Cyclin-Dependent Kinase 5 Phosphorylates Endothelial Nitric Oxide Synthase at Serine 116
Nitric oxide (NO) production in endothelial cells (EC) is regulated by multisite phosphorylation of specific serine and threonine residues in endothelial NO synthase (eNOS). Among these, eNOS-Ser116 is phosphorylated in the basal state, and its phosphorylation contributes to basal NO production. Here, we investigated the mechanism by which eNOS-Ser116 is phosphorylated during the basal state using bovine aortic EC. Although a previous study suggested that protein kinase C was involved in eNOS-Ser116 phosphorylation, overexpression of various protein kinase C isoforms did not affect eNOS-Ser116 phosphorylation. An in silico analysis using a motif scan revealed that the eNOS-Ser116 residue might be a substrate for proline-directed protein kinases. Roscovitine, a specific inhibitor of cyclin-dependent kinase (CDK), 1, 2, and 5, but not an inhibitor of mitogen-activated protein kinase kinase or glycogen synthase kinase 3β, inhibited eNOS-Ser116 phosphorylation dose dependently. Furthermore, purified CDK1, 2, or 5 directly phosphorylated eNOS-Ser116 in vitro. Ectopic expression of the dominant-negative CDK5 but not dominant-negative CDK1 or dominant-negative CDK2 repressed eNOS-Ser116 phosphorylation and increased NO production. In addition, CDK5 activity was detected in bovine aortic EC, and coimmunoprecipitation and confocal microscopy studies revealed a colocalization of eNOS and CDK5. Cotransfection of CDK5 and p25, the specific CDK5 activator, increased eNOS-Ser116 phosphorylation and decreased NO production, but its parent molecule, p35, and p39, another activator, were not detected in bovine aortic EC, which suggests the existence of a novel CDK5 activator. Overall, this is the first study to find that CDK5 is a physiological kinase responsible for eNOS-Ser116 phosphorylation and regulation of NO production.
- nitric oxide
- endothelial nitric oxide synthase
- cyclin-dependent kinase 5
- signal transduction
Endothelial nitric oxide synthase (eNOS) is an essential enzyme responsible for the production of endothelium-derived nitric oxide (NO), which is a key molecule with multiple functions, including vascular homeostasis, angiogenesis, and cell cycle regulation.1,2 The dysregulation of eNOS, therefore, contributes to the pathogenesis of certain diseases, such as atherosclerosis, hypertension, and cancer.2 It is well known that eNOS is regulated at the level of its phosphorylation.3,4 Several specific sites of phosphorylation have been identified, among which eNOS-Ser1179 (bovine sequence) and eNOS-Thr497 have been the most thoroughly evaluated. The phosphorylation of eNOS-Ser1179 increases NO production,5–7 which is mediated by several specific protein kinases, including Akt, AMP-activated protein kinase, calmodulin-dependent kinase II, and protein kinase A (PKA).8–11 Conversely, the phosphorylation of eNOS-Thr497 decreases eNOS activity,8,10 which is mediated by AMP-activated protein kinase5 and protein kinase C (PKC).10,12 This site is also dephosphorylated by phosphatases, such as protein phosphatase 1 and protein phosphatase 2B, which results in an increase in NO production.3,10 Two other sites, eNOS-Ser635 and eNOS-Ser617, have also been identified as phosphorylation targets of PKA and Akt, respectively.13 A recent study14 showed that AMP-activated protein kinase also phosphorylates eNOS-Ser635. Although the phosphorylation of eNOS-Ser635 serves as a positive regulator of NO production,15 the phosphorylation of eNOS-Ser617 does not exert a regulatory role on NO production itself.13
Phosphorylation of eNOS-Ser116 has also been documented. Although the exact function of eNOS-Ser116 phosphorylation is still the subject of debate, we demonstrated that its dephosphorylation was associated with eNOS activation and NO production by the oral antidiabetic drug troglitazone.16 Consistent with these findings, it was found that eNOS-Ser116, like eNOS-Thr497, was phosphorylated in cells in the basal state and that dephosphorylation by protein phosphatase 2B or a serine to alanine mutation mimicking dephosphorylation increased eNOS activity.17 It was also demonstrated that the PKC inhibitor calphostin C reduced phosphorylation of eNOS-Ser116, which suggests that phosphorylation of eNOS-Ser116 would be mediated by PKC. However, because calphostin C does not only inhibit PKC, PKC-mediated eNOS-Ser116 phosphorylation has not been clearly demonstrated. Therefore, studies evaluating the effects of wild-type or dominant-negative (DN) PKC constructs are warranted to provide direct evidence of the involvement of PKC in eNOS-Ser116 phosphorylation. In this study, we demonstrate for the first time that cyclin-dependent kinase (CDK) 5 is a physiological kinase responsible for eNOS-Ser116 phosphorylation and NO production in the basal state.
