2 and Protein Kinase A

J Mol Cell Cardiol 34, 749ÿ763 (2002) doi:10.1006/jmcc.2002.2014, available online at http://www.idealibrary.com on

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Phenylephrine Promotes Phosphorylation of Bad in Cardiac Myocytes Through the Extracellular Signal-regulated Kinases 1/2 and Protein Kinase A Donna M. Valks1, Stuart A. Cook2, Fong H. Pham1, Paul R. Morrison2, Angela Clerk1 and Peter H. Sugden2 1

Division of Biomedical Sciences (Cell and Molecular Biology Section), Faculty of Medicine, Imperial College of Science, Technology and Medicine, London SW7 2AZ, UK and 2 National Heart and Lung Institute (NHLI) Division (Cardiac Medicine Section), Faculty of Medicine, Imperial College of Science, Technology and Medicine, London SW3 6LY, UK (Received 16 January 2002, accepted for publication 25 March 2002) D. M. VALKS, S. A. COOK, F. H. PHAM, P. R. MORRISION, A. CLERK AND P. H. SUGDEN. Phenylephrine Promotes Phosphorylation of Bad in Cardiac Myocytes Through the Extracellular Signal-regulated Kinases 1/2 and Protein Kinase A. Journal of Molecular and Cellular Cardiology (2002) 34, 749ÿ ÿ763. Studies in non-cardiomyocytic cells have shown that phosphorylation of the Bcl-2 family protein Bad on Ser-112, Ser-136 and Ser-155 decreases its pro-apoptotic activity. Both phenylephrine (100 mM) and the cell membrane-permeating cAMP analog, 8-(4-chlorophenylthio)-cAMP (100 mM), protected against 2-deoxy-D-glucose-induced apoptosis in neonatal rat cardiac myocytes as assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). In cardiac myocytes, phenylephrine primarily stimulates the a-adrenoceptor, but, at high concentrations (100 mM), it also increases the activity of the cAMP-dependent protein kinase, protein kinase A (PKA) through the b-adrenoceptor. Phenylephrine (100 mM) promoted rapid phosphorylation of Bad(Ser-112) and Bad(Ser-155), though we were unable to detect phosphorylation of Bad(Ser-136). Phosphorylation of Bad(Ser-112) was antagonized by either prazosin or propranolol, indicating that this phosphorylation required stimulation of both a1- and b-adrenoceptors. Phosphorylation of Bad(Ser-155) was antagonized only by propranolol and was thus mediated through the b-adrenoceptor. Inhibitor studies and partial puri®cation of candidate kinases by fast protein liquid chromatography showed that the p90 ribosomal S6 kinases, p90RSK2/3 [which are activated by the extracellular signal-regulated kinases 1 and 2 (ERK1/2)] directly phosphorylated Bad(Ser-112), whereas the PKA catalytic subunit directly phosphorylated Bad(Ser-155). However, ef®cient phosphorylation of Bad(Ser-112) also required PKA activity. These data suggest that, although p90RSK2/3 phosphorylate Bad(Ser-112) directly, phosphorylation of this site is enhanced by phosphorylation of Bad(Ser-155). These phosphorylations potentially

Please address all correspondence to: Peter H. Sugden, NHLI Division (Cardiac Medicine), Faculty of Medicine, Imperial College of Science, Technology and Medicine, Dovehouse Street, London SW3 6LY, UK. Tel: ‡44 20 7351 8144; Fax: ‡44 20 7823 3392; E-mail: [email protected] Abbreviations used: CPT-cAMP, 8-(4-chlorophenylthio)-cAMP; 2-DOG, 2-deoxy-D-glucose; DTT, dithiothreitol; ERK, extracellular signal-regulated kinase; FPLC, fast protein liquid chromatography; GST, glutathione-S-transferase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; p90RSK, 90 kDa ribosomal subunit S6 kinase; p70S6K, 70 kDa ribosomal subunit S6 kinase; PDK1, PtdlnsP3-dependent kinase 1; PE, phenylephrine; PI3K, phosphatidylinositol 3 0 -kinase; PtdInsP3, phosphatidylinositol 3 0 ,4 0 ,5 0 trisphosphate; PKA, PKB and PKC, protein kinases A, B and C; PKI, peptide inhibitor of PKA catalytic subunit; PMSF, phenylmethylsulfonyl ¯uoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; E64, trans-epoxy succinyl-Lleucylamido-(4-guanidino)butane; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

0022ÿ ÿ2828/02/070749 ‡ 15 $35.00/0

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2002 Elsevier Science Ltd. All rights reserved.

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diminish the pro-apoptotic activity of Bad and contribute to the cytoprotective effects of phenylephrine and # 2002 Elsevier Science Ltd. All rights reserved. 8-(4-chlorophenylthio)-cAMP. Key Words: Cardiac myocytes; Adrenoceptors; Bcl-2 proteins; Mitogen-activated protein kinases; cAMP; Apoptosis.

