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Am J Physiol Cell Physiol 291: C1308-C1317, 2006. First published May 24, 2006; doi:10.1152/ajpcell.00618.2005
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RECEPTORS AND SIGNAL TRANSDUCTION

ERK1/2 regulates ANG II-dependent cell proliferation via cytoplasmic activation of RSK2 and nuclear activation of elk1

Michael D. Godeny and Peter P. Sayeski

Department of Physiology and Functional Genomics, University of Florida College of Medicine, Gainesville, Florida

Submitted 12 December 2005 ; accepted in final form 18 May 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In a concurrently submitted article, we show that ANG II-induced ERK1/2 activation is mediated by both c-Src/Yes/Fyn and heterotrimeric G protein/PKC{zeta}-dependent signaling. Furthermore, we show that heterotrimeric G protein/PKC{zeta}-activated ERK1/2 is destined for the nucleus while ERK1/2 activated by c-Src/Yes/Fyn-dependent signaling remains in the cytoplasm. Interestingly, both mechanisms of activation are required for maximum ANG II-induced cell proliferation. In this study, we sought to determine the mechanisms by which ERK1/2 facilitate cell proliferation via these distinct nuclear and cytoplasmic events, using cells that were lacking either c-Src/Yes/Fyn or heterotrimeric G protein/PKC{zeta}-dependent ERK1/2 activation. A loss of c-Src/Yes/Fyn blocked ANG II-dependent RSK2 activation, RSK2 nuclear translocation, serum-response factor (SRF) phosphorylation, a portion of c-fos transcriptional activity and c-Fos phosphorylation. Blocking ANG II-induced heterotrimeric G protein/PKC{zeta} activity resulted in a loss of ERK1/2 nuclear translocation, elk1 phosphorylation, and the remaining portion of c-fos transcriptional activity not dependent on c-Src/Yes/Fyn. Inhibition of RSK with the potent and selective inhibitor, SL0101, attenuated ANG II-induced cell proliferation, and, in combination with a PKC{zeta} pseudosubstrate, completely attenuated cell proliferation. Thus we conclude that ERK1/2 mediate ANG II-dependent cell proliferation via distinct cytoplasmic and nuclear signaling events, which are in turn governed by c-Src/Yes/Fyn and heterotrimeric G protein/PKC{zeta}-dependent signaling, respectively.

SL0101; angiotensin II; ribosomal S6 kinase

THE ANGIOTENSIN II (ANG II) type 1 receptor (AT1R) is like other traditional seven-transmembrane spanning receptors in that it couples to and activates heterotrimeric G proteins, subsequently initiating downstream signaling events. More recent work (10, 15), however, has shown that the AT1R can also recruit and activate kinases independent of heterotrimeric G protein signaling, including the well-known tyrosine kinases of the Jak and Src families. These findings initiated a wave of research aimed at characterizing the intracellular signaling events associated with ANG II-induced tyrosine kinase activation. Generally, the activation of tyrosine kinase vs. heterotrimeric G protein-dependent signaling cascades through the AT1R have been described as separate events leading to specific cellular outcomes, with little focus placed upon the integration of these signaling events inside the cell.

Extracellular signal-related kinase 1 and 2 (ERK1/2) are examples of two proteins whose activities are regulated by both heterotrimeric G protein and tyrosine kinase-dependent signaling (9, 18, 20, 23, 28). In our companion paper (7; see p. 1297 of this issue), we show that in response to angiotensin II, ERK1/2 activation is mediated independently by either Src family tyrosine kinase-dependent signaling or heterotrimeric G protein/PKC{zeta}-dependent signaling in a variety of cell types. Both of these signaling mechanisms accounted for roughly 50% of ANG II-induced ERK1/2 activation. Interestingly, heterotrimeric G protein/PKC{zeta} signaling regulates ERK1/2 nuclear translocation, while c-Src/Yes/Fyn-dependent signaling influences cytoplasmic ERK1/2 activation in response to ANG II. Furthermore, these two pathways were both implicated in ANG II-induced cell proliferation, with inhibition of both signaling cascades necessary to achieve complete attenuation of ANG II-induced cell proliferation. These data were striking since previous reports had shown that ERK1/2 nuclear translocation is a critical step in initiating the transcription of early response genes such as c-fos (18). However, it appears here that cytoplasmic ERK1/2, under the control of c-Src/Yes/Fyn, also mediates ANG II-induced cell proliferation independent of nuclear ERK1/2.

One explanation for how cytoplasmic ERK1/2 can influence early response gene transcription is that it phosphorylates cytoplasmic substrates, which in turn translocate into the nucleus and regulate transcriptional activity. Members of the ribosomal S6 kinase (RSK) family of proteins are well-known cytoplasmic targets of ERK1/2, and have been shown to promote the transcription and translation of selected mRNAs important for cell growth (11). Previous reports (19, 25, 27) have shown that ERK1/2 activates members of the RSK family of proteins, however, it is not clear if RSK represents a possible pathway by which ANG II-activated ERK can initiate the events leading to cell proliferation.

