Coordinated Control of Endothelial Nitric-oxide Synthase Phosphorylation by Protein Kinase C and the cAMP-dependent Protein Kinase*

Endothelial nitric-oxide synthase (eNOS) is an important regulatory enzyme in the cardiovascular system catalyzing the production of NO from arginine. Multiple protein kinases including Akt/PKB, cAMP-dependent protein kinase (PKA), and the AMP-activated protein kinase (AMPK) activate eNOS by phosphorylating Ser-1177 in response to various stimuli. During VEGF signaling in endothelial cells, there is a transient increase in Ser-1177 phosphorylation coupled with a decrease in Thr-495 phosphorylation that reverses over 10 min. PKC signaling in endothelial cells inhibits eNOS activity by phosphorylating Thr-495 and dephosphorylating Ser-1177 whereas PKA signaling acts in reverse by increasing phosphorylation of Ser-1177 and dephosphorylation of Thr-495 to activate eNOS. Both phosphatases PP1 and PP2A are associated with eNOS. PP1 is responsible for dephosphorylation of Thr-495 based on its specificity for this site in both eNOS and the corresponding synthetic phosphopeptide whereas PP2A is responsible for dephosphorylation of Ser-1177. Treatment of endothelial cells with calyculin selectively blocks PKA-mediated dephosphorylation of Thr-495 whereas okadaic acid selectively blocks PKC-mediated dephosphorylation of Ser-1177. These results show that regulation of eNOS activity involves coordinated signaling through Ser-1177 and Thr-495 by multiple protein kinases and phosphatases.

Protein kinases involved in the regulation of endothelial NO production and eNOS activity include AMPK, 1 PKA, PKB/Akt, PKC, and the calmodulin-dependent kinase II. Initially AMPK was shown to mediate ischemia-induced activation of eNOS (1), but multiple stimuli including vascular endothelial growth factor (VEGF) (2,3), insulin-like growth factor-1 (IGF-1) (2), estrogen (4,5), and fluid shear stress (6,7) signal through Akt/ PKB kinase to activate eNOS by Ser-1177 phosphorylation. Other vasoactive substances that elevate intracellular calcium (Ca 2ϩ ) also regulate eNOS activity through Ca 2ϩ -calmodulin (CaM) binding (8). In addition to activating Akt/PKB, VEGF also activates PKC in endothelial cells (9). Activation of both PLC and PLD by VEGF is accompanied by an early influx of Ca 2ϩ , which is inhibited by reduced extracellular Ca 2ϩ , PKC inhibitors, and tyrosine kinase inhibitors (10). Previously we found phosphorylation of Thr-495 by AMPK in vitro attenuated eNOS activity (1) and recently reported that bradykinin activates eNOS in endothelial cells by triggering dephosphorylation at this site (11). Endothelial cell NOS activity is inhibited following phorbol 12,13-dibutyrate treatment (12,13). In the present study we show PKC signaling causes eNOS phosphorylation at Thr-495 as well as promoting dephosphorylation of Ser-1177. In contrast, PKA signaling results in phosphorylation of Ser-1177 and dephosphorylation of Thr-495 in endothelial cells. The dephosphorylation events are catalyzed by phosphatases PP1 and PP2A acting selectively on these two sites.

EXPERIMENTAL PROCEDURES
Cell Culture and NOS Activity Assay-Bovine aortic endothelial cells (BAEC) and human umbilical vein endothelial cells (HUVEC) were serum-starved in 0.1% fetal bovine serum in Dulbecco's modified Eagle's medium for 20 h or M199 for 8 h, respectively, prior to addition of activators or inhibitors. Cells were harvested in lysis buffer (50 mM Hepes, pH 7.5, 2 mM EDTA, 50 mM NaF, 5 mM Na 4 P 2 O 7 , 1 mM dithiothreitol, 1% Nonidet P-40, 10 g/ml trypsin inhibitor, 10 g/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride), and eNOS was purified by ADP-Sepharose chromatography (2). The purification of eNOS, activity assays (1,14), SDS-polyacrylamide gel electrophoresis and Western blot analysis were as described previously (2). To investigate the changes in eNOS activity with phosphorylation, EGTA buffering was used to shift the CaM dose response range from 0 -100 nM to 0 -500 nM (1).
Antibodies and Western Blotting-Polyclonal antibodies raised against synthetic phosphopeptides to the eNOS phosphorylation sites, Ser-1177 and Thr-495, were used to detect eNOS phosphorylation by Western blotting as described previously (1). Blots were probed with the anti-phospho-Ser-1177 and anti-phospho-Thr-495 antibodies and were stripped and re-probed for total eNOS (Transduction Laboratories) and quantitated using a scanning densitometer (Molecular Dynamics). PP1 and PP2A were detected in Western blots and immunoprecipitated from BAEC using antibodies against the catalytic subunits (15).
* This research was supported by grants from the NHMRC Australia, National Heart Foundation, and Diabetes Australia. 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.

