Published online before print August 28, 2008, doi: 10.1161/CIRCRESAHA.108.175877
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© 2008 American Heart Association, Inc.
Cellular Biology |
Protein Kinase B/Akt Phosphorylates and Inhibits the Cardiac Na+/H+ Exchanger NHE1
From the King’s College London British Heart Foundation Centre, Cardiovascular Division, The Rayne Institute, St. Thomas’ Hospital, London, UK.
Correspondence to Dr Andrew K. Snabaitis, Cardiovascular Division, King’s College London, The Rayne Institute, St. Thomas’ Hospital, London SE1 7EH, United Kingdom. E-mail andrew.snabaitis{at}kcl.ac.uk
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Abstract |
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Sarcolemmal Na+/H+ exchanger (NHE) activity is mediated by NHE isoform 1 (NHE1), which is subject to regulation by protein kinases. Our objectives were to determine whether NHE1 is phosphorylated by protein kinase B (PKB), identify any pertinent phosphorylation site(s), and delineate the functional consequences of such phosphorylation. Active PKB
phosphorylated in vitro a glutathione S-transferase (GST)-NHE1 fusion protein comprising amino acids 516 to 815 of the NHE1 carboxyl-terminal regulatory domain. PKB-mediated phosphorylation of GST-NHE1 fusion proteins containing overlapping segments of this region localized the targeted residues to the carboxyl-terminal 190 amino acids (625 to 815) of NHE1. Mass spectrometry and phosphorylation analysis of mutated (Ser
Ala) GST-NHE1 fusion proteins revealed that PKB-mediated phosphorylation of NHE1 occurred principally at Ser648. Far-Western assays demonstrated that PKB-mediated Ser648 phosphorylation abrogated calcium-activated calmodulin (CaM) binding to the regulatory domain of NHE1. In adult rat ventricular myocytes, adenovirus-mediated expression of myristoylated PKB (myr-PKB) increased cellular PKB activity, as confirmed by increased glycogen synthase kinase 3β phosphorylation. Heterologously expressed myr-PKB was present in the sarcolemma, colocalized with NHE1 at the intercalated disc regions, increased NHE1 phosphorylation, and reduced NHE1 activity following intracellular acidosis. Conversely, pharmacological inhibition of endogenous PKB increased NHE1 activity following intracellular acidosis. Our data suggest that NHE1 is a novel PKB substrate and that its PKB-mediated phosphorylation at Ser648 inhibits sarcolemmal NHE activity during intracellular acidosis, most likely by interfering with CaM binding and reducing affinity for intracellular H+.
Key Words: PKB • Akt • Na+/H+ exchanger • calmodulin • acidosis • phosphorylation
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Na+/H+ exchanger (NHE) isoform 1 (NHE1) is a membrane glycoprotein encoded by the NHE1/SLC9A1 gene1 and is expressed in all tissues. In cardiac myocytes, NHE1 protein contributes significantly to the control of intracellular pH (pHi), particularly in response to intracellular acidosis.2 Although basal activity of the sarcolemmal NHE is low under physiological conditions,2 increased exchanger activity may mediate inotropic responses to neurohormonal stimuli, such as endothelin-1,3 angiotensin II,4 and
1-adrenoceptor agonists,5 principally through NHE1-mediated increases in intracellular sodium.6,7 The regulation of NHE1 activity in such settings involves modification of the intracellular carboxyl-terminal regulatory domain of the exchanger, either by the binding of accessory proteins (such as calcineurin homologous protein,8 carbonic anhydrase II,9 and calmodulin [CaM]10) and/or by phosphorylation by protein kinases (such as the extracellular signal-regulated kinases [ERKs],11 p90 ribosomal S6 kinase [RSK],12,13 Rho-associated kinase [p160-ROCK)],14 and p38 mitogen-activated protein kinase [p38-MAPK]15). Although the majority of studies have linked protein kinase–mediated phosphorylation of the NHE1 carboxyl-terminal regulatory domain to the upregulation of NHE1 activity,11–15 there are reports that certain protein kinase pathways can inhibit NHE1.16,17 Furthermore, although the functionally important phosphorylation sites in NHE1 have been established for some kinases (eg, RSK12,18), they remain to be confirmed for many others.
