Angiotensin II Stimulates p90rsk in Vascular Smooth Muscle Cells
A Potential Na+-H+ Exchanger Kinase
Abstract Angiotensin II is a multifunctional agonist for vascular smooth muscle cells (VSMCs), stimulating increases in signal events, cell growth, and ion flux. We previously defined protein kinase C (PKC)–dependent and –independent mechanisms by which angiotensin II stimulated activity of the Na+-H+ exchanger isoform-1 (NHE-1) and identified a 90-kD kinase that exhibited increased activity in VSMCs isolated from genetically hypertensive rats. To determine whether this 90-kD kinase was p90rsk (RSK), VSMCs were stimulated with 100 nmol/L angiotensin II, and NHE-1 kinase activity was measured by phosphorylation of recombinant NHE-1 (a glutathione S-transferase fusion protein containing amino acids 516 to 815 of the cytoplasmic carboxyl tail) in vitro. NHE-1 kinase (90 kD) activity was markedly decreased by immunodepletion of RSK. Characterization of RSK activation by angiotensin II revealed many similarities to the 90-kD NHE-1 kinase, including time course and NHE-1 domain phosphorylation, as well as regulation by extracellular signal–regulated kinases (ERK1/2), intracellular Ca2+, and PKC. Specifically, angiotensin II stimulated a rapid and transient (peak, 5 minutes) increase in RSK activity. Analysis of several NHE-1 fusion proteins revealed that only proteins containing amino acids 670 to 714 were phosphorylated by RSK. Inhibiting ERK1/2 (30 μmol/L PD098059 for 30 minutes) or chelating intracellular Ca2+ prevented RSK activation. In contrast, downregulating PKC (1 μmol/L phorbol dibutyrate for 24 hours) had little effect. These findings establish RSK as a putative NHE-1 kinase and potential mediator of increased Na+-H+ exchange in hypertension.
The growth factor–activated Na+-H+ exchanger (NHE-1) is a member of a multigene family whose activity is increased in tissues of hypertensive humans and animals. Because NHE-1 is activated by both hyperplastic (eg, platelet-derived growth factor) and hypertrophic (eg, angiotensin II) agonists, it has been proposed that abnormal NHE-1 function may be involved in the pathophysiology of hypertension. Increased activity of NHE-1 hypertension could be caused by three nonexclusive mechanisms: mutation in the gene, increased expression of the gene product, and altered posttranslational regulation of the exchanger. Previous studies comparing SHR VSMCs with normotensive WKY VSMCs, including analysis of NHE-1 cDNA sequence,1 steady state mRNA levels,2 and protein expression,3 have shown no significant differences. However, there is clearly an increase in NHE-1 phosphorylation in cells derived from the SHR.4 Because changes in phosphorylation of NHE-1 itself or of an associated regulatory protein have been implicated in activation and regulation of transport kinetics,5 these findings suggest that increased Na+-H+ exchange in the SHR is caused by an alteration in the kinases that regulate NHE-1 activity.
Several protein kinases have been proposed to regulate NHE-1, including Ca2+-calmodulin–dependent kinases, cAMP-dependent kinase, and members of the MAP kinase family.6 We recently reported that a 90-kD NHE-1 kinase was more active in SHR than WKY VSMCs under both growth-arrested and angiotensin II–stimulated conditions.7 Characterization of the kinase indicated that it was Ca2+ dependent and PKC independent. A recent study with the MEK-1 inhibitor PD098059 showed that ERK1/2 was involved in the regulation of NHE-1 in platelets.8
ERK1/2 has been proposed to regulate many different intracellular events, including other protein kinases.9 One of the kinases that has been demonstrated to be regulated by ERK1/2 is RSK, which exists as three isoforms, termed RSK1, RSK2, and RSK3. Several features of RSK suggest it may be a candidate VSMC NHE-1 kinase. Specifically, RSK is activated by ERK1/2, its activation is Ca2+ dependent and PKC independent, and it is universally expressed similar to NHE-1. RSK has pleiotropic functions: it has been suggested to participate in oncogenic transformation,10 stimulation of the G0/G1 transition,11 12 13 T-cell activation,14 differentiation of PC12 cells,15 platelet activation,16 cellular responses to heat shock,17 and ionizing radiation.18 RSK has been localized to the cell nucleus and is thought to phosphorylate nuclear substrates, including transcription factors.13 RSK is activated by serine and threonine phosphorylation mediated by ERK1/2, as well as by additional autophosphorylation.11 In the present study, we have characterized the ability of RSK to phosphorylate NHE-1 and have demonstrated that RSK is a candidate NHE-1 kinase activated by angiotensin II.
