A role for PYK2 in ANG II-dependent regulation of the PHAS-1-eIF4E complex by multiple signaling cascades in vascular smooth muscle

Petra Rocic,1 Hanjoong Jo,2 and Pamela A. Lucchesi1

1Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005; and 2Coulter Department of Biomedical Engineering, Georgia Tech and Emory University, Atlanta, Georgia 30322

Submitted 24 February 2003 ; accepted in final form 26 July 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Regulation of the PHAS-1-eukaryotic initiation factor-4E (eIF4E) complex is the rate-limiting step in the initiation of protein synthesis. This study characterized the upstream signaling pathways that mediate ANG II-dependent phosphorylation of PHAS-1 and eIF4E in vascular smooth muscle. ANG II-dependent PHAS-1 phosphorylation was maximal at 10 min (2.47 ± 0.3 fold vs. control). This effect was completely blocked by the specific inhibitors of phosphatidylinositol 3-kinase (PI3-kinase, LY-294002), mammalian target of rapamycin, and extracellular signal-regulated kinase 1/2 (ERK1/2, U-0126) or by a recombinant adenovirus encoding dominant-negative Akt. PHAS-1 phosphorylation was followed by dissociation of eIF4E. Increased ANG II-induced eIF4E phosphorylation was observed at 45 min (2.63 ± 0.5 fold vs. control), was maximal at 90 min (3.38 ± 0.3 fold vs. control), and was sustained at 2 h. This effect was blocked by inhibitors of the ERK1/2 and p38 mitogen-activated protein (MAP) kinase pathways, but not by PI3-kinase inhibition, and was dependent on PKC, intracellular Ca2+, and tyrosine kinases. Downregulation of proline-rich tyrosine kinase 2 (PYK2) by antisense oligonucleotides led to a near-complete inhibition of PHAS-1 and eIF4E phosphorylation in response to ANG II. Therefore, PYK2 represents a proximal signaling intermediate that regulates ANG II-induced vascular smooth muscle cell protein synthesis via regulation of the PHAS-1-eIF4E complex.

tyrosine kinase; antisense oligonucleotides; protein synthesis


A MAJOR CHARACTERISTIC of angiotensin II (ANG II)-induced hypertrophic growth is the increase in protein synthesis. Regulation of the complex between the eukaryotic initiation factor-4E (eIF4E) and its inhibitory protein PHAS-1 (also called EIF-4E- or eIF4E-binding protein or 4E-BP1) has been shown to be critical for translation initiation, the rate-limiting step for protein synthesis in many cell types (26). eIF4F is a protein complex consisting of an ATP-dependent helicase (eIF4A), an eIF4G, a linker subunit, and eIF4E, which allows the complex to bind to the mRNA cap structure (36). eIF4E has been a target for studies on the regulation of translation initiation, because it is present in rate-limiting amounts relative to the other factors and its RNA cap-binding ability correlates with its phosphorylation on Ser209 (36). Activity of eIF4E is upregulated on treatment with growth factors, mitogens, and phorbol esters, and this activation results in an increase in protein translation and changes in cell growth rate (10, 39, 42, 45). Recently, Rao et al. (34, 35) demonstrated that ANG II and oxidative stress increase the phosphorylation of eIF4E in cultured vascular smooth muscle cells (VSMC).

The function of eIF4E is also tightly regulated by interaction with a binding protein termed PHAS-1, a phosphorylated, heat- and acid-stable protein of apparent molecular weight 22,000 that acts as a translational repressor (28). Nonphosphorylated PHAS-1 binds tightly to eIF4E and inhibits protein synthesis, presumably by preventing eIF4E from binding to the mRNA cap structure. PHAS-1 phosphorylation results in its dissociation from eIF4E and the subsequent phosphorylation of eIF4E, which allows for mRNA binding and translation initiation (42).

The signaling pathways that regulate the phosphorylation state of the PHAS-1-eIF4E complex in response to ANG II in VSMC have not been fully characterized. Two signaling pathways that may converge on the regulation of protein synthesis are the extracellular signal-regulated kinase 1/2 (ERK1/2) MAP kinase and the phosphatidylinositol 3-kinase (PI3-kinase) pathway. Translation initiation and protein synthesis have been shown, in other cell types, to be dependent on PI3-kinase and its downstream effectors Akt (5) and ribosomal p70 S6 kinase (6, 8, 12, 18). Recently, the activation of PI3-kinase, Akt, and p70 S6 kinase has been shown to be critical for protein synthesis in VSMC. For example, p70 S6 kinase is thought to be the major in vivo mediator of ribosomal S6 protein phosphorylation, a necessary step in ANG II-mediated protein synthesis in VSMC (43).