Interestingly, the carboxyl-terminal regulatory domain of NHE1 contains 3 putative phosphorylation sites that conform to the optimal protein kinase B (PKB) target motif (RxRxxS/T19), which suggests a potential regulatory interaction between PKB and NHE1. In the heart, 3 isoforms of PKB (PKB/Akt1, PKBβ/Akt2, and PKB
/Akt3) are differentially expressed (=β>), and each constitutes a phosphoprotein of 
57 kDa that consists of an amino-terminal pleckstrin homology (PH) domain,20,21 a central kinase domain,22,23 and a carboxyl-terminal hydrophobic motif.24 The PH domain is crucial for the activation of PKB as it facilitates the phosphatidylinositol 3,4,5-triphosphate–dependent translocation of PKB to the inner surface of the cell membrane, where dual phosphorylation at Thr308 and Ser473 by phosphoinositide-dependent kinase 125 and the mTORC2 protein complex,25,26 respectively, achieves full activation. In the heart, active PKB plays a role in several physiological and pathological cellular processes. For example, increased myocardial PKB activity stimulates glucose uptake,27,28 regulates glycogen metabolism through the phosphorylation and inhibition of glycogen synthase kinase (GSK)3,29 and is regarded as central to the initiation of cellular survival pathways.27,30 PKB activity has also been implicated in the regulation of both exercise-induced physiological cardiac hypertrophy31 and maladaptive cardiac hypertrophy and its progression to heart failure.32 Although PKB has been suggested to target multiple substrates in the heart,33 whether NHE1 is phosphorylated and regulated by PKB is unknown. The objectives of the present study, therefore, were to determine whether NHE1 is a PKB substrate, identify any pertinent phosphorylation site(s), and determine the functional consequences of their PKB-mediated phosphorylation in adult myocardium.
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Materials and Methods |
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An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org. Key techniques were adapted from previously published studies, including the isolation, culture and adenoviral infection of adult rat ventricular myocytes (ARVMs),34 generation of adenoviral vectors,35 in vitro phosphorylation assays and Western immunoblot analysis,36 measurement of sarcolemmal NHE activity,13 determination of cellular NHE1 phosphorylation,36 and immunocytochemistry and confocal microscopy.36
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Results |
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PKB-Mediated Phosphorylation of NHE1
The carboxyl-terminal regulatory domain of NHE1 contains 3 sites that conform to the optimal PKB phosphorylation motif RxRxxS/T,19 namely Ser648 (RQRLRS), Ser703 (RARIGS), and Ser796 (RIQRCLS), as predicted by a motif scanning algorithm.37 Therefore, we initially determined whether active PKB
would phosphorylate recombinant NHE1, in an in vitro kinase assay using 32P-labeled ATP. Autoradiography revealed that PKB progressively phosphorylated a recombinant GST-NHE1(516–815) fusion protein, encompassing the final 300 amino acids(516–815) of the carboxyl-terminal regulatory domain of human NHE1, in a time-dependent manner (Figure 1A). In parallel, identical in vitro kinase assays were performed in the presence of unlabeled ATP and used a phospho-PKB substrate antibody, which detects phosphorylated Ser or Thr residues within the PKB phosphorylation consensus motif RxRxxS/T, to monitor PKB-mediated phosphorylation of the GST-NHE1(516–815) fusion protein. This nonradioactive approach revealed a similar pattern of PKB-mediated phosphorylation of GST-NHE1(516–815) (Figure 1B). Notably, PKB-mediated phosphorylation was absent when either assay used recombinant GST protein as substrate (data not shown). These data suggest that the carboxyl-terminal regulatory domain of NHE1 is a novel substrate for PKB-mediated phosphorylation and that the phospho-PKB substrate antibody is able to detect such phosphorylation. We then used truncated GST-NHE1 