula Laboratories. Monoclonal anti-FLAG antibodies were from Sigma. Polyclonal anti-phospho-MAP kinase antibodies were from New England Biolabs. The HeLa cells (ATCC) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mm glutamine, 100 units/ml streptomycin, 100 units/ml penicillin, and 10% fetal bovine serum (FBS). The primary culture of rat aortic smooth muscle cells (RASMC) from Wistar-Kyoto rats was kindly provided by Dr. Sergei Orlov (University of Montreal, Montreal, Canada). The RASMC were cultured for up to 10 passages in DMEM supplemented with 10% FBS, 2 mmglutamine, 100 units/ml streptomycin, and 100 units/ml penicillin as described elsewhere (22Orlov S.N. Tremblay J. Hamet P. Hypertension. 1996; 27: 774-780Crossref PubMed Google Scholar). For transient overexpression of proteins, the HeLa cells or RASMC were transfected with desired DNA in the presence of serum, using LipofectAMINE-2000 or LipofectAMINE-Plus reagents (Life Technologies, Inc.), respectively, following the manufacturer's protocol. The cells were serum-starved in 0.2% FBS for 24 h before the experiment. Phosphorylation-induced electrophoretic mobility shift of the VASP is a highly sensitive functional assay for the activity of cyclic nucleotide-dependent protein kinases in intact cells (23Halbrugge M. Friedrich C. Eigenthaler M. Schanzenbacher P. Walter U. J. Biol. Chem. 1990; 265: 3088-3093Abstract Full Text PDF PubMed Google Scholar,24Butt E. Abel K. Krieger M. Palm D. Hoppe V. Hoppe J. Walter U. J. Biol. Chem. 1994; 269: 14509-14517Abstract Full Text PDF PubMed Google Scholar) and was used in this study. The specificity of PKA-mediated phosphorylation of VASP was confirmed by overexpression of the dominant negative mutant of PKA, δR1α, which abolished VASP phosphorylation induced by isoproterenol (see Fig. 1 C) or by 8-bromo-cAMP (25Browning D.D. McShane M.P. Marty C. Ye R.D. J. Biol. Chem. 2000; 275: 2811-2816Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) but not by 8-bromo-cGMP (25Browning D.D. McShane M.P. Marty C. Ye R.D. J. Biol. Chem. 2000; 275: 2811-2816Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The assay involved transient transfection of cells with FLAG-tagged VASP cDNA, stimulation of quiescent cells with desired agonists, cell lysis followed by immunoblotting of cell lysates with FLAG antibodies (see below), and monitoring the phosphorylation-dependent electrophoretic mobility shift of VASP, as described previously (25Browning D.D. McShane M.P. Marty C. Ye R.D. J. Biol. Chem. 2000; 275: 2811-2816Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). After stimulation of quiescent cells with desired agonists, the cells were lysed in the buffer containing 25 mm HEPES (pH 7.5), 150 mm NaCl, 1% Triton X-100, 0.1% SDS, 5 mm EDTA, 1 mm NaF, 200 μm sodium orthovanadate, and protease inhibitors (1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 mmphenylmethylsulfonyl fluoride). The lysates were cleared from insoluble material by centrifugation at 20,000 × g for 10 min, subjected to polyacrylamide gel electrophoresis, transferred to nitrocellulose, and analyzed by Western blotting with 0.5 μg/ml primary antibodies followed by 0.3 μg/ml horseradish peroxidase-conjugated secondary antibodies and developed by ECL (Amersham Pharmacia Biotech). Cyclic AMP accumulation was determined as described previously (26Voyno-Yasenetskaya T.A. Conklin B.R. Gilbert R.L. Hooley R. Bourne H.R. Barber D.L. J. Biol. Chem. 1994; 269: 4721-4724Abstract Full Text PDF PubMed Google Scholar). Briefly, cells were serum-starved and labeled with 3 μCi/ml [3H]adenine for 24 h, washed twice with serum-free DMEM, and stimulated with desired agonists for various times at 37 °C. Reactions were terminated by aspiration of medium followed by addition of ice-cold 5% trichloroacetic acid. Acid-soluble nucleotides were separated on ion-exchange columns and subjected to scintillation spectroscopy. The radioactivity of cAMP-containing fractions was normalized on the total (cAMP + ATP) radioactivity in each sample and finally expressed as -fold increase over control (zero time point). Fig.1 