Introduction Phenylephrine (PE), an agonist that acts primarily at a-adrenoceptors (members of the 7-transmembrane spanning Gq protein-coupled receptor family), induces a powerful hypertrophic response in cardiac myocytes in primary culture.1 This is, at least in part, attributable to increased gene transcription and protein synthesis, effects which are mediated by the activation of one or more intracellular signaling pathways.2 Three mitogenactivated protein kinase (MAPK) signaling cascades are activated by PE, namely the extracellular signal-regulated kinase1/2 (ERK1/2) cascade,3,4 the c-Jun N-terminal kinase (JNK) cascade,5 and the p38-MAPK cascade.6 The activation of the ERK1/2 cascade by Gq protein-coupled receptors involves hydrolysis of phosphatidylinositol 4 0 ,5 0 bisphosphate and activation of diacylglyceroldependent isoforms of protein kinase C (PKC).3,4 Although the topic of active debate as to their individual importance, all of these MAPK cascades (as well as the less-well characterized ERK5 cascade7) have been implicated in the hypertrophic responses2 Following their activation, MAPKs phosphorylate a wide range of signaling molecule substrates to modulate their biological activities, and it is assumed that the ability of MAPKs to modulate the activities of these molecules underlies their ability to induce hypertrophy. Signaling molecules phosphorylated by ERK1/2 include transcription factors such as Elk-1,8,9 and other protein kinases such as the p90-ribosomal S6 kinases (p90RSKs).10,11 Of particular relevance to the cardiac myocyte is the ®nding that ERK1/2 are responsible for increasing the transactivating and DNA binding activity of the transcription factor GATA-4 in these cells.12 GATA-4 is a transcription factor that is involved in the regulation of transcription of a number of genes whose expression is associated with hypertrophic responses in cardiac myocytes.13 Other signaling pathways in addition to the MAPKs have been implicated in the hypertrophic response (e.g. calcineurin),14 but there is no clear evidence that they are activated by PE. In addition to direct effects on the hypertrophic response, MAPKs may also modulate apoptosis in

myocytes. Thus, activation of the ERK1/2 cascade appears to be cytoprotective,15,16 whereas activation of p38-MAPKs (especially p38-MAPKa) is ÿ18 The role of JNKs is not probably proapoptotic.16ÿ clear since JNK activation is associated with myocyte apoptosis in a simulated ischemia/reperfusion model,16 but is cytoprotective against apoptosis induced by NO.19 Bad is a proapoptotic Bcl-2 family protein that participates in the mitochondrial pathway of apoptosis.20,21 Given the highly aerobic nature of the cardiac myocyte, this pathway is likely to predominate over other apoptotic pathways here. Bad promotes apoptosis by binding to antiapoptotic Bcl-2 proteins (e.g. Bcl-XL and Bcl-2) in the outer mitochondrial membrane. This results in the release of cytochrome c from the intermembrane space of the mitochondria into the cytoplasm where it promotes the formation of an apoptosome complex leading to cleavage and activation of procaspase 9. In earlier overexpression studies using cell lines, phosphorylation of Bad on three sites, (Ser-112,22,23 Ser-136,22,23 and Serÿ28 of murine Bad) diminishes its proapop15524ÿ totic activity. Very recently, Bad(Ser-170) has also been identi®ed as a phosphorylation site and this phosphorylation is also anti-apoptotic.29 The numbering of the residues in rat Bad differs slightly from murine Bad (see the Appendix for further details of the amino-acid sequence of rat Bad), but, for the sake of simplicity, we will use the murine numbering system here. Ser-136 was originally thought to be phosphorylated by protein kinase B ÿ32 but more recent (PKB, also known as Akt),30ÿ evidence has indicated that p70 ribosomal subunit S6 kinase (p70S6K) is responsible.33 Both PKB and p70S6K are activated by the phosphatidylinositol 3 0 -kinase (PI3K) ! phosphatidylinositol 3 0 ,4 0 ,5 0 trisphosphate (PtdInsP3) ! PtdInsP3-dependent kinase 1 (PDK1) signaling pathway.22,23 Ser-112 ÿ37 Phosphoryis phosphorylated by p90RSKs.34ÿ lation of these sites promotes association of Bad with 14-3-3 proteins and this prevents it from interacting with Bcl-2/Bcl-XL by sequestering it away from the mitochondria.30 Ser-155 is phosphorylated by protein kinase A (PKA), and, whilst phosphorylation of this site may promote the

Phosphorylation of Bad in Cardiac Myocytes

interaction of Bad with 14-3-3 proteins, it also interferes directly with the interaction of Bad with ÿ28 The kinase responsible for the Bcl-2/Bcl-XL.24ÿ phosphorylation of Ser-170 has not been identi®ed.29 Here, we show that PE promotes phosphorylation of endogenous Bad on Ser-112 and Ser-155 in cardiac myocytes. Phosphorylation of Ser-155 is mediated through the b-adrenoceptor and PKA, and, although phosphorylation of Ser-112 is mediated through ERK1/2 and p90RSKs, ef®cient phosphorylation of this site requires signals from both the a- and b-adrenoceptors.