ERK1/2 MAP kinases are thought to be involved in the regulation of translation initiation through phosphorylation of the PHAS-1-eIF4E complex. On dissociation, eIF4E undergoes phosphorylation by ERK1/2 (42) directly or via an intermediate kinase termed Mnk-1 (36). eIF4E can be phosphorylated in response to ANG II and oxidative stress in VSMC (34, 35).

The proximal signaling events that control phosphorylation of PHAS-1 and eIF4E and VSMC translation initiation in response to ANG II remain to be determined. The tyrosine kinases proline-rich tyrosine kinase 2 (PYK2) (14, 38), janus kinase (41), src (2, 25), and focal adhesion kinase (21), as well as epidermal growth factor (EGF) receptor transactivation (2, 13) and the NADH/NADPH oxidase (23), have been implicated in the regulation of ANG II-induced VSMC protein synthesis. We recently demonstrated that PYK2 links ANG II type 1 (AT1) receptor activation to the ERK1/2 and PI3-kinase signaling pathways (37, 38), suggesting that PYK2 may represent an upstream signaling molecule that will regulate parallel pathways involved in the regulation of translation initiation.

The aim of the present study is to identify the proximal signaling events that link AT1 receptor activation to the regulation of the PHAS-1-eIF4E complex as well as to clearly establish the roles of ERK1/2 and PI3-kinase signaling cascades in the regulation of PHAS-1 and eIF4E phosphorylation. The results from this study indicate that PYK2 is a key regulator of PHAS-1 and eIF4E phosphorylation via regulation of multiple signaling pathways.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials. BAPTA-AM was obtained from Alexis Laboratories; chelerythrine, U-0126, LY-294002, SB-203580, rapamycin, and genistein from Calbiochem; anti-total PHAS-1 antibodies from Oncogene; anti-total eIF4E and PYK2 antibodies from Transduction Laboratories; antiphospho-PHAS-1 (Thr70 and Ser65) and antiphospho-eIF4E (Ser209) antibodies from Cell Signaling; and anti-phospho- and total p38 antibodies from Promega. PYK2 antisense oligonucleotides and scrambled control oligonucleotides were custom-designed by Biognostik (Göttingen, Germany).

Cell culture and adenoviral transfection. VSMC from 10- to 12-wk-old male Sprague-Dawley rat thoracic aortas were isolated by enzymatic digestion as described elsewhere (37) and used at passages 3-6. The adenoviral construct dominant-negative (DN)-Akt-Adv (Thr308Ala and Ser473Ala) was used to overexpress DN-Akt mutant. VSMC at 80% confluency were treated at 50 multiplicity of infection adenovirus in serum-free DMEM for 1 h, washed with DMEM, and incubated for 48 h under serum-free conditions. Recombinant adenovirus encoding bacterial {beta}-galactosidase (Ad-LacZ) was used as a negative control. Infection efficiency was close to 100% as determined by immunocytochemical staining of {beta}-galactosidase (data not shown).

PYK2 antisense oligonucleotide incorporation. VSMC were grown in 10% calf serum (CS)-DMEM to ~60% confluency. Cells were washed three times in Opti-MEM (GIBCO-BRL) 1 h before antisense treatment. VSMC were treated with PYK2 antisense oligonucleotides (0.75 µM) for 8 h. LipofectAMINE Plus (10 µg/ml; GIBCO-BRL) was used as a transfection reagent. After 8 h, the medium was replaced with 0.2% CS-DMEM and left overnight. On the next day, 0.2% CS-DMEM was replaced with serum-free DMEM for >=1 h before treatment with ANG II.

Immunoblotting. PHAS-1 immunoprecipitates (800 µg) or VSMC cell lysates (100 µg) were separated by SDS-PAGE and transferred to nitrocellulose. Immunoblot analysis was performed using monoclonal antiphospho-PHAS-1 antibodies, antiphospho-eIF4E antibodies, antiphospho-p38, antiphospho-Akt, total p38, and total PYK2. All antibodies were used at a 1:1,000 dilution. Bands were visualized by enhanced chemiluminescence (ECL, Amersham) and quantified using NIH Image software.