Materials and Methods
VSMCs were isolated from the thoracic aorta of 200- to 250-g male Sprague-Dawley rats (Harlan Sprague Dawley, Inc) and maintained in DMEM supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% calf serum (Hyclone) as described previously.19 SHR VSMCs were obtained from thoracic aortas and maintained as described previously.7 Sprague-Dawley VSMCs (at passages between 5 and 13) and SHR VSMCs (at passages 3 and 5) were growth-arrested at 70% to 80% confluence by incubation in 0.4% calf serum/DMEM for 48 hours before use.
Preparation of Cell Lysates and Western Blot Analysis
Cells were incubated at 37°C in HEPES-buffered DMEM containing 100 nmol/L angiotensin II or vehicle alone for various times. After agonist stimulation, cells were harvested by aspirating the medium and washing twice with ice-cold PBS. Cells were lysed by the addition of ice-cold lysis buffer (1.0 mL per 100-mm dish) consisting of (mmol/L) NaCl 50, NaF 50, sodium pyrophosphate 50, EDTA 5, EGTA 5, Na3VO4 2, phenylmethylsulfonyl fluoride 0.5, and HEPES 10, along with 0.1% Triton X-100 and 10 μg/mL leupeptin, pH 7.4, followed by immediate freezing on ethanol/dry ice. The cell lysates were then thawed on ice, scraped, sonicated, and centrifuged at 14 000g at 4°C for 30 minutes. Supernatants were used immediately or stored at −80°C. Protein concentrations were determined using a Bio-Rad protein assay kit. For Western blot analysis, equal amounts of protein cell lysates were subjected to SDS-PAGE, transferred to nitrocellulose (Hybond-ECL, Amersham Corp), and immunoblotted with primary antibodies (Santa Cruz Biotechnology). After incubation with horseradish peroxidase–conjugated secondary anti-goat antibody (Sigma Chemical Co), the blots were developed using ECL (Amersham Corp).
GST–NHE-1 Fusion Protein Construction and Purification
As described previously,7 bacterial expression plasmids containing different domains of the human NHE-1 were prepared by subcloning polymerase chain reaction–generated EcoRI cDNA (cloned pBluescript) into pGEX-KG. Cytoplasmic domains of the human NHE-1 (amino acids 516 to 815) and three overlapping constructs were prepared by polymerase chain reaction: NHE-1 (516 to 630), NHE-1 (625 to 747), and NHE-1 (748 to 815). The orientation and reading frames of all constructs were confirmed by sequencing. Two additional fusion proteins (NHE-1 [625 to 670] and NHE-1 [625 to 714]) were prepared from NHE-1 (625 to 747) by digesting NHE-1 (625 to 747) with Bse AI and Psp5II, respectively. After blunting the ends, the fragments were cloned in pGEX-KG.
After transformation of GST–NHE-1 constructs into the BL21 strain of Escherichia coli, cultures were grown to the sublog phase and induced and grown for 3 hours at 37°C with 0.5 mol/L isopropyl-β-d-thiogalactopyranoside (Sigma). Cells were collected, sonicated, and centrifuged. For GST–NHE-1 (516 to 815), cells were incubated for 16 hours at 30°C before harvest. The supernatants were incubated with glutathione-agarose beads (Sigma) for 60 minutes at 4°C. Bound fusion proteins were washed extensively and eluted with 20 mmol/L reduced glutathione, 100 mmol/L Tris-HCl, pH 7.4, and 100 mmol/L NaCl. Protein concentrations were checked by Coomassie staining of SDS-PAGE–separated proteins.