Materials and Methods Preparation of neonatal rat cardiac myocytes Myocytes were dissociated from the ventricles of 1- to 3-day-old Sprague-Dawley rat hearts essentially6,38 by the method of Iwaki et al.39 and plated at a density of 1.4  103 cells/mm2 in 60 or 35 mm plastic culture dishes. Serum was withdrawn for 24 h before use. Myocytes were exposed to PE or 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) with or without pretreatment with inhibitors (30 min) or receptor antagonists (1 min). Immunoblot analysis Myocytes were washed, extracted in Buffer A [20 mM b-glycerophosphate, pH 7.5, 50 mM NaF, 2 mM microcystin LR, 2 mM EDTA, 0.2 mM Na3VO4, 10 mM benzamidine, 200 mM leupeptin, 10 mM trans-epoxy succinyl-L-leucylamido-(4-guanidino)butane (E64), 5 mM dithiothreitol (DTT), 300 mM phenylmethylsulfonyl ¯uoride (PMSF), 1% (v/v) Triton X-100] and centrifuged as described previously.6 Proteins in myocyte supernatant extracts or fast protein liquid chromatography (FPLC) fractions were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using 15% (w/v) (for Bad) or 10% (w/v) (for all other proteins) polyacrylamide gels and transferred to nitrocellulose as described previously.6 Blots were probed with antibodies to phospho-Bad(Ser-112), phospho-Bad (Ser-136) or phospho-Bad(Ser-155), (Cell Signaling Technology, New England Biolabs, Hitchin, UK, 1/500 dilution), phospho-PKB (Ser-473) or phospho-PKB(Ser-308) (Cell Signaling Technology, 1/1000), PKB or total Bad (Transduction Laboratories, now BD biosciences, Oxford, UK, 1/1000 or 1/500, respectively), p90RSK1, p90RSK2, p90RSK3 or the PKAa catalytic subunit (Santa Cruz Biotechnology, supplied by Autogen Bioclear,

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Calne, UK, each at 1/200), or ERK5 (Chemicon, Chandlers Ford, UK, 1/300). Bands were detected by enhanced chemiluminescence and were quanti®ed by scanning densitometry. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analysis Following serum starvation, myocytes were exposed to 5 mM 2-deoxy D-glucose (2-DOG) in the absence or presence of 100 mM PE or 100 mM CPT-cAMP for a further 48 h. Myocytes showing unequivocal nuclear TUNEL staining (performed as described previously40) in conjunction with chromatin condensation and staining for b-myosin heavy chain were counted as apoptotic. PKB assay Myocytes were exposed to agonists and scraped into 100 ml ice-cold Buffer B [20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 10% (v/v) glycerol, 100 mM KCl, 5 mM NaF, 0.2 mM Na3VO4, 5 mM MgCl2 0.05% (v/v) 2-mercaptoethanol, 4 mM microcystin LR, 0.2 mM leupeptin, 10 mM E64, 5 mM DTT, 0.3 mM PMSF, and 1% (v/v) Triton X100]. Extracts were centrifuged (5 min, 10 000  g, 4 C), the supernatants were incubated with 0.8 mg PKBa/b/g antibody (Santa Cruz Biotechnology) prebound to protein A-Sepharose (1 h, 4 C). Pellets were washed in Buffer B (2  300 ml) and then in 300 ml PKB kinase assay buffer [25 mM HEPES (pH 7.6), 15 mM b-glycerophosphate, 50 mM NaF, 1.2 mM EDTA, 0.18 mM Na3VO4 2.5 mM DTT]. Pellets were resuspended in 50 ml 1.2-fold strength PKB kinase assay buffer containing 300 mM PKB peptide substrate (RPRAATF, Severn Biotech Ltd., Kidderminster, UK) and 10 ml of 0.12 mM ATP/120 mM MgCl2 containing 1 mCi/ assay [g-32P]ATP was added. After incubation (30 C), assays were terminated by cooling to 0 C, centrifuged, and 50 ml of supernatant were spotted onto P81 papers squares (Whatman International Ltd., Maidstone, Kent) which were then washed in 75 mM orthophosphoric acid (3  50 ml, 1 h). Radioactivity incorporated was determined by Cerenkov counting. Puri®cation of protein kinases by fast protein liquid chromatography (FPLC) and their assay Myocytes were washed, extracted in Buffer A and centrifuged as described previously.6 The