fusion proteins, encompassing residues 516 to 630, 625 to 747, and 748 to 815 of the carboxyl-terminal regulatory domain of NHE1, to perform an additional series of in vitro phosphorylation experiments and revealed that PKB-mediated 32P incorporation occurred at residues within 625 to 747 and 748 to 815, but not 516 to 630 (Figure 2A). Again, parallel experiments using the phospho-PKB substrate antibody revealed a similar pattern (Figure 2B). These data suggest that PKB-mediated phosphorylation of the carboxyl-terminal regulatory domain of NHE1 occurs among the final 190 residues, which contain Ser648, Ser703, and Ser796. We then performed a series of experiments to identify the pertinent PKB phosphorylation site(s) by liquid chromatography–tandem mass spectrometry. Tryptic digestion of phosphorylated GST-NHE1(516–815) yielded peptide fragments that provided 70% coverage of the NHE1 portion of the fusion protein (see Figure I in the online data supplement). Mass spectrometric analysis of the tryptic peptides revealed that Ser703 and Ser796 underwent PKB-mediated phosphorylation (see supplemental Figure II). However, the third putative phosphorylation site, Ser648, was outside of the coverage of this analysis (see supplemental Figure I). We then constructed a new series of GST-NHE1(625–815) fusion proteins in wild-type (WT) form or containing a mutated NHE1 component, in which Ser648, Ser703, and Ser796 were replaced by nonphosphorylatable alanine (Ala), in isolation and in all possible combinations. Figure 3A shows that WT GST-NHE1(625–815), containing all 3 putative PKB phosphorylation sites, was phosphorylated by PKB in vitro. Interestingly, mutation of Ser648 to Ala (Ser648Ala) markedly reduced PKB-mediated phosphorylation of GST-NHE1(625–815). In contrast, the Ser703Ala mutation had no discernible effect and the Ser796Ala mutation resulted in only a small reduction in phosphorylation (Figure 3A). The double mutations Ser648/703Ala and Ser648/796Ala also dramatically reduced PKB-mediated phosphorylation, whereas the double mutation Ser703/796Ala produced only a small reduction (Figure 3A). The triple mutation Ser648/703/796Ala yielded a nonphosphorylatable GST-NHE1(625–815) fusion protein. Parallel immunoblot analysis using the phospho-PKB substrate antibody revealed a similar pattern of phosphorylation (Figure 3B). Taken together, these data suggest that Ser648 is the major PKB phosphorylation site in the NHE1 regulatory domain and that its phosphorylation is detected by the phospho-PKB substrate antibody.
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We next performed in vitro kinase assays using the WT and double mutant (Ser648/703Ala, Ser648/796Ala, and Ser703/796Ala) GST-NHE1(625–815) fusion proteins as PKB substrates to study the kinetics of individual phosphorylation of Ser648, Ser703, or Ser796. PKB-mediated incorporation of 32P into the pertinent GST-NHE1(625–815) fusion protein substrates showed that the phosphorylation of Ser648 proceeded at a rate similar to that of the targeted residues within the WT protein, reaching saturation within 5 to 10 minutes (Figure 4A). In contrast, under the same assay conditions, PKB-mediated phosphorylation of Ser703 and Ser796 occurred at a much slower rate and did not reach saturation even after 60 minutes (Figure 4A). Again, parallel experiments using the phospho-PKB substrate antibody revealed a similar pattern, with the exception that Western immunoblotting with the phospho-PKB substrate antibody was not as sensitive as 32P autoradiography in detecting Ser703 phosphorylation (Figure 4B). These data provide further support for our finding that Ser648 within the NHE1 carboxyl-terminal regulatory domain is a novel substrate for PKB-mediated phosphorylation. The detection of phosphopeptides containing pSer703 and pSer796 by mass spectrometry analysis likely reflects the fact that, for these studies, the substrate fusion protein was exposed to PKB for 60 minutes, after which time detectable phosphorylation of these sites also occurs (Figure 4A).