In this study, we sought to define the mechanisms whereby ERK1/2 activated by either c-Src/Yes/Fyn or heterotrimeric G proteins/PKC{zeta}-dependent signaling generates the proliferative response associated with the high-affinity binding of ANG II to the AT1R. We hypothesized that c-Src/Yes/Fyn-activated ERK1/2 mediates ANG II-induced cell proliferation through RSK, whereas heterotrimeric G protein/PKC{zeta} signaling regulates cell proliferation through control of ERK1/2 nuclear translocation and subsequent elk1 activation. To examine this, we utilized cells devoid of Src kinases or cells lacking the ability to couple to and activate heterotrimeric G proteins. Both cell lines were stably transfected with the AT1R and were the same as those utilized in our accompanying manuscript (7). We found that ERK1/2, activated via c-Src/Yes/Fyn-dependent signaling, phosphorylates ribosomal S6 kinase 2 (RSK2), which subsequently translocates into the nucleus and modulates c-fos activity at the transcriptional and posttranslational levels. These events partially mediate cell proliferation since pretreatment with SL0101, a potent and specific inhibitor of RSK, significantly attenuated ANG II-induced cell proliferation. ERK1/2 activated by heterotrimeric G protein/PKC{zeta} signaling localizes to the nucleus, where it phosphorylates the transcription factor elk1 and regulates c-fos transcription. Together with ERK1/2-RSK signaling, these events mediate ANG II-induced cell proliferation. As such, this study demonstrates that the AT1R coordinately utilizes both hetrotrimeric G protein and Src family tyrosine kinase signaling to achieve a common cellular outcome via two different mechanisms acting in distinct cellular compartments.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Antibodies and pharmacological inhibitors. A cocktail of phosphospecific ERK1/2 antibodies (Promega and Santa Cruz Biotechnologies) were used to increase signal:noise ratio. Both of these antibodies are specific for phosphothreonine 202 and phosphotyrosine 204 within the conserved TEY motif. The phospho-specific RSK polyclonal antibody [{alpha}RSK(P)-pAb] was purchased from Cell Signaling Technologies and recognizes the phosphothreonine 356/phosphoserine 360 motif. The phospho-specific SRF antibody [SRF(P)] recognizes the phosphorylated Ser103 residue; the phospho-specific elk1 [elk1(P)] monoclonal antibody recognizes phosphoserine 383. Both of these antibodies were purchased from Cell Signaling Technologies. The RSK1 antibody ({alpha}RSK1-pAb) and the RSK2 antibody ({alpha}RSK2-pAb) were obtained from Santa Cruz Biotechnology. A cocktail of ERK1/2 antibodies ({alpha}ERK1/2-Abs) were used to measure total ERK1/2 protein levels. This cocktail consisted of ERK1/2 monoclonal and polyclonal antibodies from Santa Cruz Biotechnology. The ERK1/2 monoclonal antibody was used separately for immunofluorescence. The {alpha}Tyr(P) mAb (PY20) was from BD Transduction Laboratories. The c-Fos polyclonal antibody ({alpha}cfos-pAb) was from Santa Cruz Biotechnology. The phosphoserine pAb [{alpha}Ser(P)-pAb] was purchased from AnaSpec. The SL0101 compound was purchased from Toronto Research Pharmaceuticals. The PKC{zeta} myristoylated pseudo-substrate (PKC{zeta} MP) was purchased from Biomol Laboratories. PP2 and PD-98059 compounds were obtained from Calbiochem. Leptomycin B (LMB) was purchased from Sigma.

Cell lines and cell culture. The Chinese hamster ovary cell lines (CHO/AT1-WT and CHO/AT1-M5 cells) were a gift from Dr. Kenneth Bernstein, and have an equal abundance of AT1R as well as affinity for ANG II (5). The WT/AT1 and SYF/AT1 mouse embryonic fibroblast cells have also been described (7).

Cell lysate preparation and Western blot analysis. Proteins were detected using enhanced chemiluminescence exactly as described (21). For Western blots, the cells were lysed in RIPA buffer containing protease inhibitors (1).

Immunoprecipitation. Cells were washed with 2 volumes of ice-cold PBS containing 1 mM Na3VO4 and lysed in 1.0 ml of ice-cold RIPA buffer plus protease inhibitors (1). The samples were immunoprecipitated exactly as described previously using the indicated antibodies (21).

Densitometric analysis. Western blots were scanned and densitized using UnScanIt Gel Analysis (Silk Scientific). The average pixel value minus background was obtained for each cell type and normalized to the average pixel value for the respective non-ANG II-treated cells.

Immunofluorescence. WT/AT1 and SYF/AT1 cells were plated onto four-chambered slides (Lab-Tek) and grown to 70% confluency. Cells were serum starved in serum-free DMEM supplemented with BSA for 48 h. Following starvation, all cells were ligand treated with 10–7 M ANG II. Slide chambers were removed from slides, and cells were washed once with PBS (pH 7.4). Cells were fixed in 4% paraformaldehyde (Fisher) for 10 min at room temperature, rinsed in PBS, and permeabilized for 3 min in acetone (Fisher) at –20°C. Cells were rinsed three times in PBS, and blocked for 15 min in 3% BSA/PBS at room temperature in a homemade hydration chamber to prevent evaporation. Cells were incubated with the primary antibody indicated for each experiment (1:500 in 3% BSA/PBS) for 1 h. All cells were next rinsed three times in PBS. Cells were incubated with the appropriate fluorochrome-conjugated secondary antibody (1:100 in 3% BSA/PBS) for 1 h at room temperature in a hydration chamber. In the case of the {alpha}RSK2-pAb, the {alpha}rabbit IgG-FITC secondary antibody (Sigma) was used. For the {alpha}ERK2-mAb and {alpha}elk1(P)-mAb, the {alpha}mouse IgG-Texas Red secondary antibody (Sigma) was used, while {alpha}Goat IgG-FITC secondary antibody (Santa Cruz) was utilized in conjunction with the {alpha}SRF(P)-pAb. Upon completion of incubation with the appropriate secondary antibody, cells were washed three times in PBS. A glass coverslip was mounted to each slide using Vectashield + DAPI mounting medium (Vector Labs). The edges of the slides were sealed with nail polish sealant (Maybelline) and allowed to dry. All dry slides were stored at –20°C until viewed. Slides were viewed on a Zeiss Axioplan II Fluorescence microscope.

Quantification of nuclear and cytoplasmic fluorescence. Fluorescence in the nucleus or cytoplasm was quantified using the NIH Image J Progam. Pixel intensity readings were taken from either the nucleus or cytoplasm of multiple cells within each field. Values were averaged for each treatment group representative of three independent experiments. Nuclear fluorescence was then normalized to cytoplasmic fluorescence by dividing the average nuclear pixel intensity by the average cytoplasmic pixel intensity from each treatment group.

c-fos transcriptional activity. SREw/Luc, mSRF/Luc, murine ternaru complex factor (mTCF)/Luc, and TK/Luc plasmids were kindly provided by Dr. Jessica Schwartz (12). WT/AT1 and SYF/AT1 cells were plated onto four-chambered slides (LabTek) and transiently-transfected with each individual plasmid using 8 µl of Lipofectin (Invitrogen). All transfected cells were incubated for 5 h at 37°C. The transfection was stopped by washing cells in PBS, and incubating the cells in serum-containing DMEM for 12–16 h. Cells were serum starved in DMEM + BSA (0.5% wt/vol) for 12–16 h, and treated with 10–7 M ANG II for the amount of time indicated in each experiment. Cells were placed in 1x Reporter Lysis Buffer (Promega), and exposed to one –80°C freeze/thaw cycle (30 min each) to aid in the disruption of cell membrane integrity. Cells were placed on a shaker at room temperature for one additional hour to ensure complete lysis, and then transferred to a microcentrifuge tube. Cells were centrifuged at 12,000 g for 2 min at 4°C, and 20 µl of cell lysate was combined with 100 µl of luciferin substrate (Promega). Luciferase activity was measured with a Monolight 3010 luminometer (PharMingen) at 10-s intervals.

Cell count. WT/AT1 and SYF/AT1 cells were plated onto 100 mm culture dishes and grown to 80% confluency. Cells were serum starved and treated with 10–7 M ANG II as indicated. Both cell types were then counted using a hemocytometer as described (23).