An expanded Materials and Methods section is available in the online Data Supplement at http://hyper.ahajournals.org.
PKC inhibitors (Go6976 and Ro318220); mitogen-activated protein kinase kinase inhibitors (PD98059 and U0126); a glycogen synthase kinase 3β inhibitor (LiCl); a CDK1, 2, and 5 inhibitor (roscovitine); a proteasome inhibitor (MG132); and recombinant bovine eNOS were purchased from Calbiochem (Darmstadt, Germany). Another glycogen synthase kinase 3β inhibitor (SB216763) was purchased from BIOMOL (Plymouth Meeting, PA). CDK1/cyclin B; CDK2/cyclin A; CDK5/p25; CDK5/p35; and antibodies against CDK1, CDK2, CDK5, and p35 were obtained from Cell Signaling Technology (Beverly, MA). Antibodies against eNOS and p-eNOS-Ser116 were purchased from Transduction Laboratories (Lexington, KY) and Upstate Biotechnology Inc. (Lake Placid, NY), respectively. Antibody against hemagglutinin was obtained from Covance (Berkeley, CA). Minimal essential medium, Dulbecco’s PBS, newborn calf serum, penicillin and streptomycin, l-glutamine, trypsin–EDTA solution, and plasticware used for cell culture were purchased from Gibco-BRL (Gaithersburg, MD). All other chemicals were of the purest analytic grade.
Cell Culture, Drug Treatment, and Transfection
Bovine aortic EC (BAEC) were isolated and cultured as described,18 and they were transfected with cDNAs encoding the wild-type or DN genes using Lipofectin reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Protocols for culture of BAEC and other cells, drug treatment, and transfection are described in detail in the online Data Supplement.
Western Blot Analysis
Western blot analysis is described in detail in the online Data Supplement.
In Silico Analysis of the eNOS Protein Sequence
The eNOS protein sequence was obtained from SWISS-PROT (locus NOS3_BOVIN, accession no. P29473), and its sequence motifs most likely to be phosphorylated by specific protein kinases were identified using the motif scan program Scansite, available at http://scansite.mit.edu.
In Vitro Phosphorylation Assay
The in vitro CDK phosphorylation assay was performed as described,19 with minor modifications, and is described in detail in the online Data Supplement.
In Vitro CDK5 Activity Assay
The in vitro CDK5 activity assay was performed as described,19 with minor modifications, and is described in detail in the online Data Supplement.
The coimmunoprecipitation assay was carried out as described,19 with minor modifications, and is described in detail in the online Data Supplement.
BAEC grown on coverslips were fixed with 4% (wt/vol) paraformaldehyde in Dulbecco’s PBS, followed by a 10-minute permeabilization in 0.2% (vol/vol) Triton X-100 in Dulbecco’s PBS at 25°C. After permeabilization, the cells were blocked in 5% goat serum in Dulbecco’s PBS for 30 minutes. The presence of eNOS and CDK5 was detected by appropriate dilutions of the primary antibodies (anti-eNOS, 1:200; anti-CDK5, 1:200) and with a 1:200 dilution of Alexa Fluor 488- or 594-conjugated secondary antibody (Invitrogen). Images were photographed using a confocal microscope (Radiance 2000, Bio-Rad, Hercules, CA).
Cloning of Bovine p25
Polymerase chain reaction was conducted using bovine p25 forward primer, bovine p25 reverse primer, and cDNA from BAEC as a template. The polymerase chain reaction products were subcloned into the EcoRI-XhoI site of the pcDNA3.1 vector (Invitrogen). TaKaRa Ex Taq HS polymerase (Takara Bio Inc., Shiga, Japan) was used for all polymerase chain reactions. DNA sequence analyses were conducted by Solgent Co. Ltd. (Daejeon, South Korea). The following primers were used for cloning: Bovine p25 forward primer, 5-AAATTTGAATTCATGGCCCAGCCCCCGCCG-3; Bovine p25 reverse primer, 5-ATATCTCGAGTCACCGGTCCAGCCCGAGGA-3.
Measurements of the level of CDK5, p35, and p39 mRNAs are described in detail in the online Data Supplement.
Measurement of NO Release
NO production by transfected BAEC was measured as nitrite (stable metabolite of NO) concentration in cell culture supernatants, as described,20 and is described in detail in the online Data Supplement.