Immunoprecipitation. Cell lysates were prepared for immunoprecipitation as described elsewhere (40). Equal amounts of protein (800 µg) were immunoprecipitated with polyclonal anti-PHAS-1 antibodies overnight at 4°C. Immune complexes were collected by incubation with protein A-Sepharose for 2 h at 4°C. Immunoprecipitates were separated by SDS-PAGE, and proteins were detected by immunoblotting as described above using anti-eIF4E or antiphospho-PHAS-1 antibodies (1:1,000).

Data analysis. Blots are representative of at least three experiments. One-way repeated-measures analysis of variance followed by Bonferroni's t-test was used for comparisons among multiple groups. Differences among means were considered significant at P < 0.05. Data were analyzed using InStat statistical software (GraphPad).


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ANG II-induced PHAS-1 phosphorylation is dependent on PI3-kinase and ERK1/2. The time course of PHAS-1 phosphorylation by ANG II detected by antiphospho-specific PHAS-1 antibodies is shown in Fig. 1. Maximal PHAS-1 phosphorylation occurred at 10 min (2.47 ± 0.3 fold vs. control), was sustained at 60 min, and began to decline by 90 min (Fig. 1, top). Similar results were obtained when PHAS-1 was immunoprecipitated using anti-total PHAS-1 antibodies and then subjected to immunoblotting with antiphospho-PHAS-1 antibodies (Fig. 1, bottom).



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Fig. 1. ANG II-induced PHAS-1 phosphorylation. Top: growth-arrested vascular smooth muscle cells (VSMC) were treated with 100 nM ANG II for 0-90 min. Western blot analysis was performed with antiphospho (pThr70 and pSer65)-PHAS-1 antibodies (top blot). Cell lysates were immunoprecipitated (IP) with anti-total PHAS-1 antibodies, and Western blot analysis was performed with antiphospho-PHAS-1 antibodies (bottom blot). Bottom: cumulative data from Western blot analysis of 3 experiments. *P < 0.05 vs. control.

 

Preliminary experiments demonstrated that ERK1/2 and p70 S6 kinase were able to phosphorylate recombinant PHAS-1 in in vitro kinase assays (data not shown). To determine the exact roles of ERK1/2, p38 MAP kinase, PI3-kinase, and the mammalian target of rapamycin (mTOR) effector p70 S6 kinase in the regulation of PHAS-1 phosphorylation in intact cells, we pretreated VSMC with the specific inhibitors of these pathways. ANG II-dependent PHAS-1 phosphorylation was completely blocked by inhibitors of the ERK1/2 pathway (5 µM U-0126), PI3-kinase (10 µM LY-294002), and p70 S6 kinase (10 nM rapamycin), but not by the p38 inhibitor (20 µM SB-203580; Fig. 2A, top). To gain insight into the possible upstream regulators of PHAS-1 phosphorylation, VSMC were pretreated with the PKC inhibitor chelerythrine (5 µM), a tyrosine kinase inhibitor (1 µM genistein), and an intracellular Ca2+ chelator (50 µM BAPTA-AM). Pretreatment with all three inhibitors resulted in complete inhibition of ANG II-induced PHAS-1 phosphorylation (Fig. 2, top). Recently, the specificity of chelerythrine as a PKC inhibitor has been questioned, because it has been reported to inhibit the activity of other kinases in in vitro assays with purified recombinant kinases (7) and promotes cardiac myocyte apoptosis in a PKC-independent manner (7). Therefore, we repeated these experiments with an additional PKC inhibitor Ro 31-8220 (10 µM). Pretreatment with this inhibitor completely blocked ANG II-induced PHAS-1 phosphorylation (Fig. 2, bottom).