Statistical analysis. Data were analyzed by two-way ANOVA. All data passed a normality test as well as equal variance test. Pairwise comparisons were made following the Holm-Sidak method. All data are expressed as means ± SE; P < 0.05 or P < 0.01.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
c-Src/Yes/Fyn-mediated ERK1/2 activation is required for RSK phosphorylation and ERK1/2-RSK2 co-association. Our accompanying work suggests that ERK1/2 activated by c-Src/Yes/Fyn acts upon cytosolic proteins to promote ANG II-dependent cell proliferation. We first examined whether the phosphorylation of RSK, a well known cytoplasmic substrate of ERK1/2 (18, 25, 27), was dependent upon c-Src/Yes/Fyn and ERK1/2 signaling.

WT/AT1 and SYF/AT1 cells were stimulated with ANG II, and RSK activation assessed via Western blots using a phospho-specific RSK antibody that recognizes phosphorylated RSK1, RSK2, and RSK3. RSK activation occurred in response to ANG II in WT/AT1 cells and was maximal after 10 min of ANG II treatment and declined thereafter (Fig. 1A and data not shown). However, ANG II-induced RSK activation was completely absent from SYF/AT1 cells, indicating that c-Src/Yes/Fyn are necessary for the activation of RSK in response to ANG II. These results were recapitulated by pretreating WT/AT1 cells with PP2, a Src kinase inhibitor (Fig. 1B). In addition, ANG II-induced RSK phosphorylation in WT/AT1 cells was dependent upon ERK1/2 activity because pretreatment with the ERK activation inhibitor, PD-98059, attenuated RSK activation in these cells (Fig. 1C). RSK activation in response to ANG II is therefore mediated by both c-Src/Yes/Fyn and ERK1/2.


Figure 1
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  		Fig. 1. Ribosomal S6 kinase (RSK2) phosphorylation and RSK2-ERK1/2 co-association are decreased in SYF/ANG type I (AT1) cells. A: wild-type (WT)/AT1 cells and SYF/AT1 cells were stimulated with 10–7 M ANG II for 0, 5, and 10 min and total RSK phosphorylation assessed via Western blot (WB) using the indicated antibody (top). Total protein loading was demonstrated by stripping the membrane and reprobing for ERK1/2 (bottom). WT/AT1 cells were pretreated with either 30 µM PP2 (B) or 50 µM PD-98059 (C) for 30 min, and stimulated with 10–7 M ANG II for 0, 5, and 10 min. Total RSK phosphorylation was assessed by Western blot analysis using the indicated antibody (top). Total protein loading was demonstrated by stripping the membrane and reprobing for ERK1/2 (bottom). D: presence or absence of RSK1 and RSK2 in cells was examined by Western blotting WT/AT1 and SYF/AT1 whole cell lysates with the indicated antibodies (top). Control MDCK and NIH3T3 whole cell lysates were also run on the same gel. Total protein loading was demonstrated by stripping the membrane and reprobing for ERK1/2 (bottom). E: active RSK2-ERK co-association was assessed in WT/AT1 and SYF/AT1 whole cell lysates by immunoprecipitating (IP) and Western blot analysis with the indicated antibodies (top). Total protein loading was demonstrated by blotting whole cell lysates with the indicated antibodies (bottom). All Western blots are representative of at least 3 independent blots.

 
We then sought to determine which specific RSK isoform(s) was phosphorylated in response to ANG II. Of the three known RSK isoforms, only RSK1 and RSK2 are phosphorylated by ERK1/2 (6). We therefore prepared whole cell lysates from WT/AT1 and SYF/AT1 cells and Western blotted with RSK1 or RSK2 specific antibodies alongside positive control whole cell lysates. RSK1 was not expressed in WT/AT1 cells, but was expressed in SYF/AT1 cells (Fig. 1D). These expression levels did not change with the addition of ANG II (data not shown). However, RSK2 was expressed equally in both cell types. Therefore, we conclude that RSK2 is most likely the isoform phosphorylated in the presence of c-Src/Yes/Fyn.

Previous reports (22, 25, 27) have shown that active ERK1/2 bind RSK proteins via the ERK docking site and, once bound, modulate RSK activity. We next examined the ability of RSK and ERK to co-associate in the presence or absence of c-Src/Yes/Fyn, to determine whether the ERK1/2 activated by c-Src/Yes/Fyn co-associates with RSK. WT/AT1 and SYF/AT1 whole cell lysates were immunoprecipitated with a phospho-specific ERK1/2 antibody, and then Western blotted with the phospho-specific RSK antibody. ERK1/2-RSK co-association was evident after 5 min of ANG II treatment in WT/AT1 cells, but was completely absent from SYF/AT1 cells (Fig. 1E). As such, these data demonstrate that ERK1/2 and RSK co-associate in response to ANG II, and that ERK1/2 must be activated by c-Src/Yes/Fyn-dependent signaling for these proteins to interact.

RSK2 nuclear translocation is dependent upon c-Src/Yes/Fyn while ERK1/2 nuclear translocation occurs independent of c-Src/Yes/Fyn. Phosphorylated RSK has been shown to translocate into the nucleus and modulate the transcriptional activity of target genes in response to stimulation of various cytokine and growth factor receptors (2, 34). We next examined whether RSK nuclear translocation occurs in response to ANG II, and whether this event is regulated by c-Src/Yes/Fyn. WT/AT1 and SYF/AT1 cells were pretreated with leptomycin B (LMB) to prevent the nuclear exportation of proteins, and then stimulated with ANG II. RSK nuclear accumulation was then assessed by immunofluorescence. Translocation of RSK2 into the nucleus occurred in response to ANG II treatment in WT/AT1 cells, and the nuclear accumulation of RSK2 was confirmed by merging the RSK2 and DAPI images (Fig. 2, A, B, E, and F). However, ANG II-induced RSK2 nuclear translocation did not occur in the absence of c-Src/Yes/Fyn (Fig. 2, C, D, G, and H). Pretreatment of WT/AT1 cells with PP2, a Src family kinase inhibitor, also prevented ANG II-induced RSK2 nuclear accumulation (Fig. 2, I, J, K, and L). Quantification of nuclear and cytoplasmic fluorescence confirmed that RSK2 nuclear translocation occurred in WT/AT1 cells stimulated with ANG II, but was blocked in SYF/AT1 cells or in WT/AT1 cells pretreated with PP2 (Fig. 2M). Collectively, these data show that ANG II-induced RSK nuclear translocation is regulated by c-Src/Yes/Fyn.