shows a time course of PKA activation in response to ET1 (Fig. 1 A) and β2-adrenergic receptor agonist isoproterenol (ISO) (Fig. 1 B) after transient transfection of HeLa cells with ETA and β2-adrenergic receptor, respectively, as measured by gel retardation of the PKA substrate VASP (see “Materials and Methods”). ET1 induced a transient phosphorylation of VASP with a maximum at 5 min. In contrast, ISO-induced phosphorylation of VASP was much stronger and persisted for at least 1 h (Fig. 1 B). To confirm that phosphorylation of VASP is mediated by PKA, we employed a cAMP-unresponsive dominant negative mutant of PKAr, δR1α. As shown in Fig. 1 C, phosphorylation of VASP, induced by ET1 and ISO, was abolished by overexpression of δR1α. Confirming the specificity of δR1α, it had no effect on ET1-induced MAP kinase phosphorylation (Fig. 1 D) or on cGMP-mediated phosphorylation of VASP (25Browning D.D. McShane M.P. Marty C. Ye R.D. J. Biol. Chem. 2000; 275: 2811-2816Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Because two mechanisms of PKA activation have been described, it was important first to examine whether the effect of ET1 on PKA activity was mediated by cAMP. As shown in Fig.2, ET1 did not stimulate cAMP production but rather reduced basal levels of cAMP in ETA-transfected HeLa cells. By contrast, ISO (positive control) increased cAMP levels by more than 8-fold in β2-adrenergic receptor-transfected cells (Fig. 2). This suggests that ET1-induced activation of PKA is cAMP-independent and confirms that in our cellular model, ET1 signaling is not coupled to Gs and adenylyl cyclase. We next addressed the possibility of a cAMP-independent mechanism of ET1-induced PKA activity, described previously for LPS, wherein PKA activation was mediated by proteasome-dependent degradation of IκB (18Zhong H. SuYang H. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1997; 89: 413-424Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar). ET1 stimulated NFκB activity in HeLa cells by 35.8 ± 4.4-fold, as measured by κB-dependent expression of the luciferase gene (Fig.3). This effect of ET1 was inhibited by the proteasome inhibitor MG-132, as well as by overexpression of the phosphorylation-deficient dominant negative mutant of IκB, IκBαm (Fig. 3). These data indicate that ET1 stimulates NFκB via phosphorylation and degradation of IκB. Preincubation of cells with increasing concentrations of MG-132 resulted in a dose-dependent inhibition of ET1-induced PKA activity, reaching maximum at 15 μm MG-132 (Fig.4 A). By contrast, up to 50 μm MG-132 had no significant effect on ET1-induced phosphorylation of MAP kinase (Fig. 4 B) or the ISO-induced VASP shift (Fig. 4 F). This suggests that ET1-induced activation of PKA is mediated by proteasome-dependent protein degradation. To examine whether this PKA activation is dependent on the degradation of IκB, we employed phosphorylation-deficient dominant negative mutants of IκB. PKA was previously shown to bind IκBα, as well as IκBβ isoforms (18Zhong H. SuYang H. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1997; 89: 413-424Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar). Therefore, we examined the effects of IκBα-S32A,S36A (IκBαm) and IκBβ-S19A,S23A (IκBβm) overexpression on ET1-induced PKA activity. Overexpression of increasing amounts of IκBαm resulted in a dose-dependent inhibition of ET1-induced PKA activity (Fig. 4 C) without affecting MAP kinase phosphorylation (Fig.4 D) or the ISO-induced VASP shift (Fig. 4 F). By contrast, overexpression of IκBβm had no significant effect on ET1-induced VASP phosphorylation (Fig. 4 E). Taken together, these data suggest that proteasome-dependent degradation of IκBα mediates ET1-stimulated PKA activity in HeLa cells. It was important to confirm that cAMP-independent activation of PKA by ET1 in HeLa cells was not an artifact of ETA overexpression. Therefore, we next examined the ability of ET1 to activate PKA in a primary culture of RASMC, which express endogenous levels of ETA receptors. As shown in Fig.5 A, ET1 and ISO stimulated phosphorylation of VASP in these