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Regulation of CaM Binding by PKB-Mediated Phosphorylation of Ser648
Ser648 resides within an autoinhibitory CaM-binding region of the NHE1 regulatory domain, with previous evidence indicating that CaM binding to this region relieves the autoinhibitory effect, resulting in increased NHE1 activity.10 We therefore hypothesized that PKB-mediated phosphorylation of Ser648 may regulate CaM binding to the NHE1 regulatory domain. To test this hypothesis, we used a far-Western approach and confirmed that CaM did indeed bind to unphosphorylated WT GST-NHE1(625–815) fusion protein, in a calcium-dependent manner (Figure 5A). Such binding was unaffected by SerAla mutations (in the absence of phosphorylation) but was attenuated by the introduction of a phosphomimetic Ser648Asp substitution (Figure 5A). Interestingly, PKB-mediated phosphorylation abolished CaM binding to WT GST-NHE1(625–815) fusion protein and its mutated variants (Figure 5B), except those carrying a Ser648Ala substitution alone or in combination with other mutations (Figure 5B). These data indicate that PKB-mediated phosphorylation of Ser648 in the carboxyl-terminal regulatory domain of NHE1 inhibits CaM binding to this domain.
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PKB-Mediated Phosphorylation and Regulation of NHE1 in Cardiac Myocytes
To manipulate PKB activity in intact ARVMs, we heterologously expressed constitutively active, hemagglutinin (HA)-tagged myristoylated PKB (myr-PKB) protein by adenoviral gene transfer. Western immunoblot analysis using antibodies to the HA-tag or PKB protein revealed a "dose"-dependent increase in myr-PKB expression 24 hours after infection (Figure 6A). The constitutively active nature of the heterologously expressed myr-PKB protein was confirmed by the detection of a parallel increase in glycogen synthase kinase (GSK)3β phosphorylation at Ser9, a known cellular PKB substrate (Figure 6B). Immunocytochemical detection of HA-tag expression confirmed that infection of ARVMs with the AdV:myr-PKB adenoviral vector at a multiplicity of infection (moi) of 300 (plaque-forming units per cell) was sufficient to achieve >95% infection efficiency (see supplemental Figure III).
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Immunocytochemistry and confocal microscopy were also used to determine the cellular localization of heterologously expressed HA-tagged myr-PKB relative to native NHE1. Consistent with our previous work,36 NHE1 protein expression was localized predominantly to the intercalated disc regions, with additional staining of the nuclei and cell periphery (Figure 6C). HA-tagged myr-PKB was localized exclusively to the cell periphery, including the intercalated disc regions, confirming that the myristoylation sequence associated with the heterologously expressed protein operated as expected and anchored the myr-PKB protein to the sarcolemma (Figure 6C). Merged images of NHE1 and myr-PKB expression revealed marked colocalization of the 2 proteins, particularly at the intercalated disc regions (Figure 6C).
We then examined whether increased PKB activity in intact ARVMs, through heterologous expression of myr-PKB, could increase phosphorylation of the full-length cellular NHE1 protein. ARVMs were coinfected with an adenoviral vector encoding epitope-tagged human NHE1 to amplify the NHE1 signal for phosphoprotein analysis.13 The expression of myr-PKB was again confirmed by the presence of a HA-tagged protein of appropriate size and increased expression of PKB protein in the crude lysate of ARVMs infected with the myr-PKB vector (Figure 7A). Equal amounts of both the 80- and 105-kDa moieties of NHE1 protein, representing differentially glycosylated forms,13 were present in crude lysates from all groups (Figure 7B). Following NHE1 immunoprecipitation, equal amounts of heterologously expressed NHE1 protein were present in the immunocomplexes in all groups, as detected by an antibody to the HA-tag (Figure 7C). However, when these immunocomplexes were probed with the phospho-PKB substrate antibody, those from ARVMs infected with the myr-PKB adenoviral vector were found to contain significantly higher amounts of phosphorylated NHE1, compared to the uninfected control group or a second control group infected with a vector encoding an unrelated protein (β-galactosidase) (Figure 7C). These data show that increased cellular PKB activity in intact ARVMs leads to increased phosphorylation of NHE1; such phosphorylation most likely occurred at Ser648, because PKB-mediated phosphorylation at this site is readily detected by the phospho-PKB substrate antibody (Figures 3B and 4
B).