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supernatants were applied to a Mono Q HR 5/5 or Mono S HR 5/5 column equilibrated with 50 mM Tris-HCl (pH 7.3), 2 mM EDTA, 2 mM EGTA (Mono Q), or 25 mM b-glycerophosphate (pH 7.3), 1 mM EDTA, 1 mM EGTA (Mono S). The column buffers also contained 0.3 mM Na3VO4 1 mM DTT, 4 mg/ml leupeptin, 5% (v/v) glycerol and 0.03% (v/ v) Brij35. Proteins were eluted with a linear NaCl gradient. Samples from each fraction were assayed for Bad kinase activity or boiled with 0.33 vol SDSPAGE sample buffer6 for immunoblotting. For assay of Bad kinases in FPLC fractions, fulllength murine Bad was cloned from a pEBG plasmid encoding Bad (New England Biolabs, Hitchin, UK), coupled to glutathione-S-transferase (GST) using the pGEX-4T-l vector (AmershamPharmacia Biotech, Amersham, UK) and the BadGST fusion protein was isolated following expression in E. coli BL21(DE3)pLysS by glutathioneSepharose af®nity binding. The Bad sequence was con®rmed by automated DNA sequencing. BadGST (0.05 mg/assay) was pre-bound (1 h, 4 C) to glutathione-Sepharose equilibrated with Buffer C [50 mM Tris-HCl (pH 7.5), 125 mM NaCl, 10 mM (bmercaptoethanol and 0.03% (v/v) Brij35]. The complex was washed and resuspended in Buffer D [50 mM Tris-HCl (pH 7.5), 0.1 mM EGTA, 0.1 mM Na3VO4, 1 mg/ml BSA, 0.1% (v/v) b-mercaptoethanol and 0.03% (v/v) Brij35]. Assays (containing 50 ml FPLC fraction) were initiated by addition of 10 ml 4 mM ATP, 0.1 M Mg2‡ acetate in Buffer D, incubated at 30 C (60 min), and terminated with 20 ml EDTA (0.5 M, pH 7.5). BadGST/glutathione-Sepharose complexes were washed Buffer C, resuspended in SDS-PAGE sample buffer, boiled and analyzed by immunoblotting for phosphorylated Bad-GST. For immunokinase assays, fraction 11 from Mono Q FPLC (300 ml) was incubated with antibodies to p90RSK2, p90RSK3, ERK1/2 or nonimmune goat serum (2.5 mg, 2 h, 4 C) and then with 40 ml of a 1:1 slurry of Protein A-Sepharose/ Protein G-Sepharose in Buffer D (1 h, 4 C). Immunoprecipitates were washed and resuspended in Buffer D. Bad kinase assays were performed as described above using 0.2 mg Bad-GST. Assays were terminated by boiling with SDS-PAGE sample buffer. Phosphorylated Bad-GST was identi®ed by immunoblotting for phospho-Bad(Ser-112) or phospho-Bad(Ser-155) as above. For PKA activities, myocytes were extracted and proteins were separated by Mono S FPLC as described above. PKA activities in each fraction were assayed by phosphorylation of kemptide as previously described.41 The pro®les of activity were plotted

for each condition and the total activity determined by integrating the area under the curve.

Results Effects of PE and CPT-cAMP on cardiac myocyte apoptosis In addition to promoting cardiac myocyte hypertrophy,1 PE also inhibits the effects of pro-apoptotic stimuli in cardiac myocytes.15,42,43 We con®rmed that this was the case in our cultures when apoptosis was assessed by TUNEL analysis (Fig. 1). Here, exposure to 5 mM 2-DOG for 48 h trebled the proportion of apoptotic myocytes but inclusion of 100 mM PE in the presence of 5 mM 2-DOG decreased the proportion of apoptotic cells to control values. These effects were prevented by U0126 (10 mM) which inhibits the ERK1/2 cascade and H89 (10 mM) which inhibits PKA (and other protein kinases).44 The effects of cAMP on myocyte apoptosis are controversial, though our results with H89 imply that PKA may be anti-apoptotic. In order to examine this, we treated myocytes with

Figure 1 PE and CPT-cAMP inhibit 2-DOG-stimulated apoptosis. Myocytes were exposed to 5 mM 2-DOG in the absence or presence of 100 mM PE or 100 mM CPT-cAMP. In some experiments, 10 mM U0126 or 10 mM H89 was also present. Myocytes showing unequivocal nuclear TUNEL staining in conjunction with chromatin condensation and staining for b-myosin heavy chain were ÿ6 counted as apoptotic. Results are means  S.E.M. for 3ÿ independent experiments. * P , 0.02 for the effect of 2-DOG relative to control, y P , 0.01 for the effects of PE or CPT-cAMP relative to 2-DOG (two-tailed Student's t-test).

Phosphorylation of Bad in Cardiac Myocytes

5 mM 2-DOG in the presence of 100 mM CPT-cAMP and showed that, in our hands, CPT-cAMP completely reversed the pro-apoptotic effects of 2-DOG (Fig. 1). PE stimulates phosphorylation of Bad on Ser-112 and Ser-155 Phosphorylation of Bad on Ser-112, Ser-136 and Ser-155 inhibits its pro-apoptotic activity. We used

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antibodies which detect total Bad, or are selective for phospho-Bad(Ser-112) or phospho-Bad(Ser155) for immunoblotting to examine whether PE increased Bad phosphorylation. PE (100 mM) increased phospho-Bad(Ser-112) from 5 min, with maximal phosphorylation from 10ÿ ÿ15 min [Fig. 2(a)]. This was maintained over 4ÿ ÿ8 h [Fig. 2(b)]. Although phospho-Bad(Ser-112) remained elevated up to 48 h, total Bad also increased up to 16 h following PE stimulation [Fig. 2(c)] such that the relative amount of

Figure 2 PE promotes phosphorylation of Bad on Ser-112 and Ser-155 in cardiac myocytes. Myocytes were exposed to 100 mM PE for the times indicated and extracts were immunoblotted with antibodies to [(a) and (b)] phospho-Bad(Ser112), (c) total Bad, or [(d) and (e)] phospho-Bad(Ser-155). Bad and phospho-Bad were detected as 25 kDa bands. Representative blots of 3 [(a) and (d)] or 4 [(b), (c) and (e)] independent experiments are shown in the upper panels. Blots were analyzed by scanning densitometry (lower panels). Results are means  S.E.M.