Immunoprecipitation and Immunodepletion of RSK
For immunoprecipitation of RSK, cell lysates containing 200 μg protein were incubated with polyclonal goat antiserum against rat RSK (2 μg, Santa Cruz Biotechnology) or control goat IgG (2 μg, Santa Cruz Biotechnology) overnight at 4°C and then incubated with 20 μL of protein G–agarose beads (GIBCO) for 1 hour on a roller system at 4°C. The beads were washed two times with 1 mL of lysis buffer, two times with 1 mL of LiCl wash buffer (500 mmol/L LiCl, 100 mmol/L Tris-Cl, pH 7.6, 0.1% Triton X-100, and 1 mmol/L dithiothreitol), and two times with 1 mL of washing buffer (20 mmol/L HEPES, pH 7.2, 2 mmol/L EGTA, 10 mmol/L MgCl2, 1 mmol/L dithiothreitol, and 0.1% Triton X-100). For immunodepletion of RSK, cell lysates containing 100 μg protein were treated twice by incubation with polyclonal goat antiserum against rat RSK (3 μg, Santa Cruz Biotechnology) or control goat IgG (3 μg, Santa Cruz Biotechnology) for 1 hour at 4°C and incubation with 20 μL protein G–agarose beads (GIBCO-BRL) for 1 hour on a roller system at 4°C. After treatment, the protein concentrations were determined with a Bio-Rad protein assay kit.
NHE-1 Phosphorylation (Immune Complex Kinase Assay)
Immunoprecipitated RSK was resuspended in 25 mmol/L HEPES, pH 7.4, 10 mmol/L MgCl2, and 10 mmol/L MnCl2, and the kinase reaction was initiated by the addition of 200 pmol substrate protein, 15 μmol/L ATP, and 0.5 mCi/mL of [γ-32P]ATP. After the reaction had proceeded for 20 minutes at 30°C, it was terminated by the addition of Laemmli sample buffer, and proteins were analyzed by 10% SDS-PAGE followed by autoradiography.
NHE-1 Phosphorylation (In-Gel Kinase Assay)
Equal amounts of cell lysate protein (20 μg) were separated by 7.5% SDS-PAGE in a gel containing 0.15 mg/mL of GST–NHE-1(516 to 815) or 1.0 mg/mL of myelin basic protein. The gel was then incubated twice in buffer A (50 mmol/L HEPES, pH 7.4, and 5 mmol/L β-mercaptoethanol) containing 20% isopropyl alcohol for 30 minutes; once in buffer A for 1 hour; twice in buffer A containing 6 mol/L guanidine HCl for 30 minutes; twice in buffer A containing 0.04% Tween 20 at 4°C for 16 hours and 2 hours; once in buffer A containing 100 μmol/L Na3VO4 and 10 mmol/L MgCl2 at 30°C for 30 minutes; and once in buffer A containing 100 μmol/L Na3VO4, 10 mmol/L MgCl2, 50 μmol/L ATP, and 50 μCi of [γ-32P]ATP for 1 hour at 30°C. The reaction was terminated by washing the gel six to eight times in fixative solution containing 10 mmol/L sodium pyrophosphate and 5% trichloroacetic acid for 15 minutes. The gel was dried and subjected to autoradiography. Autoradiographic signal intensity was quantified by densitometry in the linear range of film exposure by using a LaCie scanner (LaCie Ltd) and NIH Image 1.54 software.
Values presented are mean±SEM. Student’s t test was used when appropriate. Values of P<.05 were considered statistically significant.
Polyclonal goat antiserum against rat RSK1, RSK2, and RSK3 and control goat IgG were obtained from Santa Cruz Biotechnology. PD098059, a MEK-1 inhibitor,20 was obtained from Parke Davis. BAPTA-AM was obtained from Molecular Probes.
