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

Protein kinase A (PKA) is an important effector en-zyme commonly activated by cAMP. The present study focuses on our finding that the vasoactive peptide endo-thelin-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 overex-pressing 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 k B, which was previously reported to bind and inhibit PKA. ET1 potently stimulated the nuclear factor- k B pathway, and this effect was inhibited by overexpression of the inhibitor of k B dominant negative mutant (I k B a m) and by treatment with the proteasome inhibitor MG-132. Im-portantly, I k B a m and MG-132 had similar inhibitory effects on ET1-induced activation of PKA without affecting G s -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 B, which was previously reported to bind and inhibit PKA. ET1 potently stimulated the nuclear factor-B pathway, and this effect was inhibited by overexpression of the inhibitor of B dominant negative mutant (IB␣m) and by treatment with the proteasome inhibitor MG-132. Importantly, IB␣m and MG-132 had similar inhibitory effects on ET1-induced activation of PKA without affecting G s -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 (ET1) 1 is a vasoactive peptide implicated in embryonic development and in pathophysiology of cardiovascular, renal, and respiratory systems (1,2). Two types of ET1 receptors, namely ET A and ET B , have been cloned and identified as typical G protein-coupled receptors (3,4). ET A receptors are coupled to G q/11 , G 12/13 , and G i heterotrimeric G proteins, leading to stimulation of phospholipase C, small GTPase RhoA, and inhibition of adenylyl cyclase, respectively (5)(6)(7)(8). The coupling of ET1 receptors to G s is controversial. A modest cAMP response to ET1 was reported by some investigators (9 -11), whereas no response or inhibition of cAMP levels was shown by others (5,7,(12)(13)(14)(15). 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, G s , which activate adenylyl cyclase resulting in production of cAMP. Binding of cAMP to PKAr leads to a release and activation of PKAc (16,17). Recently, a novel mechanism for PKA activation by lipopolysaccharide (LPS) has been described that is related to the nuclear factor-B (NFB) pathway (18). NFB is a transcription factor that is commonly activated during immune and inflammatory responses (19,20). Under basal conditions, NFB exists in an inactive state bound to its natural inhibitor IB. Activation of NFB occurs as a result of agonist-induced phosphorylation and degradation of IB followed by a release of free NFB. Apparently, a certain pool of PKAc also exists in a complex with IB (18). Under basal conditions, IB retains PKAc in the inactive state, presumably by masking its ATP binding site. LPS-induced phosphorylation and degradation of IB results in a release and activation of PKAc (18). 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 IB. Moreover, our data suggest that this is most likely a general phenomenon common for vasoactive peptides.

MATERIALS AND METHODS
Reagents-The cDNA for ET A 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 (␦R1␣) was a gift from Dr. Stanley McKnight (University of Washington, Seattle, WA). The cDNA for the phosphorylationdeficient S32A,S36A mutant of mouse IB␣ (IB␣m) was a gift from Dr. Inder Verma (The Salk Institute, La Jolla, CA). The phosphorylation-deficient S19A,S23A mutant of mouse IB␤ (IB␤m) was generated by polymerase chain reaction, and its identity was confirmed by sequencing. The NFB-driven luciferase reporter plasmid was described previously (21). Endothelin-1, isoproterenol, tumor necrosis factor ␣, 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.
Cell Culture and DNA Transfection-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 mM glutamine, 100 units/ml streptomycin, and 100 units/ml penicillin as described elsewhere (22). For transient * This work was supported by National Institutes of Health Grant GM56159 and a grant from American Heart Association (to T. V. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
PKA Activity in Intact Cells-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 (23,24) and was used in this study. The specificity of PKAmediated phosphorylation of VASP was confirmed by overexpression of the dominant negative mutant of PKA, ␦R1␣, which abolished VASP phosphorylation induced by isoproterenol (see Fig. 1C) or by 8-bromo-cAMP (25) but not by 8-bromo-cGMP (25). 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 (25).
Immunoblotting-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 M sodium orthovanadate, and protease inhibitors (1 g/ml leupeptin, 1 g/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride). The lysates were cleared from insoluble material by centrifugation at 20,000 ϫ g for 10 min, subjected to polyacrylamide gel electrophoresis, transferred to nitrocellulose, and analyzed by Western blotting with 0.5 g/ml primary antibodies followed by 0.3 g/ml horseradish peroxidaseconjugated secondary antibodies and developed by ECL (Amersham Pharmacia Biotech).
Cyclic AMP Assay-Cyclic AMP accumulation was determined as described previously (26). Briefly, cells were serum-starved and labeled with 3 Ci/ml [ 3 H]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 ϩ 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. 1A) and ␤ 2 -adrener-gic receptor agonist isoproterenol (ISO) (Fig. 1B) after transient transfection of HeLa cells with ET A and ␤ 2 -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. 1B). To confirm that phosphorylation of VASP is mediated by PKA, we employed a cAMP-unresponsive dominant negative mutant of PKAr, ␦R1␣. As shown in Fig. 1C, phosphorylation of VASP, induced by ET1 and ISO, was abolished by overexpression of ␦R1␣. Confirming the specificity of ␦R1␣, it had no effect on ET1-induced MAP kinase phosphorylation (Fig. 1D) or on cGMP-mediated phosphorylation of VASP (25).