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Fig. 2. ANG II-induced PHAS-1 phosphorylation is dependent on phosphatidylinositol 3-kinase (PI3-kinase), ERK1/2, mammalian target of rapamycin (mTOR), PKC, tyrosine kinases, and Ca2+. Top: growth-arrested VSMC were pretreated with 10 µM LY-294002 (LY), 20 µM SB-203580 (SB), 5 µM U-0126 (U0), 10 nM rapamycin (Rap), 5 µM chelerythrine (Chel), 1 µM genistein (Gen), or 50 µM BAPTA-AM (BAP) for 45 min before treatment with 100 nM ANG II for 15 min. Western blot analysis was performed using antiphospho (pThr70 and pSer65)-PHAS-1 antibodies. Cumulative data from 3 experiments are shown. *P < 0.05 vs. control (Ctrl). {dagger}P < 0.05 vs. ANG II. Bottom: growth-arrested VSMC were pretreated with 10 µM Ro-31-8220 (Ro) for 45 min before treatment with 100 nM ANG II for 10 min. *P < 0.05 vs. control.

 

There have been reports that Akt phosphorylation may be indirectly inhibited by SB-203580, because this inhibitor is able to block PDK1 (4), an upstream activator of Akt (47). We first determined whether SB-203580 was able to block Akt activation in response to ANG II. Pretreatment with SB-203580 attenuated ANG II-induced Akt phosphorylation but had no effect on p70 S6 kinase phosphorylation (Fig. 3A). To clarify the role for Akt, we examined whether overexpression of a DN Akt mutant, generated by substituting Ala at two major regulatory phosphorylation sites (Thr308 and Ser473), would inhibit phosphorylation of PHAS-1 stimulated by ANG II. Overexpression of DN-Akt substantially inhibited phosphorylation of PHAS-1 in response to ANG II (Fig. 3B). In contrast, LacZ-Adv did not have any effect on phosphorylation of PHAS-1, showing the specific effect of DN-Akt.



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Fig. 3. ANG II-induced PHAS-1 phosphorylation is dependent on Akt. A: VSMC were pretreated with 20 µM SB-203580 for 45 min before treatment with 100 nM ANG II for 15 min. Western blot analysis was performed using antiphospho- and anti-total p70 S6 kinase antibodies or antiphospho (pThr70 and pSer65)-PHAS-1 antibodies. B: VSMC were infected with LacZ-Adv or dominant-negative adenoviral Akt (DN-Akt-Adv) and then treated with 100 nM ANG II for 15 min. Western blot analysis was performed using antiphospho-PHAS-1 antibodies (top blot) or anti-PHAS-1 antibodies (bottom blot). Cumulative data from 3 experiments are shown. C, control. *P < 0.05 vs. control. {dagger}P < 0.05 vs. ANG II.

 

PHAS-1 phosphorylation is followed by the release of eIF4E. On phosphorylation, PHAS-1 dissociates from eIF4E. This step is critical for subsequent phosphorylation of eIF4E and its binding to the 5' mRNA cap (36). Coimmunoprecipitation analysis was used to demonstrate that PHAS-1 phosphorylation in response to ANG II results in dissociation of the PHAS-1-eIF4E complex (Fig. 4). The time course for complex dissociation closely follows that of PHAS-1 phosphorylation (Fig. 4A). Reassociation was observed at 60 min but was not complete at 90 min.



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Fig. 4. Phosphorylation of PHAS-1 is associated with release of eukaryotic initiation factor 4E (eIF4E). A: growth-arrested VSMC were treated with 100 nM ANG II for 0-90 min. Cell lysates were immunoprecipitated with anti-total PHAS-1 antibodies, and Western blot analysis was performed with anti-phospho-PHAS-1, anti-total eIF4E, or anti-total PHAS-1 antibodies. B: growth-arrested VSMC were pretreated with 10 µM LY-294002, 5 µM U-0126, 5 µM chelerythrine, 1 µM genistein, 50 µM BAPTA-AM, or 20 µM SB-203580 for 45 min before treatment with 100 nM ANG II for 30 min. Cell lysates were immunoprecipitated with anti-total PHAS-1 antibodies, and Western blot analysis was performed with anti-total eIF4E antibodies.

 

To determine whether the inhibition of PHAS-1 phosphorylation affects the dissociation of the PHAS-1-eIF4E complex, we investigated the effects of ERK1/2 and PI3-kinase inhibition. ANG II-induced complex dissociation was blocked by U-0126 and LY-294002, as well by PKC and tyrosine kinase inhibitors and intracellular Ca2+ chelation (Fig. 4B). Inhibition of p38 MAP kinase phosphorylation by SB-203580 had no effect on ANG II-induced complex dissociation, consistent with its inability to block PHAS-1 phosphorylation on Thr70 and Ser65.