Immunodetection of RSK in VSMCs
To identify which RSK isoforms were present in VSMCs and to determine the specificity of the isoform-specific RSK antibodies, Western blot analysis was performed with VSMC lysates. A predominant 90-kD protein band was readily observed in cultured rat VSMCs (Fig 1A⇓) with each RSK antibody. Assuming that the antibodies have equal ability to identify the RSK isoforms on Western blots, it appears that the relative expression of RSK in VSMCs is RSK2≫RSK3>RSK1. To determine the specificity of RSK2 antibody for immunoprecipitation, VSMC lysates were prepared and immunoprecipitated with RSK2 antibody, and proteins were detected by Western blot with the RSK2 antibody (Fig 1B⇓). A predominant 90-kD protein was readily observed without evidence for other coprecipitating proteins (Fig 1B⇓). Immunoprecipitation with control IgG showed no immunoreactive 90-kD band. However, the RSK isoform–specific antibodies showed no specificity for the individual isoforms, as demonstrated by the findings that immunoprecipitation with RSK1 antibody followed by Western blot analysis with RSK1, RSK2, and RSK3 antibodies yielded the same pattern on Western blot (RSK2≫RSK3>RSK1) as obtained with isoform-specific immunoprecipitation (data not shown). Similar patterns were observed with the RSK2 and RSK3 immunoprecipitations. These results suggest that in rat VSMCs, the isoform-specific antibodies cannot discriminate among the isoforms when used for immunoprecipitation or that only RSK2 is expressed and there is a small degree of cross-reactivity with RSK2 by the RSK1 and RSK3 antibodies. Because of these findings, we chose to use the RSK2 antibody for all subsequent studies and to define the immunoreactive protein as RSK.
Immunodepletion of RSK Decreases the Activity of a 90-kD NHE-1 Kinase in Sprague-Dawley VSMCs
Angiotensin II stimulated the activity of several NHE-1 kinases in Sprague-Dawley VSMCs, including kinases of 90, 70, 62, 60, 44, and 42 kD measured by in-gel kinase assay with GST–NHE-1 (516 to 815) as substrate, as previously reported.7 Immunodepletion of RSK with RSK2 antibody selectively decreased the activity of a 90-kD kinase present in Sprague-Dawley VSMCs (inhibition of 82±3%, n=3, P<.05) (Fig 2⇓, top). Immunodepletion with control goat IgG had no significant effect on the 90-kD kinase activity. Immunodepletion with RSK2 antibody and goat IgG caused small decreases in the activity of two other NHE-1 kinases (inhibition of 23±3% to 44±5%), but these were of significantly smaller magnitude than the decrease in RSK observed with RSK2 antibody. To prove further the specificity of immunodepletion by RSK2 antibody, we performed an in-gel kinase assay with myelin basic protein, which is an excellent RSK substrate (Fig 2⇓, bottom). The only significant difference in myelin basic protein kinase activity observed for immunodepletion with RSK2 antibody compared with goat IgG was a 90-kD kinase (Fig 2⇓, asterisk in bottom panel). Similar results were obtained using antibodies to RSK1 and RSK3. On the basis of these results, RSK is the predominant 90-kD NHE-1 kinase.
Immunodepletion of RSK Decreases the Activity of a 90-kD NHE-1 Kinase in SHR VSMCs
To prove that the 90-kD NHE-1 kinase previously identified in SHR7 was RSK, we repeated the immunodepletion experiments with RSK2 antibody and performed an in-gel kinase assay with GST–NHE-1 (516 to 815) as substrate (Fig 3⇓). Angiotensin II stimulated the activity of a 90-kD NHE-1 kinase in SHR VSMCs (Fig 3⇓). Immunodepletion with RSK2 antibody selectively decreased the activity of only this 90-kD kinase in SHR VSMCs. Immunodepletion with control goat IgG had no significant effect on the 90-kD kinase activity. Thus, the 90-kD NHE-1 kinase previously identified in SHR VSMCs is RSK.
RSK, After Stimulation by Angiotensin II, Phosphorylates NHE-1 (625 to 747) In Vitro
To determine whether RSK phosphorylates NHE-1 in vitro and to identify the amino acid(s) phosphorylated in NHE-1 (516 to 815) by RSK, we prepared three overlapping GST–NHE-1 fusion proteins (Fig 4⇓). When used as a substrate for immunoprecipitated RSK, only NHE-1 (625 to 747) was phosphorylated to a significant extent in vitro (Fig 5A⇓). This region of NHE-1 contains several potential phosphorylated serine and threonine residues. On the basis of the location of these residues, we constructed two additional GST–NHE-1 fusion proteins, NHE-1 (625 to 714) and NHE-1 (625 to 670). RSK was immunoprecipitated from angiotensin II–stimulated Sprague-Dawley VSMCs and, in an immune complex kinase assay, phosphorylated NHE-1 (625 to 747) and NHE-1 (625 to 714) but not NHE-1 (625 to 670) (Fig 5B⇓). On the basis of these results, it appears that RSK specifically phosphorylates serine and/or threonine residues located between amino acids 670 and 714 of NHE-1.