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phospho-Bad(Ser-112) declined [Fig. 2(b), lower panel]. PE stimulated a more gradual increase in phospho-Bad(Ser-155) from 10ÿ ÿ15 min, with maximal phosphorylation of this site at 2ÿ ÿ4 h [Fig. 2(d) and (e)]. As with phospho-Bad(Ser-l12), phosphorylation of Ser-155 was sustained up to 8 h and, relative to total Bad, declined to basal levels by 48 h [Fig. 2(e)]. We did not detect any signi®cant phosphorylation of Bad(Ser-136) in myocytes exposed to PE (results not shown). Phosphorylation of this site ÿ32 was initially thought to be mediated by PKB,30ÿ but, more recently, p70S6K (which, like PKB, is activated by the PI3K-PtdlnsP3-PDK1 signaling pathway)23 has been implicated.33 We therefore examined whether PE stimulated the enzymic activity of PKB and the phosphorylation of Ser473 in PKB (one of the sites in PKB which, along with Thr-308, is phosphorylated to produce the activated form of the enzyme)23 as an indication of activation of the PI3K-PtdlnsP3-PDK1 signaling pathway. Since insulin and H2O2 are powerful stimulators of PKB(Ser-473) phosphorylation in cardiac myocytes,45 we compared PE with these agonists. If PKB is activated at all by PE, it is only activated very weakly compared with the 7.5-fold activation by insulin [Fig. 3(a)]. These results are

Figure 3 Activation and stimulation of phosphorylation of PKB. (a) Myocytes were exposed to 50 mU/ml insulin or 100 mM PE and PKB activity was assayed after extraction and immunoprecipitation. Results are means  S.E.M. for 3 independent observations, (b) Myocytes were exposed to 50 mU/ml insulin, 1 mM H2O2, 5% (v/v) fetal calf serum (FCS), 100 nM endothelin-1 (ET-1), 100 mM PE or 1 mM phorbol 12-myristate 13-acetate (PMA) for 5 min. Extracts were immunoblotted for phospho-PKB(Ser-473) (upper panel) or total PKB (lower panel), which were detected as 60 kDa bands. A representative experiment (of 3 independent experiments) is shown.

con®rmed by examination of PKB(Ser-473) phosphorylation [Fig. 3(b)]. PE stimulates phosphorylation only very weakly compared with insulin or H2O2, suggesting that PE only weakly activates the PI3K-PtdInsP3-PDK1-PKB signaling pathway in cardiac myocytes. This is a property shared with another Gq protein-coupled receptor agonist, endothelin-1, which, like PE, activates PKC isoforms in myocytes.4 Furthermore, the direct activator of the diacylglycerol-dependent isoforms of PKC, phorbol 12-myristate 13-acetate did not induce any detectable phosphorylation of PKB(Ser473) [Fig. 3(b)].

Stimulation of Bad phosphorylation by PE (100 mM) is mediated through both a- and b-adrenoceptors PE is generally accepted to be a selective aadrenoceptor agonist. However, at the high concentrations which are commonly used (100 mM), it is possible that there may be some cross-stimulation of the b-adrenoceptor. The b-adrenoceptor couples through Gs to stimulate cAMP production and activate PKA. We were previously unable to detect an increase in the PKA activity ratio (activity measured ÿcAMP/‡2 mM cAMP) in crude extracts of cardiac myocytes which had been exposed to 100 mM PE,41 but this may re¯ect a relatively low sensitivity of this methodology. We therefore examined whether PE increases PKA activity using MonoS FPLC to purify the PKA catalytic subunit, a method which is likely to be more sensitive. Note that the PKA holoenzymes do not bind to cationic ion exchange matrices and elute in the isocratic wash (results not shown). The PKA catalytic subunit eluted predominantly in fractions 16ÿ ÿ18 (0.10ÿ ÿ0.14 M NaCl) [Fig. 4(a)]. A low level of PKA activity was detected in unstimulated myocytes [Fig. 4(b) and (c)] and this was increased 11.4  3.5 Fold (mean  S.D., n ˆ 2) following stimulation of myocytes with 100 mM CPT-cAMP [Fig. 4(b)] to activate PKA fully (as demonstrated by no further increase in PKA activity on addition of 2 mM cAMP to the PKA assay).41 PE (100 mM) signi®cantly increased PKA activity 2.3  0.4-fold (mean  S.E.M., n ˆ 5; P , 0.005 vs unstimulated control, paired Student's t-test) [Fig. 4, (b) and (c)]. The increase in PKA activity induced by PE was entirely inhibited by the b-adrenoceptor antagonist propranolol (1 mM), but prazosin (1 mM), an a1-adrenoceptor antagonist, had no effect [Fig. 4(c)]. These data indicate that 100 mM PE does activate the

Phosphorylation of Bad in Cardiac Myocytes

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(1 mM) or the b-adrenoceptor antagonist propranolol (1 mM) [Fig. 5(a)], suggesting that both receptors are required for ef®cient phosphorylation of this site. However, the stimulation of phospho-Bad(Ser155) was inhibited only by propranolol [Fig. 5(b)], indicating phosphorylation of Bad(Ser-155) is mediated through the b-adrenoceptor. In the absence of PE, neither prazosin nor propranolol affected the basal levels of Bad phosphorylation (results not shown). p90RSK2/3 phosphorylates Bad(Ser-112) and PKA phosphorylates Bad(Ser-155), but ef®cient phosphorylation of Bad(Ser-112) requires PKA activity