All results are expressed as mean±SD, with n indicating the number of experiments. Statistical significance was determined by a Student t test for 2 points. All differences were considered significant at P<0.05.
PKC Does Not Mediate eNOS-Ser116 Phosphorylation
Because a previous study showed that a PKC inhibitor, calphostin C, inhibited eNOS-Ser116 phosphorylation in basal and vascular endothelial growth factor–treated cells,17 we examined PKC to determine whether it is involved in eNOS-Ser116 phosphorylation. Experiments evaluating the effects of the other PKC-specific inhibitors, Go6976 and Ro318220, did not lead to changes in eNOS-Ser116 phosphorylation (see Figure S1 in the online Data Supplement). To further clarify our data, we transfected various hemagglutinin-tagged PKC isoforms, α, βI, βII, δ, ε, and ζ, into BAEC. Consistent with the results from PKC inhibitor experiment, overexpression of the PKC genes did not alter the status of eNOS-Ser116 phosphorylation in BAEC (Figure 1), which suggests that PKCs are not involved in the phosphorylation of eNOS-Ser116.
Roscovitine Represses eNOS-Ser116 Phosphorylation, and CDK1, CDK2, or CDK5 Directly Phosphorylates eNOS-Ser116 In Vitro
Next, we attempted to identify other protein kinase(s) that might be involved in eNOS-Ser116 phosphorylation. In silico analysis using the motif scan program (Scansite, http://scansite.mit.edu) revealed a PXSP/PSP motif around the eNOS-Ser116 sequence (see the amino acid sequences of eNOS from 101 to 130: 101-lgslvlprk lqtrpSpgpp paeqllsqar-130), which represents a putative substrate sequence for CMGC kinases, such as CDK, mitogen-activated protein kinase, glycogen synthase kinase, and CDK-like kinase. Treatment with U0126 and PD98059, which are specific inhibitors of mitogen-activated protein kinase kinase, had no effect on the phosphorylation of eNOS-Ser116 (Figure S2). Furthermore, treatment with the glycogen synthase kinase 3β inhibitors SB216763 and LiCl also had no effect on eNOS-Ser116 phosphorylation (Figure S2). However, we found that roscovitine, which is a specific inhibitor of CDK1, 2, and 5, dramatically decreased eNOS-Ser116 phosphorylation in a dose-dependent manner (Figure 2A). To further confirm whether CDK1, 2, or 5 directly phosphorylates eNOS-Ser116, we performed an in vitro phosphorylation assay. Compared with the control, a purified CDK1/cyclin B, CDK 2/cyclinA, or CDK5/p25 complex significantly phosphorylated eNOS-Ser116 (Figure 2B). Furthermore, this phosphorylation was almost completely blocked by roscovitine, which suggests that the eNOS-Ser116 phosphorylation is specifically mediated by CDK1, 2, or 5. Although the CDK5/p25 complex phosphorylated eNOS-Ser116, we failed to find that the CDK5/p35 complex phosphorylated it (Figure S3). These results suggest that CDKs are involved in eNOS-Ser116 phosphorylation in the basal state and that a CDK5 activator plays a critical role in determining the substrate specificity.
Ectopic Expression of DN-CDK5 Inhibits eNOS-Ser116 Phosphorylation and Increases NO Production in BAEC
We next determined which CDK isoform was a kinase responsible for phosphorylation of eNOS-Ser116 in cells. As shown in Figure 3A, ectopic expression of DN-CDK5 alone (but not of DN-CDK1 or DN-CDK2) dramatically repressed eNOS-Ser116 phosphorylation. Furthermore, ectopic expression of DN-CDK5 significantly increased basal NO production in BAEC (Figure 3B).
CDK5 Is Enzymatically Active and Interacts with eNOS in Basal BAEC
In basal BAEC, we clearly detected CDK5 activity (Figure 4A). Furthermore, CDK5 was coimmunoprecipitated with eNOS, suggesting that there was a physical interaction between CDK5 and eNOS in cells (Figure 4B). Confocal microscopy also showed colocalization of eNOS and CDK5 in the perinuclear region (Figure 4C). Taken together, these results showed that CDK5 is a physiological kinase that mediates eNOS-Ser116 phosphorylation within the cell.