Figure 2
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  		Fig. 2. RSK2 and ERK1/2 nuclear phosphorylation in response to ANG II in WT/AT1 and SYF/AT1 cells. A–Z: WT/AT1 and SYF/AT1 cells were pretreated with 0.005 µg of leptomycin B for 5 min before stimulation with 10–7 M ANG II for 10 min. All cells were incubated with antibodies specific for the indicated proteins (right) and respective fluorochrome-conjugated secondary antibody. E–H, K, L, V–Y: images were DAPI stained and merged with the respective fluorescent protein images. I–L: WT/AT1 cells were pretreated with 20 µM PP2 for 1 h before stimulation with 10–7 M ANG II. M: nuclear fluorescence from A–L was quantified and normalized to cytoplasmic fluorescence. Z: nuclear fluorescence from R–U was quantified and normalized to cytoplasmic fluorescence. All images are representative of the entire field and were taken at x40 magnification. Bar represents 15 µm.

 
Next, we planned to determine whether RSK2 nuclear translocation occurred independent of ERK1/2 nuclear translocation. WT/AT1 and SYF/AT1 cells were pretreated with LMB and then stimulated with ANG II. RSK2 and ERK1/2 nuclear translocation were then assessed via immunofluorescence. Both RSK2 and ERK1/2 translocated into the nucleus in response to ANG II in WT/AT1 cells (Fig. 2, N, O, R, and S). Merging of RSK2 and ERK1/2 images confirmed that RSK2 and ERK1/2 nuclear translocation patterns were the same in these cells (Fig. 2, V and W). However, nuclear translocation of ERK1/2 persisted in SYF/AT1 cells while RSK2 nuclear translocation was attenuated (Fig. 2, P, Q, T, and U). Merging of the ERK1/2 and RSK2 images confirmed that these two proteins exhibit different patterns of nuclear translocation in SYF/AT1 cells (Fig. 2, X and Y). Quantification of nuclear and cytoplasmic fluorescence revealed that ERK1/2 nuclear translocation was similar in WT/AT1 and SYF/AT1 cells (Fig. 2Z). Furthermore, pretreatment of cells with PKC{zeta} myristoylated pseudosubstrate (PKC{zeta} MP) attenuated ERK1/2 nuclear translocation in both cell types but had no effect on RSK2 nuclear translocation in response to ANG II (data not shown). As such, these data demonstrate that ERK1/2 nuclear translocation is regulated by PKC{zeta}-dependent signaling whereas RSK2 nuclear translocation is controlled by c-Src/Yes/Fyn.

SRF and elk1 modulate c-fos transcriptional activity in reponse to ANG II. c-fos transcription is regulated via the binding of specific transcription factors to the serum response element (SRE) within the c-fos promoter, namely the serum response factor (SRF) and ternary complex factor (TCF) (12, 14, 16, 34). RSK has been implicated in the phosphorylation of the SRF while ERK1/2 have shown to phosphorylate TCF proteins, thereby increasing the activity of these transcription factors (14, 16, 29). However, the roles of the SRF and the TCF during ANG II-induced c-fos transcription are still unknown.

We first examined whether SRF and TCF activity are regulated by either PKC{zeta}-dependent or c-Src/Yes/Fyn-dependent signaling in response to ANG II. WT/AT1 and SYF/AT1 cells were stimulated with ANG II, and SRF or TCF nuclear phosphorylation assessed via immunofluorescence. Nuclear SRF phosphorylation occurred in response to ANG II treatment in WT/AT1 cells (Fig. 3, A, B, F, and G). ANG II-induced SRF phosphorylation was completely lost in SYF/AT1 cells stimulated with ANG II (Fig. 3, D, E, I, and J). In addition, pretreatment of WT/AT1 cells with PKC{zeta} MP did not affect ANG II-induced SRF phosphorylation (Fig. 3, C and H). Quantification of nuclear and cytoplasmic fluorescence revealed that ANG II-induced SRF phosphorylation in WT/AT1 cells was not affected by PKC{zeta} MP pretreatment, whereas ANG II-induced SRF phosphorylation did not occur in SYF/AT1 cells (Fig. 3Q). Thus SRF phosphorylation is dependent upon c-Src/Yes/Fyn-dependent signaling and not PKC{zeta}.


Figure 3
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  		Fig. 3. ANG II-induced serum-response factor (SRF) and elk-1 nuclear phosphorylation in WT/AT1 and SYF/AT1 cells. A–P: WT/AT1 or SYF/AT1 cells were stimulated with 10–7 M ANG II for 10 min. All cells were incubated with antibodies specific for the indicated proteins (right) and their respective fluorochrome-conjugated secondary antibody. F–J, N–P: protein fluorescence images were merged with DAPI-stained images. C, H, M, and P: cells were pretreated with 1 µM PKC{zeta} MP for 1 h before ANG II treatment. Q: nuclear fluorescence from A–E was quantified and normalized to cytoplasmic fluorescence. R: nuclear fluorescence from K–M was quantified and normalized to cytoplasmic fluorescence. These results are representative of three independent experiments. All images are representative of the entire field and were taken at x40 magnification. Bar represents 15 µm.

 
The nuclear phosphorylation of elk, a TCF activated in response to ERK1/2, was assessed in the same manner. Elk1 phosphorylation occurred in response to ANG II in WT/AT1 cells (Fig. 3, K, L, N, and O). In addition, ANG II-induced elk1 phosphorylation persisted in SYF/AT1 cells (data not shown). Pretreatment with PKC{zeta} MP attenuated elk1 nuclear phosphorylation (Fig. 3, M and P). Quantification of nuclear and cytoplasmic fluorescence revealed that ANG II-induced elk1 phosphorylation in WT/AT1 cells was blocked by PKC{zeta} MP pretreatment (Fig. 3R). Thus TCF phosphorylation appears to be mediated by PKC{zeta}-dependent signaling and not by c-Src/Yes/Fyn.

Both SRF and TCF have been shown to modulate c-fos transcriptional activity in response to growth hormone treatment (12). We therefore sought to determine whether SRF and TCF binding of the c-fos SRE occurred in response to ANG II, and whether these events were altered by the absence of c-Src/Yes/Fyn. WT/AT1 and SYF/AT1 cells were transiently transfected with wild-type or mutated SRE-luciferase plasmids and then stimulated with ANG II. The wild-type SRE plasmid (SREw/Luc) alone mediated ANG II-induced luciferase expression in both cell types (Fig. 4) . ANG II-induced luciferase activity was reduced by ~50% in SREw/Luc transfected SYF/AT1 cells relative to transfected WT/AT1 cells, indicating that c-fos transcriptional activity is in part dependent upon c-Src/Yes/Fyn. Furthermore, mutation of either the SRF or TCF binding sites only partially blocked ANG II-induced luciferase expression in WT/AT1 cells. In addition, ANG II-induced luciferase expression was significantly reduced in SYF/AT1 cells transfected with either the mSRF/Luc or mTCF/Luc plasmids. Note that the reporter plasmid alone (TK/Luc) consistently failed to respond to ANG II in both cell types. These data therefore indicate that the SRF and TCF transcription factors both partially modulate c-fos transcriptional activity in either a c-Src/Yes/Fyn or PKC{zeta}-dependent manner, respectively.