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We then used the interventions described above to determine the effects of increased cellular PKB activity on sarcolemmal NHE activity in intact ARVMs. There were no significant differences between infected groups in cell dimensions or estimated volumes (see supplemental Table I) or the basal intracellular pH (pHi) and the degree of intracellular acidosis achieved after NH4Cl washout (see supplemental Table II). Intrinsic buffering capacity was determined in all 3 groups and used to calculate sarcolemmal NHE activity in the appropriate group (see supplemental Figure IV). Figure 7D shows that sarcolemmal NHE activity, as reflected by the H+ efflux rate across the sarcolemma (JH), was significantly lower (by 60% to 70%) in ARVMs expressing myr-PKB, when compared to either control group. These gain-of-function data indicate that, in intact ARVMs, increased PKB activity results in a significant reduction in sarcolemmal NHE activity in response to intracellular acidosis.
Finally, we adopted a complementary loss-of-function pharmacological approach and determined the role of endogenous PKB/β activity in the regulation of sarcolemmal NHE activity in ARVMs using a recently characterized specific PKB inhibitor [1,3-dihydro-1-(1-((4-(6-phenyl-1H-imidazo[4,5-g]quinoxalin-7-yl)phenyl)methyl)-4-piperidinyl)-2H-benzimidazol-2-one], referred to as Akt inhibitor-1/2 (Akti-1/2).38,39 Akti-1/2 interacts with the PH domains of PKB/β, thereby preventing the conformational change required for their phosphorylation and activation by upstream kinases.40 Consistent with this, pretreatment of ARVMs with 1 µmol/L Akti-1/2 inhibited insulin-induced phosphorylation of endogenous PKB at Ser473 and the PKB substrate GSK3β at Ser9 (Figure 8A). In subsequent experiments, such pretreatment with Akti-1/2 was found not to alter basal pHi and the degree of intracellular acidosis achieved after NH4Cl washout (see supplemental Table II) but to significantly increase sarcolemmal NHE activity (Figure 8B), through an apparent increase in affinity for intracellular H+ (Figure 8C).
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Taken together, our complementary gain-of-function and loss-of-function data indicate that PKB activity in cardiac myocytes inhibits sarcolemmal NHE activity during intracellular acidosis, most likely through phosphorylation of Ser648 in the NHE1 regulatory domain and inhibition of CaM binding to this domain.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The original findings of the present study are that: (1) NHE1 is a novel PKB substrate; (2) the principal PKB-mediated phosphorylation site in NHE1 is Ser648, which has not been previously identified as a kinase target; (3) PKB-mediated phosphorylation of Ser648 inhibits binding of Ca2+-activated CaM to the NHE1 regulatory domain in vitro; (4) PKB-mediated phosphorylation of NHE1 in intact ventricular myocytes markedly depresses H+ extrusion via sarcolemmal NHE activity, in response to intracellular acidosis.