ANG II-induced eIF4E phosphorylation is dependent on ERK1/2 and p38 MAP kinases but not on PI3-kinase signaling. On dissociation, eIF4E is phosphorylated, increasing its binding to the 5' mRNA cap. We next determined the time course of eIF4E phosphorylation by ANG II. eIF4E phosphorylation was significantly increased at 30 min (2.68 ± 0.2 fold vs. control), reached a maximum at 90 min (3.38 ± 0.3 fold vs. control), and was sustained at 2 h (Fig. 5). ANG II-induced eIF4E phosphorylation was significantly blocked by ERK1/2 and p38 MAP kinase inhibition but was unaffected by PI3-kinase inhibition (Fig. 5B). PKC inhibition, tyrosine kinase inhibition, and Ca2+ chelation also led to a complete inhibition of ANG II-induced eIF4E phosphorylation (Fig. 5B).



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Fig. 5. ANG II-induced eIF4E phosphorylation. A: growth-arrested VSMC were treated with 100 nM ANG II for 0-120 min, and Western blot analysis was performed with anti-phospho (Ser209)-eIF4E antibodies. Cumulative data from 3 experiments are shown. *P < 0.05 vs. control. B: growth-arrested VSMC were pretreated with 10 µM LY-294002, 20 µM SB-203580, 5 µM U-0126, 5 µM chelerythrine, 10 µM Ro-31-8220, 1 µM genistein, or 50 µM BAPTA-AM for 45 min before treatment with 100 nM ANG II for 45 min, and Western blot analysis was performed using anti-phospho (Ser209)-eIF4E antibodies. Cumulative data from 3-4 experiments are shown. *P < 0.05 vs. control. {dagger}P < 0.05 vs. ANG II.

 

PYK2 is required for PHAS-1. Our results suggest that a Ca2+- and PKC-dependent tyrosine kinase may be an upstream regulator of PHAS-1 phosphorylation by ANG II. An attractive candidate is PYK2, because we recently reported that PYK2 is required for ANG II-induced VSMC protein synthesis (38). We previously showed a >80% inhibition of PYK2 expression with PYK2 antisense oligonucleotides that did not effect expression of the closely related tyrosine kinase focal adhesion kinase, ERK1/2, and p70 S6 kinase (38). Using a similar strategy, we determined the effects of PYK2 antisense oligonucleotides on ANG II-induced PHAS-1 and eIF4E phosphorylation. PYK2 downregulation led to near-complete inhibition of ANG II-induced PHAS-1 and eIF4E phosphorylation (Fig. 6). Control, scrambled oligonucleotides had no effect on PHAS-1 or eIF4E phosphorylation in response to ANG II. We next determined whether PYK2 antisense oligonucleotides had an effect on ANG II-induced p38 MAP kinase activation, because the p38 MAP kinase inhibitor SB-203580 blocked eIF4E phosphorylation. PYK2 downregulation led to a significant inhibition of ANG II-induced p38 MAP kinase phosphorylation (Fig. 7).



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Fig. 6. Proline-rich tyrosine kinase 2 (PYK2) antisense oligonucleotides (AS-ODN) block PHAS-1 and eIF4E phosphorylation by ANG II. Lysates from control VSMC, cells treated with LipofectAMINE alone, cells treated with PYK2 sense oligonucleotides (S-ODNs), or cells treated with PYK2 antisense oligonucleotides for 8 h were placed in 0.2% calf serum-DMEM overnight and then treated with 100 nM ANG II for 15 min (PHAS-1) or 30 min (eIF4E). Representative Western blots from 3 experiments using antiphospho-PHAS-1, anti-total PHAS-1, or antiphospho-eIF4E antibodies are shown.

 


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Fig. 7. PYK2 antisense oligonucleotides decrease p38 MAP kinase activation by ANG II. Lysates from control VSMC, cells treated with LipofectAMINE alone, or cells treated with PYK2 antisense oligonucleotides for 8 h were placed in 0.2% calf serum-DMEM overnight and then treated with 100 nM ANG II for 30 min. Representative Western blots from 3 experiments using antiphospho-p38 antibodies are shown.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
These data are the first to demonstrate a requirement for the tyrosine kinase PYK2 in the regulation of translation initiation by PHAS-1 and eIF4E. In addition, our results demonstrate that the PI3-kinase pathway and the ERK1/2 MAP kinase pathway, but not p38 MAP kinase, are responsible for the ANG II-dependent phosphorylation of PHAS-1 on Thr70 and Ser65, which then leads to the release of eIF4E. On the other hand, ANG II-induced eIF4E phosphorylation is regulated by ERK1/2 and p38 MAP kinase, but not by PI3-kinase (Fig. 8).