Figure 4
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  		Fig. 4. c-fos transcriptional activity in WT/AT1 and SYF/AT1 cells in response to ANG II. WT/AT1 or SYF/AT1 cells were transfected with the indicated plasmid and then stimulated with ANG II for 0 or 5 h. Data are expressed as percentage of luciferase activity relative to unstimulated cells. These results are representative of 3 independent experiments.

 
Inhibition of PKC{zeta} in addition to the elimination of c-Src/Yes/Fyn, completely attenuated ANG II-induced c-Fos protein synthesis. We next wanted to determine whether a loss of PKC{zeta}-dependent and/or Src kinase-dependent signaling caused a reduction in c-Fos protein levels. WT/AT1 and SYF/AT1 cells were stimulated with ANG II and c-Fos protein levels assessed via Western blot. c-Fos protein exhibited an ANG II-dependent increase in both cell types, and was maximal after 60 min of ANG II treatment (Fig. 5A). However, ANG II-induced c-Fos protein production was reduced in SYF/AT1 cells relative to WT/AT1 cells. Infact, there was about a 50% reduction in c-Fos in SYF/AT1 cells compared with WT/AT1 cells (Fig. 5B). Pretreatment of WT/AT1 cells with PKC{zeta} MP also reduced c-Fos protein amounts by 50%, while PKC{zeta} MP addition to ANG II-stimulated SYF/AT1 cells resulted in a complete loss of ANG II-induced c-Fos protein production. These data demonstrate that ANG II-induced c-Fos protein synthesis is partially influenced by PKC{zeta} and partially by Src kinase-dependent signaling.


Figure 5
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  		Fig. 5. c-Fos protein levels in response to ANG II in WT/AT1 and SYF/AT1 cells. A: WT/AT1 or SYF/AT1 cells were stimulated with 10–7 M ANG II for 0 or 60 min, and c-Fos protein production and total protein levels assessed by Western blot using the indicated antibodies. Some cells were pretreated with 1 µM PKC{zeta} MP for 1 h as indicated. B: these data are expressed as the percentage of maximum c-Fos protein production in response to ANG II. Protein amounts were densitized and values were normalized to the amount of c-Fos protein in WT/AT1 cells treated with ANG II. These results are representative of three independent experiments.

 
c-Fos phosphorylation at serine residues is attenuated in SYF/AT1 cells. Previous reports (2) have shown that c-fos activity is not only regulated at the transcriptional level, but also post-translationally via specific phosphorylation events. Specifically, phosphorylation of c-Fos by RSK at Ser residues within the COOH-terminal tail increases the stability of c-Fos and the subsequent growth-promoting effects associated with extended c-fos activity (2). Therefore, we next wanted to examine if a loss of c-Src/Yes/Fyn mediated ERK/RSK2 activation also impaired c-Fos phosphorylation in addition to c-fos transcription.

WT/AT1 and SYF/AT1 cells were stimulated with ANG II, and cell lysates were immunoprecipitated with anti-phosphoserine antibody and Western blotted with a c-Fos-specific antibody to assess for changes in c-Fos phosphorylation at Ser residues. A marked fourfold increase in c-Fos phosphorylation was observed in WT/AT1 cells after 60 min of ANG II treatment (Fig. 6A). Phosphorylated c-Fos levels remained elevated after 120 min of ANG II treatment, and began to decline by 240 min. In contrast, a comparatively small increase in c-fos phosphorylation was observed in ANG II-treated SYF/AT1 cells after 60 min, and phosphorylated c-Fos levels declined to baseline amounts by 120 min. These results are displayed graphically, and were normalized to total c-Fos protein amounts to account for differences in total c-Fos protein (Fig. 6B). Collectively, these data suggest that c-Fos phosphorylation is reduced in the absence of c-Src/Yes/Fyn and subsequent ERK/RSK2 activation, in response to ANG II.


Figure 6
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  		Fig. 6. ANG II-induced c-Fos phosphorylation in WT/AT1 and SYF/AT1 cells. WT/AT1 or SYF/AT1 cells were stimulated with 10–7 M ANG II for 0, 60, 120, or 240 min. A: c-Fos phosphorylation was then assessed by immunoprecipitating and Western blot analysis with the indicated antibodies. B: protein bands were densitized and values were expressed as a fold change in c-Fos phosphorylation relative to unstimulated cells as a function of total c-Fos protein amounts. These results are representative of 3 independent experiments.

 
Selective RSK inhibition partially reduced ANG II-induced cell proliferation. We next wanted to assess the consequence of selective RSK inhibition on ANG II-induced cell proliferation since RSK activation, under the influence of Src kinase activated ERK1/2, appears to modulate c-Fos levels and its biological activity. Recently, a highly selective and potent RSK inhibitor, SL0101, was isolated by Lannigan and colleagues (26). Sl0101 has already been shown to selectively inhibit RSK without interfering with upstream activators of RSK like ERK, MEK, EGFR, and PKC (13). Furthermore, this compound has been shown to prevent cell proliferation in cancer cells and thus has established RSK as a target for therapeutic intervention and SL0101 as an anti-cancer agent (3). However, a role for RSK in ANG II-induced cell proliferation remains to be established.

WT/AT1 and SYF/AT1 cells were stimulated with ANG II, and cell proliferation assessed via a direct cell count. A marked threefold increase in cell number was observed in WT/AT1 cells treated with ANG II (Fig. 7A). ANG II-induced increases in cell number were reduced by 1.5-fold in SYF/AT1 cells relative to WT/AT1 cells. Pretreatment with SL0101 reduced WT/AT1 cell number to levels found in SYF/AT1 cells treated with ANG II. However, SL0101 pretreatment did not affect ANG II-induced increases in SYF/AT1 cell number, suggesting that ANG II-induced cell proliferation occurring independent of c-Src/Yes/Fyn is not dependent upon RSK2 and also that SL0101 exhibits low toxicity. Finally, PKC{zeta} MP pretreatment completely attenuated ANG II-induced increases in SYF/AT1 cell number but only partially attenuated ANG II-induced increases in WT/AT1 cell number. Furthermore, the addition of PKC{zeta} MP and SL0101 to WT/AT1 cells blocked all ANG II-induced increases in cell number. These data therefore demonstrate that PKC{zeta} partially regulates ANG II-induced cell proliferation independent of RSK2. Furthermore, RSK2 mediates ANG II-induced cell proliferation downstream of c-Src/Yes/Fyn since the addition of SL0101 to SYF/AT1 cells did not further lower the already reduced amount of ANG II-induced cell proliferation exhibited by these cells.