Overexpression of both CDK5 and p25 Increases eNOS-Ser116 Phosphorylation and Decreases NO Production
In an attempt to examine the regulatory mechanism by which CDK5 induces eNOS-Ser116 phosphorylation in cells, we transfected either CDK5, p25, or both constructs in BAEC. We found that overexpression of CDK5 alone had no effect on eNOS-Ser116 phosphorylation (Figure 5A). However, transfection with p25 alone led to a significant increase in eNOS-Ser116 phosphorylation, which increased further when both CDK5 and p25 were cotransfected in the cells. Furthermore, a significant decrease of NO release (to 72.5±7.4% of the control) was also observed when BAEC were transfected with both CDK5 and p25 (Figure 5B), suggesting a physiological role for CDK5 activity in NO production.
p25 or p35 Protein Is Not Detected in BAEC
Because it has been well established that p35 and p39 are CDK5 activators in neurons, we evaluated these activators to determine whether they are also expressed and play a role in regulation of CDK5 activity in BAEC. RT-PCR clearly demonstrated that p35 mRNA was expressed in several types of EC, including BAEC (Figure 6A). However, the use of commercially available anti-p35 antibody, which is also known to recognize the proteolytic peptide p25, did not detect p35 or p25 protein expression in any of the EC tested, whereas it was clearly expressed in neuronal cells (Figure 6B). These results suggest that posttranscriptional or posttranslational regulation of neuronal p35 occurs in EC. Furthermore, another neuronal CDK5 activator, p39, was not detected in EC, even at the mRNA level (Figure 6A).
Previously, we reported that the PPARγ ligand, troglitazone, decreased eNOS-Ser116 phosphorylation in a PPARγ-independent manner, which led to increased NO production.16 However, to date, no kinases responsible for eNOS-Ser116 phosphorylation have been identified. This study clearly shows that CDK5 phosphorylates eNOS-Ser116 in BAEC that are in the basal state. We believe that this provides the first solid evidence that CDK5 is a physiological kinase that mediates eNOS-Ser116 phosphorylation and that the modulation of CDK5 activity may play an important role in regulating basal NO production in EC.
There has been only 1 study conducted to date that has demonstrated that pretreatment with the PKC inhibitor calphostin C represses basal eNOS-Ser116 phosphorylation.17 However, the authors in this previous study did not provide direct evidence that PKC itself was indeed a kinase responsible for eNOS-Ser116 phosphorylation. Calphostin C has a rather broad ability to inhibit kinases in addition to PKC, such as myosin light chain kinase, PKA, and protein kinase G; therefore, it is not surprising that calphostin C inhibited protein kinase activities other than PKC under the experimental conditions in the previously conducted study. On the basis of this notion, we evaluated various isoforms of PKCs to determine whether they directly phosphorylated eNOS-Ser116 and which isoforms, if any, were responsible for the phosphorylation. Evaluation of the effects of more specific inhibitors (Figure S1) and isoform-specific PKC gene transfection (Figure 1) revealed that the PKC isoforms did not play a significant role in eNOS-Ser116 phosphorylation. These findings indicate that PKCs are not kinases responsible for eNOS-Ser116 phosphorylation. Furthermore, in silico analysis using the motif scan program (Scansite) available at http://scansite.mit.edu revealed that the eNOS-Ser116 site was a putative substrate for the CMGC kinase superfamily, but not for the AGC kinase superfamily, such as PKA, protein kinase G, and PKC.21
Evaluation of several members of the CMGC kinase superfamily revealed that CDK5 was a physiological kinase capable of phosphorylation of the eNOS-Ser116 in basal EC. Unlike other types of CDKs, CDK5 exhibits functions that are not involved in the cell cycle.22 CDK5 activity is critical for neuronal functions such as axonal guidance, neuronal migration, membrane transport, dopamine signaling, and cytoskeletal dynamics.23 Recently, several studies have shown that CDK5 plays an important role in the exocytosis, differentiation, and senescence of nonneuronal cells such as pancreatic β cells,24,25 monocytes,26 muscle cells,27 and testis.28 For example, CDK5 regulates glucose-induced insulin exocytosis by phosphorylating Munc1825 or the L-type voltage-dependent calcium channel24 in pancreatic β cells. In muscle cells, CDK5 phosphorylates nestin, the intermediate filament protein, which plays an important role in muscle development and regeneration.27 In EC, there has been only 1 report demonstrating that roscovitine, a selective inhibitor of CDK5, inhibited cell proliferation and induced apoptosis by blocking CDK5 expression, suggesting that CDK5 plays a role in the regulation of apoptosis.29 Although the molecular mechanism by which CDK5 mediates the growth inhibition of EC has not been fully defined, it is likely that it can be attributed to CDK5-mediated phosphorylation in the substrate(s) responsible for EC proliferation and apoptosis. In this regard, the present study shows that eNOS-Ser116 is a physiological substrate in nonneuronal EC. Because NO plays an important role in cell proliferation and apoptosis,30 the results of this study suggest that CDK5 is involved in the proliferation of EC through the suppression of NO production in the basal state. However, further study is needed to elucidate the relationship between increased eNOS-Ser116 phosphorylation, reduced NO production, and EC proliferation.