Several protein kinases including ERK,11 RSK,12 p160-ROCK,14 and p38-MAPK15 have been shown to stimulate NHE1 activity through phosphorylation of its carboxyl-terminal regulatory domain. The targeted amino acid residues have been identified for ERK (Ser770 and Ser771),11 RSK (Ser703),12 and p38-MAPK (Thr717, Ser722, Ser725 and Ser728)15 but remain unidentified for p160-ROCK.14 Furthermore, although the functional importance of ERK-mediated phosphorylation of Ser770 and Ser771 and RSK-mediated phosphorylation of Ser703 has been established,11,12,18 that of p38-MAPK–mediated phosphorylation of the pertinent target sites in NHE1 remains unknown. With specific regard to cardiac physiology, recent studies from our laboratory have confirmed the functional importance of RSK-mediated phosphorylation of NHE1 in 1-adrenoceptor–induced stimulation of sarcolemmal NHE activity, through the use of a novel RSK inhibitor in ARVMs.41 Furthermore, heterologous expression of a dominant negative RSK mutant in neonatal rat ventricular myocytes has been shown to inhibit oxidative stress-induced stimulation of sarcolemmal NHE activity.42 In the same study, transgenic expression of the dominant negative RSK mutant in the mouse heart attenuated myocardial injury and left ventricular dysfunction following ischemia and reperfusion, potentially reflecting the pathophysiological significance of phosphorylation-mediated regulation of NHE1 activity.42 In this context, the present study is the first to identify PKB as an NHE1 kinase, both in vitro and in intact cells, and also the first to identify Ser648 as a phosphorylation site in NHE1. Furthermore, our data suggest that, unlike most kinases that phosphorylate the NHE1 carboxyl-terminal regulatory domain, PKB inhibits NHE1 activity.
Our findings also provide a potential molecular mechanism through which PKB inhibits NHE1 activity. As noted earlier, Ser648, which we have identified as the principal PKB-mediated phosphorylation site in NHE1, resides in the middle of a high-affinity CaM binding region that comprises amino acids 636 to 656.10 Previous evidence indicates that, in the absence of activated CaM, this region of the NHE1 regulatory domain is unoccupied and exerts an autoinhibitory effect on the NHE1 transport domain; on its Ca2+-induced activation, CaM binds to this region, abolishing the autoinhibitory interaction between the regulatory and transport domains and increasing NHE1 activity.43 Our novel data show that PKB-mediated phosphorylation of Ser648 inhibits the binding of Ca2+-activated CaM to the NHE1 regulatory domain (Figure 5). In the intact cell, this mechanism is likely to sustain the autoinhibitory effect of the CaM binding region on NHE1 activity, even in the presence of activated CaM. Notably, we did not see a significant difference in basal pHi between ARVMs with heterologous expression of myr-PKB and control cells that were either uninfected or infected to heterologously express β-galactosidase in our gain-of-function studies or between ARVMs with or without pretreatment with Akti-1/2 in our loss-of-function experiments (see supplemental Table II). A possible explanation for this observation is the fact that sarcolemmal NHE activity is very low at physiological pHi in unstimulated cells,2 in which the CaM-binding site is likely to be unoccupied.43 Under such conditions, PKB-mediated phosphorylation of Ser648 would be expected to have little impact on CaM binding to the NHE1 regulatory domain and, thereby, on NHE1-mediated H+ extrusion.
In contrast to the above, we observed a markedly reduced sarcolemmal NHE activity in response to intracellular acidosis in ARVMs with heterologous expression of myr-PKB, which occurred concomitantly with increased NHE1 phosphorylation (Figure 7). This increased phosphorylation most likely occurred at Ser648, as determined by the incorporation of 32P and the phospho-PKB substrate antibody that we used to detect such phosphorylation (Figures 3 and 4). Furthermore, inhibition of endogenous PKB activity in uninfected ARVMs led to a significantly increased sarcolemmal NHE activity in response to intracellular acidosis (Figure 8). Taken together with our finding that PKB-mediated phosphorylation of Ser648 inhibits the binding of Ca2+-activated CaM to the NHE1 regulatory domain (Figure 5), this observation suggests a potential new mechanism for NHE1 stimulation by acute intracellular acidosis, through enhanced CaM binding. In this regard, intracellular acidosis has long been known to increase free intracellular [Ca2+] in cardiac cells,44 and has been proposed to activate another CaM target, Ca2+/CaM-dependent kinase II.45 Thus, during intracellular acidosis, Ca2+-activated CaM would be expected to bind to the CaM-binding region of the NHE1 regulatory domain, thereby overcoming its autoinhibitory effect and increasing NHE1 activity. On the basis of our findings, PKB-mediated phosphorylation of Ser648 is likely to inhibit such CaM binding, thereby suppressing NHE1 activity in response to intracellular acidosis. Interestingly, inhibition of phosphodiesterase 5A (which hydrolyzes cGMP) has also been shown recently to inhibit sarcolemmal NHE activity during intracellular acidosis without affecting basal pHi,46 suggesting the possibility that cGMP-responsive pathways, such as the cGMP-dependent protein kinase, may regulate NHE1 activity through an analogous mechanism.