Figure 7
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  		Fig. 7. ANG II-induced cell proliferation in response to RSK and PKC{zeta} inhibition. A: WT/AT1 and SYF/AT1 cells were stimulated with ANG II for 0 (–) or 24 (+) h. Some cells were pretreated with either 30 µM SL0101, 1 µM PKC{zeta} MP, or both inhibitors in combination for 1 h as indicated. Cells were counted and fold changes in cell number plotted relative to unstimulated cells (left). Right, the intact endogenous ERK1/2 activation pathway in the SYF/AT1 cells. B: Chinese hamster ovary (CHO)/AT1 and CHO/AT1-M5 cells were stimulated with ANG II for 0 (–) or 24 (+) h. Some cells were pretreated with either 30 µM SL0101, 1 µM PKC{zeta} MP, or both inhibitors in combination for 1 h as indicated. Cells were counted and fold changes in cell number plotted relative to unstimulated cells (left). The drawing on the right illustrates the intact endogenous ERK1/2 activation pathway in CHO/AT1-M5 cells. These results are representative of 3 independent experiments.

 
To recapitulate these findings in another cell type, we examined the effect of SL0101 addition on cells devoid of heterotrimeric G protein activation, the CHO/AT1-M5 cells. A marked threefold increase in cell number was observed in CHO/AT1 cells treated with ANG II (Fig. 7B). ANG II-induced increases in cell number were reduced by ~0.5 day in CHO/AT1-M5 cells relative to CHO/AT1 cells. Pretreatment of CHO/AT1-M5 cells with SL0101 completely blocked ANG II-induced increases in cell number, whereas SL0101 partially attenuated cell number in CHO/AT1 cells stimulated with ANG II to levels found in ANG II-treated CHO/AT1-M5 cells. Thus RSK2 does not regulate ANG II-induced cell proliferation through heterotrimeric G protein-dependent mechanisms. The pretreatment of CHO/AT1 cells with PKC{zeta} MP partially reduced ANG II-induced cell proliferation; however, CHO/AT1-M5 cell number was not affected by the addition of PKC{zeta} MP. Therefore, PKC{zeta} appears to partially mediate ANG II-induced cell proliferation, and it does so downstream of heterotrimeric G proteins. Finally, all ANG II-induced cell proliferation in CHO/AT1 cells was blocked by pretreatment with PKC{zeta} MP and SL0101. Collectively, these data further demonstrate that ANG II-induced cell proliferation is regulated by heterotrimeric G protein/PKC{zeta} and c-Src/Yes/Fyn/RSK-dependent signaling.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In our accompanying report (7), we show that intracellular ERK1/2 activation is mediated by both c-Src/Yes/Fyn-dependent and heterotrimeric G protein/PKC{zeta}-dependent signaling and that both of these signaling pathways contribute equally to cell proliferation in response to ANG II. In this report, we extend these findings by defining the mechanism as to how this occurs (summarized in Fig. 8). The key to these findings is that heterotrimeric G protein/PKC{zeta}-dependent signaling dictates that ERK1/2 translocates into the nucleus and phosphorylates specific transcription factors like elk1, leading to increased c-fos transcriptional activity. c-Src/Yes/Fyn signaling on the other hand, phosphorylates ERK1/2 in the cytoplasm, where ERK1/2 remains and complexes with RSK2. RSK2 becomes activated, and then translocates into the nucleus to modulate c-fos transcription and c-Fos protein activity. In combination, these two signaling events coordinately regulate proliferation in response to ANG II.


Figure 8
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  		Fig. 8. Mechanistic diagram illustrating how c-Src/Yes/Fyn and PKC{zeta}-dependent ERK1/2 activation pathways dually regulate ANG II-induced cell proliferation. ERK1/2 activation is separately mediated by Src family tyrosine kinase and heterotrimeric G protein/PKC{zeta} signaling in response to ANG II. With regard to PKC{zeta} regulation, ERK1/2 translocates into the nucleus upon stimulation of the AT1R. Here, ERK1/2 phosphorylates elk1, which binds to the c-Fos SRE and partially regulates c-fos transcriptional activity. c-fos transcriptional activity is also regulated by binding of the SRF. The SRF is phosphorylated in response to nuclear RSK2, which translocates into the nucleus after being phosphorylated by ERK1/2 in the cytoplasm. Cytoplasmic ERK1/2 phosphorylation is regulated by c-Src/Yes/Fyn-dependent signaling, independent of heterotrimeric G protein/PKC{zeta} activity. In addition, nuclear RSK2 directly phosphorylates c-Fos and increases the activity and stability of this protein. Thus two independent pathways of ERK1/2 activation coordinately regulate ANG II-induced cell proliferation by inducing c-fos transcription and increasing c-Fos activity through the posttranslational modification of this protein.

 
These findings support the idea that two separate pools of ERK1/2 exist within the cell; a pool of ERK1/2, which complexes with and activates RSK2 and a pool of ERK1/2, which translocates directly into the nucleus. Previous studies (8) support this idea. In Xenopus laevis oocytes, as much as 50% of the total pool of ERK2 complexed with RSK. However, ERK2 activation resulted in the dissociation of the ERK2-RSK complex within these cells. Other studies in COS-7 cells ectopically expressing the three RSK isoforms demonstrated that ERK-RSK complexes did not dissociate when ERK1/2 was activated by EGF (33). Here, we show that ERK1/2 and RSK2 rapidly co-associate in response to ANG II, with maximum co-association occurring after 10 min of ANG II treatment. This co-association is completely dependent upon c-Src/Yes/Fyn signaling. Other pools of ERK1/2 do not co-associate with RSK because we see nuclear accumulation of ERK1/2 occurring well before RSK2 nuclear translocation happens, indicating that some ERK1/2 does not complex with RSK2. In addition, this nuclear translocation of ERK1/2 occurs at the same time as a portion of ERK1/2 complexes with RSK2. Note that RSK2-ERK1/2 complexes do not translocate into the nucleus together since pretreatment of cells with PKC{zeta} MP blocked all ERK1/2 nuclear translocation but did not affect RSK2 nuclear translocation (data not shown). Therefore, ERK1/2 and RSK2 nuclear translocation occur separately in response to ANG II. Further supporting the idea that two different pools of ERK exist inside the cell, our data show that in response to ANG II, some ERK1/2 directly enters the nucleus whereas another portion complexes with RSK2. Previous studies (12) have also shown that a pool of ERK complexes with RSK, though RSK-ERK co-association patterns may differ depending upon the receptor activated.