Figure 4 PE activates PKA through the b-adrenoceptor. Myocytes were unstimulated (Control), or exposed to 100 mM PE or 100 mM CPT-cAMP for 10 min in the absence or presence of 1 mM prazosin or 1 mM propranolol. Proteins were separated by Mono S FPLC. Fractions were (a) immunoblotted with antibodies to the PKA catalytic subunit (detected as a 40 kDa band), or [(b) and (c)] assayed for PKA activity using Kemptide as a substrate, (a) Elution pro®le of PKA catalytic subunit assessed by immunoblotting. A representative blot is shown from myocytes exposed to PE. (b) Elution pro®le of PKA activities from unstimulated, PE-stimulated or CPTcAMP-stimulated myocytes. The experiment was repeated twice with similar results, (c) PKA activities were calculated by measuring the peak area following Mono S puri®cation and PKA assay. Results are means  S.D. for 5 (Control), 6 (PE) or 2 (PE ‡ prazosin or PE ‡ propranolol) independent experiments.

b-adrenoceptor and increase PKA activity in cardiac myocytes, albeit to a relatively low level. We investigated whether a- and/or b-adrenoceptors are involved in the stimulation of Bad phosphorylation induced by PE. The stimulation of phospho-Bad(Ser-l12) was completely inhibited by either the a1-adrenoceptor antagonist prazosin

Experiments in other cells indicate that PKA phosphorylates Bad(Ser-155), whereas p90RSKs (1, 2 or 3), which are activated by the ERK1/2 cascade, phosphorylate Bad(Ser-112). Consistent with this, stimulation of Bad(Ser-155) phosphorylation by PE in cardiac myocytes was attenuated by H89 (10 mM) which inhibits PKA (and other protein kinases),44 but not U0126 (10 mM) which inhibits the ERK1/2 cascade [Fig. 6(a)]. Bad(Ser112) phosphorylation was inhibited by U0126, an alternative ERK1/2 cascade inhibitor PD98059 (50 mM), or GF109203X (10 mM), an inhibitor of PKC and p90RSKs46 [Fig. 6(b)]. However, consistent with the requirement for both a- and b-adrenoceptors [Fig. 5(a)], phosphorylation of Bad(Ser-112) was also inhibited by H89 [Fig. 6(b)]. In spite of the fact that H89 inhibits other protein kinases,44 this evidence taken together with the demonstration of inhibition of PE-stimulated Bad(Ser-l12) phosphorylation by propranolol, [Fig. 5(a)] suggests that PKA activity is also required for ef®cient phosphorylation of this site. Inhibition of PI3K (LY294002, 50 mM) did not affect phosphorylation of either Bad(Ser-112) or Bad(Ser-155) [Fig. 6(a) and (b)], suggesting a lack of involvement of either PKB or p70S6K. Positive controls with 50 mU/ml insulin showed that 50 mM LY294002 essentially completely inhibited phosphorylation of PKB (results not shown and47). Since PKA activity and the ERK1/2 cascade were both required for Bad(Ser-l12) phosphorylation, we examined which kinases directly phosphorylate the Bad(Ser-l12) and Bad(Ser-155) by using FPLC ionexchange chromatography for partial puri®cation of candidate kinases. The activities of Bad kinases were measured using an in vitro kinase assay with recombinant Bad-GST as substrate. Phosphorylation of Bad(Ser-112) or Bad(Ser-155) was assessed by immunoblotting of Bad-GST with site-speci®c

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Figure 5 Stimulation of Bad(Ser-112) and Bad(Ser-155) phosphorylation by PE is mediated through the a- and badrenoceptors. Myocytes were unstimulated (Control) or exposed to 100 mM PE (10 min) in the absence or presence of 1 mM propranolol or 1 mM prazosin. Myocyte extracts were immunoblotted with antibodies to (a) phospho-Bad(Ser-112) or (b) phospho-Bad(Ser-155). Bad and phospho-Bad were detected as 25 kDa bands. Representative blots of 4 (a) or 3 (b) independent experiments are shown in the upper panels. Blots were analyzed by scanning densitometry (lower panels). Results are means  S.E.M. * P , 0.05, relative to PE alone (two-tailed Student's t-test).

phospho-Bad antibodies. Using Mono Q anion exchange chromatography, the major peak of Bad(Ser-112) kinase activity eluted in fractions 10ÿ ÿ12 (0.12 M NaCl), with a minor peak of activity eluting at the start of the gradient (fraction 6) [Fig. 7(a)]. These activities were not inhibited by inclusion of the peptide PKA inhibitor (PKI, 8 mM) in the assay [Fig. 7(a)]. Immunoblotting showed that the activities co-eluted with p90RSK2 and p90RSK3 [Fig. 7(b)], and ERK2 (results not shown), but not with p90RSKl, ERK1, PKB or ERK5 (results not shown). The lack of co-elution of ERK5 with the Bad(Ser-112) kinase activity precluded the possibility that the effects of PD98059 and U0126 shown in Fig. 6(a) and (b) were the result of inhibition of the ERK5 cascade (which is inhibited by PD98059 and U0126, albeit with a greater IC50),48,49 rather than inhibition of the ERK1/2 cascade. Antibodies to p90RSK2 or p90RSK3 immunoprecipitated both p90RSK2 and p90RSK3 from fraction 11, but not ERK2 (results not shown), and immunokinase assays