Time Course for Activation of NHE-1 by RSK in VSMCs Stimulated by Angiotensin II
To compare the characteristics of RSK and the 90-kD NHE-1 kinase previously reported,7 the time course for activation by 100 nmol/L angiotensin II was determined. RSK was immunoprecipitated, and its ability to phosphorylate NHE-1 (625 to 747) was measured by the in vitro immune complex kinase assay. After agonist stimulation, RSK activity rapidly increased with a peak at 5 minutes and a return to baseline at 60 minutes (Fig 6⇓), similar to the time course reported previously for the 90-kD NHE-1 kinase.
PD098059, an MEK-1 Inhibitor, Inhibits Phosphorylation of NHE-1 by RSK in VSMCs Stimulated by Angiotensin II
We previously reported that the 90-kD NHE-1 kinase was inhibited by PD098059, suggesting that this kinase was regulated by the MEK-1/ERK1/2 pathway.7 ERK1 does not readily phosphorylate recombinant NHE-1 (625 to 747) as shown by a stoichiometry of <0.05 (M. Kusuhara et al, unpublished data, 1996), suggesting that ERK1/2 is not an NHE-1 kinase. To inhibit ERK1/2 activity specifically, we studied the effect of the MEK-1 inhibitor PD098059 on RSK activity. Treatment of VSMCs with 30 μmol/L PD098059 completely inhibited the activation of ERK1/2 by angiotensin II without a significant effect on the activation of JNK (not shown). This concentration of PD098059 also significantly inhibited (89±2% inhibition, n=3, P<.05) angiotensin II–stimulated phosphorylation of NHE-1 (625 to 747) by immunoprecipitated RSK (Fig 7⇓). The decrease in RSK NHE-1 kinase activity correlated well with the decrease in ERK1/2 activity (data not shown). Thus, both RSK and the 90-kD NHE-1 kinase are dependent on MEK-1 and ERK1/2 for activation.
Ca2+ Chelation Inhibits Phosphorylation of NHE-1 by RSK in VSMCs Stimulated by Angiotensin II
We previously found that the 90-kD NHE-1 kinase required increases in intracellular Ca2+ for activation by angiotensin II,7 as shown by the ability of Ca2+ chelation (incubation in Ca2+-free solution containing 1 mmol/L EGTA and 75 μmol/L BAPTA-AM) to block activity. Chelation of intracellular Ca2+ also completely inhibited (94±9% inhibition, n=3, P<.05) activation of RSK, measured by phosphorylation of NHE-1 (625 to 747) (Fig 8⇓). Thus, both the 90-kD NHE-1 kinase and RSK are activated by increases in intracellular Ca2+.
PKC Downregulation Does Not Inhibit RSK Activity Stimulated by Angiotensin II
We previously found that the 90-kD NHE-1 kinase did not require the activity of phorbol ester–responsive PKC isoforms for stimulation by angiotensin II,7 as shown by the failure of PKC downregulation (incubation with 1 μmol/L PDBU for 24 hours) to inhibit activity. Downregulation of PKC also failed to inhibit (22±3% inhibition, n=3, P=.28) activation of RSK by angiotensin II (Fig 9⇓), demonstrating that both the 90-kD NHE-1 kinase and RSK are not regulated by a phorbol ester–responsive PKC isoform. In contrast, PMA-mediated activation of RSK was completely inhibited by PDBU treatment.