The duration as well as the magnitude of ERK1/2 activation has also been proposed to regulate gene expression and other specific intracellular responses. Catt and colleagues have shown that the magnitude and duration of ERK1/2 activation in response to gonadotropin-releasing hormone (GnRH) depends upon the mechanism whereby ERK1/2 is activated, with a more sustained pattern of ERK activation occurring through G{alpha}q/PKC-dependent signaling, and a more transient pattern of ERK activation caused by transactivation of the EGFR by the GnRH receptor (24). Sustained GnRH-induced ERK1/2 activation lead to ERK1/2 nuclear accumulation, whereas ERK1/2 activated transiently failed to accumulate in the nucleus. Other reports have shown that sustained vs. transient patterns of ERK1/2 lead to different cellular responses, including cell death or cell growth/proliferation (17). We previously showed that more sustained patterns of ANG II-induced ERK1/2 activation occurred in cells, in which both c-Src/Yes/Fyn and heterotrimeric G protein/PKC{zeta} signaling were intact (7). More transient patterns of ERK1/2 activation are evident when either of these pathways were disrupted. Here, we demonstrate that a longer and more robust ERK1/2 activation pattern achieved through the simultaneous activation of both signaling cascades resulted in greater amounts of ANG II-induced cell proliferation. Less robust and transient patterns of ERK1/2 activation achieved by the disruption of one pathway resulted in a diminished amount of cell proliferation in response to ANG II. We found cell proliferation to be dependent upon the amount of c-fos transcribed and phosphorylated, with a greater amount of c-fos transcription occurring when both heterotrimeric G protein/PKC{zeta} and c-Src/Yes/Fyn signaling pathways were dually activated. Furthermore, c-fos stability was extended when c-Src/Yes/Fyn-dependent signaling was intact. Thus the magnitude and duration of ERK1/2 activation affect ANG II-induced cell proliferation as well.

ANG II-induced cell proliferation is associated with aberrant ANG II release and subsequent cell growth and proliferation during the progression of various cardiovascular diseases and cancers (4, 28, 30–32). Here, we show that both heterotrimeric G protein and Src family tyrosine kinase signaling must be blocked to completely attenuate ANG II-induced ERK1/2 activation. RSK2 can also be inhibited, as we show this to be the downstream target for ERK1/2 activated by c-Src/Yes/Fyn-dependent signaling. Thus, SL0101, a compound previously shown to be an anti-cancer agent, may also provide possible therapeutic benefits to patients suffering from certain cardiovascular diseases.

In summary, we show that ANG II-induced cell proliferation is mediated by two mechanistically different signaling pathways, both dependent on ERK1/2. Whether ERK1/2 activates RSK2 or translocates into the nucleus to mediate cell proliferation, is determined by c-Src/Yes/Fyn or heterotrimeric G protein/PKC{zeta} signaling, respectively. Interestingly, both of these pathways positively regulate c-fos transcription and c-Fos protein activity via the phosphorylation of different transcription factors, which initiate cell proliferation. Thus, this work demonstrates that the AT1R, a prototypical GPCR, coordinately utilizes both heterotrimeric G protein and Src family tyrosine kinase-dependent signaling pathways to achieve angiotensin II-induced proliferation.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a Biomedical Research Support Program for Medical Schools Award to the University of Florida College of Medicine by the Howard Hughes Medical Institute, an American Heart Association Florida/Puerto Rico Affiliate Grant in Aid 0555359B, and National Institutes of Health Awards K01-DK60471 and R01-HL67277. M. Godeny was supported by a predoctoral research fellowship from the Florida/Puerto Rico Affiliate of the American Heart Association (0515138B).


   ACKNOWLEDGMENTS
 
We are indebted to Dr. Philippe Soriano for providing us with the mouse embryonic fibroblasts and Dr. Kenneth Bernstein for the Chinese hamster ovary cells. We also thank Dr. Jessica Schwartz for kindly providing us with the c-fos SRE plasmids.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. P. Sayeski, Dept. of Physiology and Functional Genomics, Univ. of Florida, College of Medicine, PO Box 100274, Gainesville, FL 32610 (e-mail: psayeski{at}phys.med.ufl.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, and Struhl K. Current Protocols in Molecular Biology. New York: Wiley, 1995.

2. Chen RH, Sarnecki C, and Blenis J. Nuclear localization and regulation of erk- and rsk-encoded protein kinases. Mol Cell Biol 12: 915–927, 1992.[Abstract/Free Full Text]

3. Clark DE, Errington TM, Smith JA, Frierson HF Jr,. Weber MJ, and Lannigan DA. The serine/threonine protein kinase, p90 ribosomal S6 kinase, is an important regulator of prostate cancer cell proliferation. Cancer Res 65: 3108–3116, 2005.[Abstract/Free Full Text]

4. Deshayes F and Nahmias C. Angiotensin receptors: a new role in cancer? Trends Endocrinol Metab 16: 293–299, 2005.[CrossRef][ISI][Medline]

5. Doan TN, Ali MS, and Bernstein KE. Tyrosine kinase activation by the angiotensin II receptor in the absence of calcium signaling. J Biol Chem 276: 20954–20958, 2001.[Abstract/Free Full Text]

6. Frodin M and Gammeltoft S. Role and regulation of 90 kDa ribosomal S6 kinase (RSK) in signal transduction. Mol Cell Endocrinol 151: 65–77, 1999.[CrossRef][ISI][Medline]

7. Godeny MD and Sayeski PP. ANG II-induced cell proliferation is dually mediated by c-Src/Yes/Fyn-regulated ERK1/2 activation in the cytoplasm and PKC{zeta}-controlled ERK1/2 activity within the nucleus. Am J Physiol Cell Physiol 291: C1297–C1307, 2006.[Abstract/Free Full Text]

8. Hsiao KM, Chou SY, Shih SJ, and Ferrell JE Jr. Evidence that inactive p42 mitogen-activated protein kinase and inactive Rsk exist as a heterodimer in vivo. Proc Natl Acad Sci USA 91: 5480–5484, 1994.[Abstract/Free Full Text]

9. Ishida M, Ishida T, Thomas SM, and Berk BC. Activation of extracellular signal-regulated kinases (ERK1/2) by angiotensin II is dependent on c-Src in vascular smooth muscle cells. Circ Res 82: 7–12, 1998.[Abstract/Free Full Text]

10. Ishida M, Marrero MB, Schieffer B, Ishida T, Bernstein KE, and Berk BC. Angiotensin II activates pp60c-src in vascular smooth muscle cells. Circ Res 77: 1053–1059, 1995.[Abstract/Free Full Text]