indicated that immunoprecipitated p90RSK2 and/ or p90RSK3, rather than ERK2, were indeed the Bad(Ser-112) kinases in fraction 11 [Fig. 7(c)]. In contrast to Bad(Ser-112) kinases, Bad(Ser-155) kinase(s) eluted in the ¯ow-through from Mono Q FPLC (fractions 1 and 2) [Fig. 7(d)]. This activity was inhibited by inclusion of PKI in the assay indicating that the PKA catalytic subunit is the kinase involved. The PKA catalytic subunit did not phosphorylate Bad(Ser-112) [Fig. 7(a)]. Equally, p90RSK2/3 did not phosphorylate Bad(Ser-155) (results not shown since blot was blank). To con®rm these results, Bad kinases were also separated on Mono S FPLC. Bad(Ser-112) kinase(s) eluted as a broad peak across fractions 17ÿ ÿ22 (0.15ÿ ÿ0.26 M NaCl) and none of the activity was inhibited by PKI [Fig. 8(a)]. As with Mono Q FPLC, the Bad(Ser-112) kinase(s) co-eluted with p90RSK2 [Fig. 8(b)] and p90RSK3 (results not shown). The Bad(Ser-155) kinase(s) eluted in fractions 15ÿ ÿ18 (0.13ÿ ÿ0.18 M NaCl) [Fig. 8(c)]. This activity was inhibited by PKI and co-eluted

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Figure 6 Stimulation of Bad(Ser-112) and Bad(Ser-155) phosphorylation by PE requires the ERK1/2 cascade and PKA activity. Myocytes were unstimulated (Control) or exposed to 100 mM PE (30 min) in the absence or presence of 50 mM PD98059, 10 mM U0126, 10 mM GF109203X, 50 mM LY294002 or 10 mM H89. Myocyte extracts were immunoblotted with antibodies to (a) phospho-Bad(Ser-155) or (b) phospho-Bad(Ser-112). Bad and phospho-Bad were detected as 25 kDa bands. Representative blots of 4 independent experiments are shown in the upper panels. Blots were analyzed by scanning densitometry (lower panels). Results are means  S.E.M. P , 0.05, relative to PE alone (two-tailed Student's t-test).

with the catalytic subunit of PKA [Fig. 4(a)]. These data indicate clearly that, consistent with studies in other cells, the kinases which directly phosphorylate Bad(Ser-112) and Bad(Ser-155) are p90RSK2/ 3 (but not p90RSKl) and PKA, respectively.

Discussion Research into cardiac myocyte apoptosis is an active and controversial area because of the possible involvement of apoptosis myocardial

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Figure 7 Partial puri®cation of Bad kinases by Mono Q FPLC. Myocytes were exposed to 100 mM PE (10 min) and extracts were applied to a Mono Q FPLC column. Fractions 1ÿ ÿ5 represent the ¯ow-through and wash from the column. ÿ20). Fractions were assayed for Bad kinase Proteins were eluted with a 0ÿ ÿ0.3 M NaCl gradient (15 ml, fractions 6ÿ activity using Bad-GST as substrate. Phosphorylation of Bad-GST on (a) Ser-112 or (d) Ser-155 was detected by immunoblotting with phospho-Bad(Ser-112) or phospho-Bad(Ser-155) antibodies, respectively. Phospho-Bad-GST was detected as a 48 kDa band. Assays were conducted in the absence or presence (*) of 8 mM PKI. (b) Fractions were immunoblotted with antibodies to p90RSK2 (upper panels) or p90RSK3 (lower panels), (c) Proteins were immunoprecipitated with non-immune goat serum (GS), or antibodies to p90RSK2, p90RSK3 or ERK1/2. Immunoprecipitates were assayed for Bad(Ser-112) kinase activity using Bad-GST as substrate. Phospho-Bad-GST was immunoblotted with phospho-Bad(Ser-112) antibodies to measure Bad(Ser-112) kinase activity. All results are representative of at least 3 independent experiments.

pathologies such as congestive heart failure.50,51 In other cells, phosphorylation of Bad on Ser-112, Ser-136 and Ser-155 decreases its pro-apoptotic activity by preventing its interaction with antiapoptotic Bcl-2 or Bcl-XL in the outer mitochondrial membrane. This is probably achieved by promoting sequestration of Bad by 14-3-3 proteins in the cytoplasm,30 and by interfering directly with its interaction with Bcl-2 and Bcl-XL25 The mechanism by which phosphorylation of Bad on Ser-170 inhibits apoptosis is not clear. Because the Ser-170 phosphorylation does not disrupt the ability of Bad to form heterodimers with Bcl-XL, Dramsi et al.29 speculate that the phosphorylation of Ser-170 may enhance the anti-appoptotic activity of Bcl-XL. The consensus from work with other cells in which Bad is overexpressed is that Bad(Ser-112) is phosphorylated by p90RSK isoÿ37 forms (p90RSKl, p90RSK2 and p90RSK3),34ÿ