It is well known that the CDK5 monomer has no kinase activity and that its binding with specific activators is necessary and sufficient for the maximal enzyme activity to occur. We found that there is CDK5 activity in EC (Figure 4A) but that ectopic expression of wild-type CDK5 did not further increase eNOS-Ser116 phosphorylation (Figure 5A). These results suggest that the level of CDK5 activator limits the kinase function of CDK5 on eNOS-Ser116 in basal EC. Although overexpression of p25 alone or of both CDK5 and p25 led to a significant increase in eNOS-Ser116 phosphorylation (Figure 5A), the neuronal p35 protein, a parent molecule of p25, was not expressed (Figure 6). Nevertheless, the mRNA of p35 was clearly detected in EC. These results suggest that posttranscriptional or posttranslational regulation of p35, such as mRNA stability, translational efficacy, stability of the protein product, or other posttranslational modifications, occurred. Because it was previously reported that p35 and p25, as well as cyclins and other CDK activators, underwent ubiquitination–proteasome degradation,31 we evaluated the effects of treatment with 10 μmol/L of MG132, a proteasome inhibitor, for 6 hours on the protein expression of p35 in BAEC. However, we were still unable to detect p35 protein in MG132-treated EC (Figure S4), which suggests that the absence of p35 protein in BAEC does not result from protein degradation via the ubiquitin-proteasome pathway. Furthermore, another neuronal CDK5 activator, p39, was not found in EC at the mRNA level, which suggests that neither p39 nor its metabolite, p29, is involved in regulation of CDK5 in these cells. Taken together, these results suggest that there is an unknown CDK5 activator(s) in EC other than the previously established neuronal p35 or p39. In this regard, a novel gene named IC53, as an isoform of the C53 gene, which encodes a CDK5-binding protein, was cloned32 and was later found to be expressed mainly in EC.33 Most recently, Zhuo et al reported a significantly decreased eNOS activity in aorta and serum NO production from transgenic mice in which IC53 was specifically overexpressed in EC.34 In the same study, they also found that knockdown of IC53 mRNA by small hairpin RNA in human umbilical vein endothelial cells increased eNOS activity by 126.3% without an alteration of eNOS mRNA level. Taken together, it is likely that the IC53 protein may be a potential candidate for the CDK activator responsible for eNOS-Ser116 phosphorylation. However, further study is needed to clarify this issue.
Considering that eNOS is a physiological substrate of CDK5 in EC, it is likely that the 2 proteins interact with each other in cells at certain levels. In this regard, unlike the nuclear localization of other CDK isoforms, such as CDK1 and 2, CDK5 has been detected in the perinuclear region of neuronal cells.35–37 Furthermore, some studies have shown that eNOS is localized in the perinuclear region of cultured EC and intact human blood vessels,38,39 which indicates that it may be cell cycle–dependent (or cell density–dependent). In the present study, abundant eNOS was found in the perinuclear regions in BAEC (Figure 4C). Moreover, eNOS was clearly colocalized with CDK5. These findings, together with the coimmunoprecipitation of CDK5 with eNOS, further demonstrate that eNOS is a physiological substrate of CDK5.
This study is the first to show that multifunctional CDK5 directly phosphorylates eNOS-Ser116. Previous studies have shown that CDK5 plays a pivotal role in regulation of cortical differentiation, Alzheimer’s disease, and cellular motility in the brain.40 However, there are few data available regarding nonneuronal CDK5 functions. On the basis of the results of the present study, CDK5 is likely to be registered as an eNOS-Ser116 kinase in EC. Because phosphorylation at eNOS-Ser116 reduces NO production, our data provide the molecular mechanism through which NO is maintained at minimal levels in EC in the basal state, and therefore CDK5 may contribute to the pathogenesis of diseases associated with dysregulation of NO release in EC. Furthermore, our study also suggests the existence of novel CDK5 activator(s) other than neuronal p35 or p39. However, further study is needed to clarify this issue.
Sources of Funding
This work was supported primarily by a Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2008-331-E00053).
D.-H.C. and J.S. contributed equally to this study.
- Received August 3, 2009.
- Revision received August 21, 2009.
- Accepted December 5, 2009.
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