11. Jefferies HB, Fumagalli S, Dennis PB, Reinhard C, Pearson RB, and Thomas G. Rapamycin suppresses 5'TOP mRNA translation through inhibition of p70s6k. EMBO J 16: 3693–3704, 1997.[CrossRef][ISI][Medline]

12. Liao J, Hodge C, Meyer D, Ho PS, Rosenspire K, and Schwartz J. Growth hormone regulates ternary complex factors and serum response factor associated with the c-fos serum response element. J Biol Chem 272: 25951–25958, 1997.[Abstract/Free Full Text]

13. Maloney DJ and Hecht SM. Synthesis of a potent and selective inhibitor of p90 Rsk. Org Lett 7: 1097–1099, 2005.[CrossRef][ISI][Medline]

14. Marais R, Wynne J, and Treisman R. The SRF accessory protein Elk-1 contains a growth factor-regulated transcriptional activation domain. Cell 73: 381–393, 1993.[CrossRef][ISI][Medline]

15. Marrero MB, Schieffer B, Paxton WG, Heerdt L, Berk BC, Delafontaine P, and Bernstein KE. Direct stimulation of Jak/STAT pathway by the angiotensin II AT1 receptor. Nature 375: 247–250, 1995.[CrossRef][Medline]

16. Misra RP, Rivera VM, Wang JM, Fan PD, and Greenberg ME. The serum response factor is extensively modified by phosphorylation following its synthesis in serum-stimulated fibroblasts. Mol Cell Biol 11: 4545–4554, 1991.[Abstract/Free Full Text]

17. Pearson G, Robinson F, Beers Gibson T, Xu BE, Karandikar M, Berman K, and Cobb MH. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev 22: 153–183, 2001.[Abstract/Free Full Text]

18. Sadoshima J and Izumo S. The heterotrimeric Gq protein-coupled angiotensin II receptor activates p21 ras via the tyrosine kinase-Shc-Grb2-Sos pathway in cardiac myocytes. EMBO J 15: 775–787, 1996.[ISI][Medline]

19. Sadoshima J, Qiu Z, Morgan JP, and Izumo S. Angiotensin II and other hypertrophic stimuli mediated by G protein-coupled receptors activate tyrosine kinase, mitogen-activated protein kinase, and 90-kD S6 kinase in cardiac myocytes. The critical role of Ca2+-dependent signaling. Circ Res 76: 1–15, 1995.[Abstract/Free Full Text]

20. Sandberg EM, Ma X, VonDerLinden D, Godeny MD, and Sayeski PP. Jak2 tyrosine kinase mediates angiotensin II-dependent inactivation of ERK2 via induction of mitogen-activated protein kinase phosphatase 1. J Biol Chem 279: 1956–1967, 2004.[Abstract/Free Full Text]

21. Sayeski PP, Ali MS, Harp JB, Marrero MB, and Bernstein KE. Phosphorylation of p130Cas by angiotensin II is dependent on c-Src, intracellular Ca2+, and protein kinase C. Circ Res 82: 1279–1288, 1998.[Abstract/Free Full Text]

22. Scimeca JC, Nguyen TT, Filloux C, and Van Obberghen E. Nerve growth factor-induced phosphorylation cascade in PC12 pheochromocytoma cells. Association of S6 kinase II with the microtubule-associated protein kinase, ERK1. J Biol Chem 267: 17369–17374, 1992.[Abstract/Free Full Text]

23. Seta K, Nanamori M, Modrall JG, Neubig RR, and Sadoshima J. AT1 receptor mutant lacking heterotrimeric G protein coupling activates the Src-Ras-ERK pathway without nuclear translocation of ERKs. J Biol Chem 277: 9268–9277, 2002.[Abstract/Free Full Text]

24. Shah BH, Farshori MP, Jambusaria A, and Catt KJ. Roles of Src and epidermal growth factor receptor transactivation in transient and sustained ERK1/2 responses to gonadotropin-releasing hormone receptor activation. J Biol Chem 278: 19118–19126, 2003.[Abstract/Free Full Text]

25. Smith JA, Poteet-Smith CE, Malarkey K, and Sturgill TW. Identification of an extracellular signal-regulated kinase (ERK) docking site in ribosomal S6 kinase, a sequence critical for activation by ERK in vivo. J Biol Chem 274: 2893–2898, 1999.[Abstract/Free Full Text]

26. Smith JA, Poteet-Smith CE, Xu Y, Errington TM, Hecht SM, and Lannigan DA. Identification of the first specific inhibitor of p90 ribosomal S6 kinase (RSK) reveals an unexpected role for RSK in cancer cell proliferation. Cancer Res 65: 1027–1034, 2005.[Abstract/Free Full Text]

27. Sturgill TW, Ray LB, Erikson E, and Maller JL. Insulin-stimulated MAP-2 kinase phosphorylates and activates ribosomal protein S6 kinase II. Nature 334: 715–718, 1988.[CrossRef][Medline]

28. Touyz RM, He G, Wu XH, Park JB, Mabrouk ME, and Schiffrin EL. Src is an important mediator of extracellular signal-regulated kinase 1/2-dependent growth signaling by angiotensin II in smooth muscle cells from resistance arteries of hypertensive patients. Hypertension 38: 56–64, 2001.[Abstract/Free Full Text]

29. Treisman R, Marais R, and Wynne J. Spatial flexibility in ternary complexes between SRF and its accessory proteins. EMBO J 11: 4631–4640, 1992.[ISI][Medline]

30. Uemura H, Nakaigawa N, Ishiguro H, and Kubota Y. Antiproliferative efficacy of angiotensin II receptor blockers in prostate cancer. Curr Cancer Drug Targets 5: 307–323, 2005.[CrossRef][ISI][Medline]

31. Xu Q, Liu Y, Gorospe M, Udelsman R, and Holbrook NJ. Acute hypertension activates mitogen-activated protein kinases in arterial wall. J Clin Invest 97: 508–514, 1996.[ISI][Medline]

32. Yamazaki T, Komuro I, Shiojima I, and Yazaki Y. Angiotensin II mediates mechanical stress-induced cardiac hypertrophy. Diabetes Res Clin Pract 30, Suppl: 107–111, 1996.[Medline]

33. Zhao Y, Bjorbaek C, and Moller DE. Regulation and interaction of pp90(rsk) isoforms with mitogen-activated protein kinases. J Biol Chem 271: 29773–29779, 1996.[Abstract/Free Full Text]

34. Zhao Y, Bjorbaek C, Weremowicz S, Morton CC, and Moller DE. RSK3 encodes a novel pp90rsk isoform with a unique N-terminal sequence: growth factor-stimulated kinase function and nuclear translocation. Mol Cell Biol 15: 4353–4363, 1995.[Abstract]




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