VEGF and PKA Signaling in Endothelial Cells-Both VEGF
and IGF-1 stimulate Akt/PKB kinase in BAEC to phosphorylate and activate eNOS (2). In HUVEC but not in BAEC, we observed that VEGF stimulation led to a transient increase in Ser-1177 phosphorylation that was accompanied by a decrease in Thr-495 phosphorylation (Fig. 1A). Human eNOS Ser-1177 and Thr-495 correspond to bovine eNOS Ser-1179 and Thr-497, respectively. Treatment of BAEC with the phosphodiesterase inhibitor IBMX (Fig. 1B) or forskolin, but not 8-bromo-cGMP (data not shown), caused dephosphorylation of Thr-497 and enhanced phosphorylation of Ser-1179 resulting in increased eNOS activity (Fig. 1C). The PKA-stimulated phosphorylation/ dephosphorylation is maintained for at least 30 min (longest period tested) whereas the VEGF-stimulated phosphorylation/ dephosphorylation is transient and reverses within 10 min. Thus, signaling through either the VEGF receptor or via PKA activates eNOS by the coordinated phosphorylation of Ser-1179 and dephosphorylation of Thr-497.
PKC Signaling in Endothelial Cells-PMA treatment of BAEC increased phosphorylation of Thr-497 and decreased Ser-1179 phosphorylation ( Fig. 2A), inhibiting eNOS activity. The partially specific PKC inhibitor Ro-318220 (17) enhanced Ser-1179 phosphorylation and suppressed Thr-497 phosphorylation while the inactive isomer Ro-310645 did not (Fig. 2B), consistent with PKC involvement. Further, chronic PMA treatment of BAEC decreases Thr-497 phosphorylation and increased Ser-1179 phosphorylation (results not shown), consistent with the down-regulation of expression of the PMA-responsive PKC isoforms (18).
HUVEC were treated with VEGF over a time course of 0, 2, 10, and 30 min with either the PKC inhibitor Ro-318220 or the inactive isomer Ro-310645. Inhibition of PKC prolonged VEGF-induced stimulation of Ser-1177 phosphorylation consistent with inhibition of a PKC-dependent phosphatase responsible for dephosphorylation of Ser-1177. The VEGF-induced dephosphorylation of Thr-495 was also prolonged by inhibition of PKC providing further evidence that PKC phosphorylates this site (Fig. 2C). Thus, signaling through PKC inhibits eNOS activity by phosphorylation of Thr-497 and dephosphorylation of Ser-1179. In contrast to the results obtained in endothelial cells, PKC phosphorylates both Thr-497 and Ser-1179 in vitro. The site phosphorylated depends on the presence of calmodulin, with Thr-497 phosphorylated in the presence of EGTA and Ser-1179 in the presence of Ca 2ϩ /CaM (Fig. 2D). Phosphoryla- tion of Thr-497 by PKC is associated with inhibition of eNOS activity as was found for the AMPK (1). Other than Thr-497 and Ser-1179, PKC did not phosphorylate other sites on eNOS to a significant extent in vitro (Fig. 2D).
PP1 and PP2A Dephosphorylation of eNOS-Both phosphatases PP1 and PP2A are associated with affinity-purified eNOS. However, there is no detectable change in their association with phosphorylation of eNOS when endothelial cells were treated with either IBMX or PMA (Fig. 3A). We therefore investigated whether these phosphatases preferably dephosphorylated either Thr-497 or Ser-1179. Recombinant eNOS phosphorylated predominantly at either Thr-497 or Ser-1179 was incubated with immunoprecipitates of PP1 and PP2A (Fig.  3B). The Thr-497 site was preferentially dephosphorylated by PP1, whereas Ser-1179 was preferentially dephosphorylated by PP2A. PP1 dephosphorylated the Thr-497 site by more than 80% whereas PP2A caused less than 40% dephosphorylation. In contrast, PP1 dephosphorylated the Ser-1179 site by ϳ30% whereas PP2A caused more than 70% dephosphorylation.
Synthetic phosphopeptides corresponding to the two phosphorylation sites were also tested as substrates using a MALDI-TOF mass spectrometry assay. The Thr-495 phosphopeptide was dephosphorylated by both phosphatases but more rapidly with PP1 than PP2A (Fig. 3, C and D). The Ser-1177 phosphopeptide was readily dephosphorylated by immunoprecipitates of PP2A but not PP1 (Fig. 3, E and F). The results show that PP1 and PP2A have distinct specificities with PP1 primarily responsible for Thr-495 dephosphorylation and PP2A for Ser-1177 dephosphorylation.
Selective Inhibition of Thr-497 and Ser-1179 Dephosphorylation by Calyculin and Okadaic Acid-Treatment of BAEC with okadaic acid alone increased Ser-1179 phosphorylation ϳ2-fold (Fig. 4, A and B) and calyculin alone increased Thr-497 phosphorylation 2.5-fold (Fig. 4, C and D). Calyculin A is reported to inhibit PP1 more selectively than PP2A, whereas okadaic acid inhibits PP2A at concentrations up to 1 M without inhibiting PP1 (19,20). The selective inhibition of the dephosphorylation of the two sites by okadaic acid and calyculin indicates that PP1 is responsible for dephosphorylation of Thr-497 and PP2A for dephosphorylation of Ser-1179 in full agreement with the specificity of these phosphatases for the respective sites in vitro.