cells with a striking similarity to their effects in the transiently transfected cellular model (compare Fig. 5 A and Fig. 1). Moreover, in RASMC, PKA was also stimulated by another vasoactive peptide, angiotensin II (AII) (Fig.5 A). Importantly, ET1 and AII failed to stimulate cAMP production in RASMC, whereas ISO increased cAMP levels by more than 200-fold (Fig. 5 B). This suggests that cAMP-independent activation of PKA may be a general phenomenon, common for vasoactive peptides. The present study describes for the first time cAMP-independent activation of PKA by G protein-coupled receptor agonist endothelin-1 and provides the mechanism of this signaling event. Employing two independent cellular models with overexpressed or endogenous levels of ETA receptors, we provide strong evidence for the ability of ET1 to stimulate PKA activity in a cAMP-independent manner. Moreover, this may represent a general phenomenon common for vasoactive peptides, because angiotensin II elicited similar effect on PKA in RASMC. With the exception of one study, which showed a modest, cAMP-dependent activation of PKA by ET1 in pig coronary arteries (10El-Mowafy A.M. White R.E. Biochem. Biophys. Res. Commun. 1998; 251: 494-500Crossref PubMed Scopus (18) Google Scholar), the stimulation of PKA by either ET1 or AII has not been reported. In our experiments, ET1 failed to stimulate cAMP production but rather reduced the basal levels of cAMP. This is in accord with other investigators having shown that ET1 either had no effect or inhibited basal or agonist-induced cAMP production, which is consistent with the coupling of ETAreceptors to Gi proteins (5Vogelsang M. Broede-Sitz A. Schafer E. Zerkowski H.R. Brodde O.E. J. Cardiovasc. Pharmacol. 1994; 23: 344-347Crossref PubMed Google Scholar, 7Wu-Wong J.R. Opgenorth T.J. J. Cardiovasc. Pharmacol. 1998; 31 Suppl. 1: 185-191Crossref Scopus (21) Google Scholar, 13Lin W.W. Chuang D.M. Mol. Pharmacol. 1993; 44: 158-165PubMed Google Scholar, 14James A.F. Xie L.H. Fujitani Y. Hayashi S. Horie M. Nature. 1994; 370: 297-300Crossref PubMed Scopus (80) Google Scholar, 15Zhu Y. Yang H.T. Endoh M. Am. J. Physiol. 1997; 273: H119-H127PubMed Google Scholar). However, one might still consider the possibility of compartment-specific changes in cAMP-levels in response to ET1, which have not been detected in the present study. The cAMP-independent mechanism of PKA activation, which is mediated by LPS-induced degradation of IκB, has been described previously by Zhong (18Zhong H. SuYang H. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1997; 89: 413-424Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar). However, except for bacterially derived LPS, no physiological has been reported to activate PKA by this mechanism. The present demonstrates for the first time that the ET1, which is to cardiovascular, renal, and also stimulates PKA in an manner (Fig. This suggests that this mechanism for PKA activation is more and might also be to other G protein-coupled receptors. important are still to be as the signaling which ETA receptors to the degradation of IκB and activation of PKA, as well as the functional of ET1-induced PKA activation. IκB degradation be mediated by a of protein kinase N. M. N. A. Sci. U. S. A. 2000; Google W. M.J. J. D. A. Nature. 2000; PubMed Scopus Google Scholar), mitogen-activated protein kinase K. D. K. S. 1997; PubMed Scopus Google Scholar), or kinase (21Xie P. Browning D.D. Hay N. Mackman N. Ye R.D. J. Biol. Chem. 2000; 275: 24907-24914Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). ETA receptors activate M.J. J. 1990; PubMed Scopus Google Scholar, A. Pharmacol. 1998; PubMed Scopus Google Scholar, M. S. M.J. A. 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This suggests that activation of PKA may as a regulatory mechanism in the of ET1. these We Dr. Masashi Yanagisawa for ETA receptor cDNA, Dr. Michael Uhler for cDNA, Dr. Stanley McKnight for cDNA, Dr. Inder Verma for IκBαm cDNA, Dr. Sergei Orlov for primary culture of rat aortic smooth muscle and Dr. for

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