ET1-induced Activation of PKA-
ET1-induced Activation of PKA Is Mediated by Degradation of IB-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 ET A -transfected HeLa cells. By contrast, ISO (positive control) increased cAMP levels by more than 8-fold in ␤ 2 -adrenergic receptor-transfected cells (Fig. 2). This suggests that ET1-induced activation of PKA is cAMPindependent and confirms that in our cellular model, ET1 signaling is not coupled to G s 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 proteasomedependent degradation of IB (18). ET1 stimulated NFB activity in HeLa cells by 35.8 Ϯ 4.4-fold, as measured by B-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 IB, IB␣m (Fig. 3). These data indicate that ET1 stimulates NFB via phosphorylation and degradation of IB.
Preincubation of cells with increasing concentrations of MG-132 resulted in a dose-dependent inhibition of ET1-induced PKA activity, reaching maximum at 15 M MG-132 (Fig. 4A). By contrast, up to 50 M MG-132 had no significant effect on ET1-induced phosphorylation of MAP kinase (Fig. 4B) or the ISO-induced VASP shift (Fig. 4F). This suggests that ET1induced activation of PKA is mediated by proteasome-dependent protein degradation. To examine whether this PKA activation is dependent on the degradation of IB, we employed phosphorylation-deficient dominant negative mutants of IB. cAMP-independent Activation of PKA by Vasoactive Peptides 20828 PKA was previously shown to bind IB␣, as well as IB␤ isoforms (18). Therefore, we examined the effects of IB␣-S32A,S36A (IB␣m) and IB␤-S19A,S23A (IB␤m) overexpression on ET1-induced PKA activity. Overexpression of increasing amounts of IB␣m resulted in a dose-dependent inhibition of ET1-induced PKA activity (Fig. 4C) without affecting MAP kinase phosphorylation (Fig. 4D) or the ISO-induced VASP shift (Fig. 4F). By contrast, overexpression of IB␤m had no significant effect on ET1-induced VASP phosphorylation (Fig.  4E). Taken together, these data suggest that proteasome-dependent degradation of IB␣ mediates ET1-stimulated PKA activity in HeLa cells.
Activation of PKA by ET1 and Angiotensin II in Vascular Smooth Muscle Cells-It was important to confirm that cAMPindependent activation of PKA by ET1 in HeLa cells was not an artifact of ET A overexpression. Therefore, we next examined the ability of ET1 to activate PKA in a primary culture of RASMC, which express endogenous levels of ET A receptors. As shown in Fig. 5A, 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. 5A and Fig. 1). Moreover, in RASMC, PKA was also stimulated by another vasoactive peptide, angiotensin II (AII) (Fig. 5A). Importantly, ET1 and AII failed to stimulate cAMP production in RASMC, whereas ISO increased cAMP levels by more than 200-fold (Fig. 5B). This suggests that cAMP-independent activation of PKA may be a general phenomenon, common for vasoactive peptides.

DISCUSSION
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.
Cyclic AMP-independent Activation of PKA by Vasoactive Peptides-Employing two independent cellular models with overexpressed or endogenous levels of ET A 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 (10), 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 ET A receptors to G i proteins (5,7,(13)(14)(15). 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.
ET1-induced PKA Activity Is Dependent on IB Degradation-The cAMP-independent mechanism of PKA activation, which is mediated by LPS-induced degradation of IB, has been described previously by Zhong et al. (18). However, except for bacterially derived LPS, no physiological ligand has been reported to activate PKA by this mechanism. The present work demonstrates for the first time that the physiologically relevant hormone ET1, which is central to cardiovascular, renal, and pulmonary physiology, also stimulates PKA in an IB-dependent manner (Fig. 4). This suggests that this mechanism for cAMP-independent Activation of PKA by Vasoactive Peptides 20829 PKA activation is more widespread and might also be relevant to other G protein-coupled receptors. Several important questions are still to be resolved, such as the signaling pathways, which link ET A receptors to the degradation of IB and activation of PKA, as well as the functional significance of ET1-induced PKA activation. IB degradation can be mediated by a variety of mechanisms, including protein kinase C (27,28), mitogen-activated protein kinase (29), or Akt/protein kinase B (21). ET A receptors can activate all abovementioned molecules (30 -32), suggesting several possibilities for the signaling cascades leading to ET1-induced activation of PKA. Regarding the functional significance of ET1-induced PKA activation, stimulation of PKA by isoproterenol or forskolin was shown to inhibit agonist-induced activation of phospholipase C (33), Ca 2ϩ mobilization (34), and Ca 2ϩ entry (22), as well as MAP kinase cascade (7,35), the signaling pathways commonly stimulated by G protein-coupled receptors including ET A . Moreover, it is generally accepted that activation of PKA leads to cell relaxation and regulation of cell growth (36,37), which is opposite of vasoconstrictive and proliferative effects of ET1. This suggests that activation of PKA may serve as a regulatory mechanism in the function of ET1. Future studies will address these issues.