The potential contributions of the other putative PKB phosphorylation sites that we detected by mass spectrometry, namely Ser703 and Ser796, need to be considered. Our studies with site-directed mutagenesis revealed that the NHE1 mutant carrying Ser648/796Ala substitutions, in which only Ser703 was available for phosphorylation, was actually a very poor PKB substrate. Thus, phosphorylation of Ser703 is unlikely to have contributed to PKB-mediated inhibition of sarcolemmal NHE activity. This is consistent with previous data suggesting that Ser703 phosphorylation by RSK stimulates (rather than inhibits) NHE1 activity in response to intracellular acidosis.12,13,18 Relative to the Ser648/796Ala mutant, we detected greater PKB-mediated phosphorylation of the NHE1 mutant carrying Ser648/703Ala substitutions, in which only Ser796 was available for phosphorylation (Figure 4). Li et al9 have previously reported Ser796 to reside within a carbonic anhydrase II–binding region of the NHE1 carboxyl-regulatory domain and to be phosphorylated by unidentified kinase(s) present in a heart cell extract. However, Ser796 phosphorylation had little impact on carbonic anhydrase II binding to NHE1 and phosphorylation of the NHE1 regulatory domain at other, more proximal site(s) was proposed to regulate such binding.9 Thus, the functional significance of Ser796 phosphorylation, by PKB or other kinase(s), in NHE1 regulation remains unclear. It is noteworthy, however, that Ser796 is a considerably poorer substrate than Ser648 for PKB-mediated phosphorylation in vitro, with detectable Ser796 phosphorylation becoming apparent only after more prolonged exposure to active PKB (Figure 4).
Sarcolemmal NHE activity increases during myocardial ischemia, largely as a consequence of the intracellular acidosis that develops rapidly on its onset, and is believed to contribute significantly to myocardial injury and dysfunction during ischemia and subsequent reperfusion.47 Consistent with this, NHE1-selective pharmacological inhibitors have been shown to afford marked cardioprotective benefit during ischemia and reperfusion in both animal models48 and specific clinical settings.49 Because increased PKB activity also has a marked protective effect during myocardial ischemia and reperfusion,27,50–52 our findings raise the possibility that reduced NHE1 activity may contribute to the pertinent cardioprotective mechanisms. In this regard, myocardial ischemia is associated with intracellular Ca2+ accumulation, with increased sarcolemmal NHE activity causally implicated in the occurrence of this phenomenon.47 In such a setting, CaM binding may sustain sarcolemmal NHE activity and contribute to a vicious cycle that further exacerbates intracellular Ca2+ accumulation; to the contrary, PKB-mediated NHE1 phosphorylation would be expected to terminate this cycle by disrupting CaM-mediated stimulation of sarcolemmal NHE activity. In view of the novel findings of the present study, further investigation of the role of CaM binding in the regulation of NHE1 activity and the physiological significance of the inhibition of such binding by PKB-mediated NHE1 phosphorylation appears warranted.
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Acknowledgments |
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Sources of Funding
A.K.S. was supported by British Heart Foundation Intermediate Research Fellowship FS/04/053.
Disclosures
None.
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Footnotes |
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Original received March 18, 2008; revision received August 13, 2008; accepted August 20, 2008.
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References |
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