Treatment with okadaic acid blocked the PMA-induced dephosphorylation of Ser-1179 (Fig. 4A) but not the effects of IBMX on Thr-497 phosphorylation even at concentrations up to 500 nM (Fig. 4B) consistent with PP2A dephosphorylating Ser-1179. In contrast, calyculin did not block the dephosphorylation of Ser-1179 (Fig. 4, C and D) but did block the IBMX-induced dephosphorylation of Thr-497 supporting the idea that PP1 dephosphorylates Thr-497. PMA-induced phosphorylation of Thr-497 is enhanced by calyculin and okadaic acid. The inhibition of PP1 by calyculin alone causes an increase in Thr-497 phosphorylation and enhanced the PMA effect on Thr-497 phosphorylation (Fig. 4C). Whereas, okadaic acid alone caused a slight reduction in Thr-497 phosphorylation (Fig. 4A) it enhanced the PMA-induced phosphorylation of Thr-497. These results demonstrate that the two phosphatase inhibitors have distinct inhibition patterns for the dephosphorylation of Thr-497 (calyculin) and Ser-1179 (okadaic acid) sites of eNOS.
Treatment of cells with okadaic acid elevated eNOS activity (Fig. 4E) in parallel with the increased phosphorylation of Ser-1179 and reduced Thr-497 phosphorylation (Fig. 4, A and  B). In contrast, PMA reduced eNOS activity in endothelial cells (Fig. 4E) in parallel with a 3-fold increase in Thr-497 phosphorylation and a 4-fold decrease in Ser-1179 phosphorylation (Fig. 4, A and C). DISCUSSION The regulation of eNOS activity by phosphorylation at Ser-1177 and Thr-495 is relatively complex involving at least four protein kinases (Akt, PKA, PKC, and AMPK) and two phosphatases (PP1 and PP2A). Previous studies have shown that Ser-1177 phosphorylation activates eNOS (1-3, 6, 7) whereas Thr-495 phosphorylation inhibits activity as a consequence of this site being present in the CaM binding sequence (1). During signaling events that promote phosphorylation at either of these sites, there is coordinated dephosphorylation at the alternate site. In this way the inhibition of eNOS resulting from PKC phosphorylation of Thr-495 is amplified by the simultaneous dephosphorylation of Ser-1177. Similarly, activation of eNOS in response to PKA signaling involves phosphorylation of Ser-1177 as well as dephosphorylation of Thr-495 (Fig. 5). At present it is not clear how signaling through PKA and PKC causes selective dephosphorylation of eNOS by PP1 and PP2A, respectively. Phosphorylation at one site may not be the trigger for dephosphorylation at the second site because in vitro one or other site is selectively phosphorylated rather than both suggesting that dephosphorylation of one precedes phosphorylation of the other. The dephosphorylation and phosphorylation reactions at the two sites appear independently coordinated.
Because PKA signaling activates PP1 to dephosphorylate Thr-495, one potential mechanism may involve the inactivation of a phosphatase inhibitor analogous to NIPP-1 the nuclearlocalized PP1 inhibitor that is inactivated by PKA phosphorylation (21). Other phosphatase inhibitors are activated by phosphorylation (inhibitor-1 and CPI-17 activated by PKA and PKC phosphorylation respectively, reviewed in Ref. 22). We have not detected PKA or PKC substrates in immunoprecipitates of PP1 or PP2A that could act as phosphatase inhibitors. Cyclosporin A blocks the dephosphorylation of eNOS at Thr-497 in response to bradykinin in early passage (2-6) BAEC as well as NO production (11). However, the dephosphorylation of Thr-497 triggered by PKA signaling observed here was unaffected by preincubation with the calcineurin inhibitor FK506 (1 M).
VEGF stimulates at least two protein kinases (Akt and PKC) that ensure the tight control of eNOS activation. Signaling through PKC attenuates VEGF-induced stimulation of Ser-1177 phosphorylation by Akt. The PKC-stimulated dephosphorylation of Ser-1177 by PP2A occurs simultaneously with enhanced phosphorylation of Thr-495 and inhibits eNOS activity. In contrast, PKA directly phosphorylates Ser-1179 and stimu-lates the PP1-dependent dephosphorylation of Thr-497, activating eNOS (Fig. 5). Several other examples of PKC-stimulated dephosphorylation have been reported including dephosphorylation of the cadherin-associated proteins P120 and p100 in epithelial and endothelial cells (24,25) and in the attenuation of the signaling of activated guanylyl cyclaselinked natriuretic peptide receptors, GC-A and -B where PP2A may also be involved (23).
The inhibition of eNOS following activation of PKC by VEGF or phorbol esters illustrates that signaling through PKC can suppress NO production from eNOS. These results add a new dimension to our understanding of the complexities of eNOS regulation (26). Given, that NO plays such a diverse role in the cardiovascular system, it raises the possibility that one of the actions of PKC inhibitors in suppressing the vascular complications of diabetes (27) may be mediated in part by blocking PKC inhibitory signaling to eNOS.