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Fig. 8. Proposed signaling cascades involved in PHAS-1 and eIF4E phosphorylation in response to AT1 receptor activation. AUG, translation initiation site.

 

Because we previously demonstrated that the PI3-kinase and the ERK1/2 pathways are distinct, parallel signaling pathways in VSMC (37), the phosphorylation of PHAS-1 may represent one point of convergence of these pathways and may, thus, be important in translation initiation. There have been several reports that offer disagreement with these data. For example, some reports indicate that Akt is necessary and sufficient for the phosphorylation of PHAS-1 (9, 44). Other studies suggest that PHAS-1 phosphorylation in response to growth factors is mediated by a wortmannin- and rapamycin-sensitive pathway (20) that is distinct from the ERK1/2 MAP kinase pathway (17, 22). Finally, a recent study demonstrated that activation of the AktmTOR pathway and its downstream targets p70 S6 kinase and the PHAS-1-eIF4E complex was an important regulator of skeletal muscle fiber size and hypertrophy (1).

In addition to differences in cell types and agonists, it is likely that the existence of multiple phosphorylation sites on PHAS-1 and multiple isoforms of the protein (11) may explain some of the reported discrepancies. For example, FRAP/mTOR is able to phosphorylate PHAS-1 on Thr37 and Thr46 (3, 19). However, the phosphorylation of these sites may or may not be associated with the release of eIF4E from PHAS-1 (3, 19). Yet another set of data outlines the sequential phosphorylation of PHAS-1 on the rapamycin-sensitive sites Thr37, Thr46, and Thr70, which facilitated the phosphorylation on Ser65 (32). This Ser65 phosphorylation appears to be required for the dissociation of PHAS-1 from eIF4E (24). Consistent with these findings, we show that the specific PI3-kinase inhibitor LY-294402 and rapamycin block PHAS-1 phosphorylation on Thr70 and Ser65 (Fig. 2) and that p70 S6 kinase is able to phosphorylate PHAS-1 in in vitro kinase assays (data not shown). These results suggest that p70 S6 kinase mediates PI3-kinase-dependent signaling to PHAS-1. Our results also point to a role for Akt in the regulation of PHAS-1 phosphorylation. DN-Akt significantly inhibited PHAS-1 phosphorylation stimulated by ANG II (Fig. 3). Taken together, these results clearly demonstrate a role for Akt and p70 S6 kinase in PHAS-1 phosphorylation.

The specific MEK inhibitor U-0126 also attenuated PHAS-1 phosphorylation in response to ANG II (Fig. 2A). These results are consistent with previous findings that PHAS-1 is phosphorylated in an ERK1/2-dependent manner (28, 35) and suggest that ERK1/2 MAP kinases and the PI3-kinase pathway are involved in regulation of ANG II-induced PHAS-1 phosphorylation.

The results in Fig. 4, for the first time, demonstrate the functional consequences of PHAS-1 phosphorylation on Thr70 and Ser65 in VSMC. Comparison of the time courses for PHAS-1 phosphorylation and eIF4E dissociation suggest that dissociation of the PHAS-1-eIF4E complex in response to ANG II in VSMC requires PHAS-1 phosphorylation (Fig. 4). This dissociation was blocked by the inhibitors of ERK1/2 and PI3-kinase signaling as well as by Ca2+ chelation and tyrosine kinase inhibition (Fig. 4).