The major finding of the present study is the identification of RSK as an angiotensin II–stimulated kinase in VSMCs that phosphorylates the NHE-1 isoform of the Na+-H+ exchanger. RSK exhibits several features suggesting that it may be the 90-kD NHE-1 kinase we previously reported.7 First, both kinases were rapidly stimulated by angiotensin II, with peak activity at 5 minutes. Second, both kinases required increases in intracellular Ca2+, but not PKC, for activation. Third, both RSK and the 90-kD NHE-1 kinase were found to require ERK1/2 activity on the basis of inhibition by the MEK-1 inhibitor PD098059. Fourth, both RSK and the 90-kD NHE-1 kinase phosphorylated NHE-1 at residues located between amino acids 670 and 714. Fifth, immunodepletion of RSK with RSK2 antibody selectively removed the 90-kD NHE-1 activity in both Sprague-Dawley VSMCs and SHR VSMCs. In sum, these findings suggest that the VSMC 90-kD NHE-1 kinase that we previously found to be stimulated by angiotensin II is the same as RSK.
The present study defines NHE-1 as a new substrate for RSK and suggests a more general role for this kinase in cell function. The subcellular location of RSK and its preferred motif for phosphorylation (R-X-X-S), which is present in NHE-1, suggest that RSK may be a physiologically important NHE-1 kinase. Previously, the roles of RSK have been confined primarily to platelet activation,16 stimulation of protein synthesis,21 and regulation of nuclear transcription factors.13 The mechanisms that regulate subcellular location of RSK have not been defined, but it is of interest that immunohistochemical characterization of RSK in HeLa cells showed a generalized distribution within the cell, with immunoreactive kinase present not only in the nucleus and cytoplasm but in the plasma membrane as well.22 Given the relationship between ERK1/2 activity and RSK activity, it is important that both kinases have been shown to translocate from cytoplasm to nucleus during growth factor stimulation.22 Of greater potential importance, ERK1/2 was shown to translocate transiently to the plasma membrane of VSMCs after stimulation by vasoactive hormones, such as angiotensin II.6 In the present study, we determined that RSK phosphorylated NHE-1 residues located between amino acids 670 and 714. Within this sequence, there is a consensus RSK phosphorylation motif (R-X-X-S, where S is amino acid 703 of NHE-1) identical to that reported previously.23 Thus, both the subcellular location and sequence motif phosphorylated by RSK suggest that it may be a physiologically relevant NHE-1 kinase.
There are three aspects of the present study that require further experimentation. First, identification of which RSK isoform is predominantly responsible for phosphorylation of NHE-1 in response to angiotensin II is essential. Second, although inhibition of ERK1/2 has been found to decrease Na+-H+ exchanger activity,5 8 this has not been shown to be due to inhibition of RSK activity or correlated with changes in the phosphorylation of NHE-1. Thus, future experiments will be necessary with dominant-negative RSK and site-directed NHE-1 mutants (eg, S703 to A703) to verify the physiological significance of the present study. Third, the present study and other investigations24 25 26 demonstrate that ERK1/2 is an important regulator of RSK activity. However, we showed previously that there was no significant difference in the time course or magnitude of ERK1/2 activation in SHR VSMCs compared with WKY VSMCs27 despite increased NHE-1 activity in SHR VSMCs. However, there was a greater dependence on intracellular Ca2+ for ERK1/2 activation in SHR VSMCs. Because RSK is also dependent on increases in intracellular Ca2+ for activation by angiotensin II, our data suggest that another Ca2+-dependent kinase may be involved in RSK activation. Similar mechanisms have also been suggested by Chen and colleagues.13 22 Thus, future studies will focus on characterizing Ca2+-dependent NHE-1 kinases that may be altered in SHR VSMCs.
Selected Abbreviations and Acronyms
|ERK||=||extracellular signal–regulated kinase(s)|
|MAP kinase||=||mitogen-activated protein kinase|
|MEK||=||MAP kinase/ERK kinase|
|NHE-1||=||Na+-H+ exchanger isoform-1|
|PKC||=||protein kinase C|
|PMA||=||phorbol 12-myristate 13-acetate|
|RSK||=||90-kD S6 kinase (p90rsk)|
|SHR||=||spontaneously hypertensive rat(s)|
|VSMC||=||vascular smooth muscle cell|
This study was supported by a grant from the National Institutes of Health (RO1 HL-44721 to Dr Berk). Dr Berk is an Established Investigator of the American Heart Association. We thank Masatoshi Kusuhara and members of the Berk laboratory for helpful discussions.
This manuscript was sent to Laurence H. Kedes, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
- Received December 19, 1996.
- Accepted May 28, 1997.
- © 1997 American Heart Association, Inc.
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