Phosphorylation of eIF4E by ANG II occurred subsequent to its dissociation from PHAS-1 (Figs. 4 and 5). The phosphorylation of eIF4E in response to ANG II has been previously demonstrated in VSMC (35). It has, however, not been clearly established which signaling pathways provide a link between AT1 receptor activation and eIF4E phosphorylation. Here we show that ANG II-induced phosphorylation of eIF4E on Ser209 is mediated by ERK1/2 and p38 MAP kinases but is PI3-kinase independent, because LY-294002 had no effect on ANG II-induced eIF4E phosphorylation (Fig. 4). In agreement with these results, ERK1/2 MAP kinases are capable of phosphorylating eIF4E on Ser209 in other cell types (42) directly or via an intermediate kinase, Mnk-1 (45, 46). In addition, the inhibition of ERK1/2 and p38 MAP kinase completely blocks eIF4E phosphorylation by phorbol esters in T cells (31) and in response to H2O2 in human embryonic kidney 293 cells (45). On the other hand, Rao (34) reported that neither ERK1/2 nor the redox-sensitive p38 MAP kinase was involved in the phosphorylation of eIF4E in response to H2O2 in VSMC.

The proximal signaling events that link AT1 receptor activation to these downstream signaling pathways and regulation of the PHAS-1-eIF4E complex remain to be determined. We previously defined a role for PYK2 in the regulation of ERK1/2 and PI3-kinase signaling, as well in ANG II-induced VSMC protein synthesis (37, 38). In addition, Rybkin et al. (39) reported that PHAS-1 is phosphorylated via a Ca2+-sensitive and PKC-dependent pathway. Morley (31) was able to demonstrate a PKC requirement for eIF4E phosphorylation. eIF4E phosphorylation has also been shown to be Ca2+ dependent (34). Through the use of pharmacological inhibitors, the results in these studies demonstrate the involvement of PKC, Ca2+, and tyrosine kinases as upstream regulators of PHAS-1 and eIF4E (Figs. 2, 3, 4). It is also likely that the EGF receptor may be downstream of PYK2/Src activation, because it was recently shown that ERK, p38, Akt, and p70 S6 kinase activation by ANG II were downstream of the EGF receptor transactivation in VSMC (13, 15).

We previously showed that PYK2 antisense oligonucleotides block ERK1/2, p70 S6 kinase, and Akt phosphorylation in response to ANG II (38). Here we show that PYK2 is also necessary for ANG II-induced p38 MAP kinase activation (Fig. 7). There is some disagreement in the literature concerning the role of PKC (and, therefore, PYK2) in ERK1/2 activation in VSMC. Several groups, including our own, have used the PKC inhibitor Ro 31-8220 (48), downregulation with phorbol dibutyrate (29, 40), or PKC-{zeta} antisense (27) to block ANG II-induced ERK1/2 activation in cultured aortic VSMC. Matrougui et al. (30) recently reported that the PKC inhibitor Go-6976 blocked ERK1/2 activation by ANG II in intact resistance arteries.

On the other hand, neither bisindolylmaleimide (16) nor GF-109203X (33) blocked ERK1/2 activation by ANG II. The differences may reflect vascular beds (aorta vs. resistance vessels) or culture conditions (method of isolation, growth arrest protocols, and amount of glucose and serum in the medium). It is also important to note that many of the pharmacological strategies to inhibit PKC are not equally effective for all isoforms.

Finally, we examined the role of PYK2 in the regulation of PHAS-1 and eIF4E phosphorylation by using PYK2 antisense oligonucleotides (Fig. 6). This study is the first to demonstrate that PYK2 downregulation decreased ANG II-induced phosphorylation of PHAS-1 and eIF4E (Fig. 6). Thus it appears that PYK2 is an important upstream signaling molecule that coordinates the regulation of multiple signaling intermediates that ultimately converge on the regulation of translation initiation, the rate-limiting step in protein synthesis. However, these results do not rule out the possible involvement of PYK2-independent processes in the regulation VSMC translation initiation and protein synthesis activated by ANG II. Nevertheless, these findings raise the exciting possibility that PYK2 could be a target for therapeutic strategies to treat vascular diseases that involve altered VSMC growth. Future studies with PYK2 antisense are necessary to determine the role of this kinase in modulating VSMC growth in vivo.


    DISCLOSURES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-56046 and HL-63318 (to P. A. Lucchesi).


    ACKNOWLEDGMENTS
 
Present address of P. Rocic: Dept. of Physiology, Louisiana State University School of Medicine, New Orleans, LA 70119.


    FOOTNOTES
 

Address for reprint requests and other correspondence: P. A. Lucchesi, UAB Dept. of Physiology and Biophysics, MCLM-986, 1530 3rd Ave. S, Birmingham, AL 35294-0005 (E-mail: lucchesi{at}physiology.uab.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
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