ÿ32 Bad(Ser-136) is phosphorylated by PKB,30ÿ and/ 33 or p70S6K, and Bad (Ser-155) is phosphorylated ÿ28 by the PKA catalytic subunit.24ÿ Little is known about the role of Bad phosphorylation in cardiac myocyte apoptosis. To our knowledge, phosphorylation of Bad has only been clearly detected with ligands signaling through the glycoprotein 130 pathway which potently stimulates PI3K and PKB.52 Thus, cardiotrophin-1 stimulates phosphorylation of Bad(Ser-136) through a PI3K-dependent pathway (presumably through PKB or p70S6K), and increases cell survival following serum starvation.53 Similarly, leukemia inhibitory factor inhibits doxorubicininduced apoptosis in cardiac myocytes, and promotes phosphorylation of Bad through PI3K.54 The phosphorylation site was not speci®ed in this publication,54 but was presumably Ser-136. However, others have shown that although adenoviral

Phosphorylation of Bad in Cardiac Myocytes

759

Figure 8 Partial puri®cation of Bad kinases by Mono S FPLC. Myocytes were exposed to 100 mM PE (10 min) and extracts were applied to a Mono S FPLC column. Proteins were eluted with a 0ÿ ÿ0.3 M NaCl gradient (15 ml, fractions 11ÿ ÿ25). Fractions were assayed for Bad kinase activity using Bad-GST as substrate. Phosphorylation of Bad-GST on (a) Ser-112 or (c) Ser-155 was detected as 48 kDa bands by immunoblotting with phospho-Bad(Ser-112) or phosphoBad(Ser-155) antibodies, respectively. Assays were conducted in the absence or presence (*) of 8 mM PKI. (b) Fractions were immunoblotted with antibodies to p90RSK2. All results are representative of at least 3 independent experiments.

infection of constitutively-active PI3K decreased doxorubicin-induced apoptosis in cardiac myocytes, there was no detectable increase in phosphorylation of Bad(Ser-136).55 Here, we show that PE increased phosphorylation of endogenous Bad (as opposed to the overexpressed Bad often used in experiments in cell lines) on Ser-112 and Ser-155 in neonatal rat cardiac myocytes. There was no evidence of involvement of the PI3K signaling pathway in these phosphorylations as they were not inhibited by LY294002 (Fig. 6). PKB did not appear to be involved because it did not co-elute with Bad(Ser-112) or Bad(Ser-155) kinases (results not shown). Furthermore, we were unable to detect phosphorylation of endogenous Bad(Ser136) (results not shown), the site which is directly ÿ33 in phosphorylated by PKB or p70S6K,24,30ÿ response to PE. However, this is not surprising since PE only weakly activates the PI3K-PtdInsP3PDK1-PKB signaling pathway in comparison to insulin in cardiac myocytes (Fig. 3). This result emphasizes the importance of examining changes in activity of signaling pathways using a comparative approach. Thus, although activation of PI3K and p70S6K (which is activated through the PI3K-PtdInsP3-PDK1 pathway) by PE has been

demonstrated in cardiac myocytes,56,57 the signi®cance of these ®ndings is impossible to assess without comparing the magnitude of the responses with that to an agonist such as insulin. We have previously shown that PE activates ERK1/2,4 which phosphorylate and activate p90RSKs.11 In addition, although PE is principally an a-adrenergic agonist, it also activates PKA at high concentrations (100 mM) through the badrenoceptor in neonatal cardiac myocytes (Fig. 4). A scheme illustrating the signaling pathways activated by PE through the two receptors and which lead to phosphorylation of Bad is shown in Fig. 9. ÿ28 bConsistent with studies in other cells,24ÿ adrenoceptor-mediated activation of the PKA catalytic subunit was responsible for phosphorylation of Bad(Ser-155) in cardiac myocytes exposed to PE [Fig. 5(b), Fig. 6(a), Fig. 7(d), and Fig. 8(c)]. Since Ser-155 lies in the Bad BH3 domain which interacts with Bcl-2 or Bcl-XL, and phosphorylation of Bad(Ser-155) interferes with this interaction,24,25 this phosphorylation may contribute to the cytoprotective effects of PE (Fig. 1). PE also increased phosphorylation of Bad(Ser112) [Fig. 2(a) and (b)]. Phosphorylation of this site promotes the association of Bad with 14-3-3

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Figure 9 Scheme of the regulation of Bad phosphorylation in cardiac myocytes exposed to PE, which activates both a- and b-adrenoceptors (AR).

proteins,30 and this may also contribute to the cytoprotective effects of PE (Fig. 1). Phosphorylation of Bad(Ser-112) in cardiac myocytes in response to PE was mediated through the a-adrenoceptor, PKC and the ERK1/2 cascade [Fig. 5(a) and Fig. 6(b)]. Our data indicate that, ÿ37 consistent with studies in other cells,34ÿ p90RSK2/3 (but not p90RSKl) are the Bad(Ser112) kinases [Fig. 7(a)ÿ ÿ(c), and Fig. 8(a) and (b)]. However, ef®cient phosphorylation also required signaling from the b-adrenoceptor and PKA [Fig. 5(a) and Fig. 6(b)]. It is possible that Bad(Ser-155) phosphorylation in some way potentiates phosphorylation of Bad(Ser-112) by p90RSK2/3. Thus Bad(Ser-155) phosphorylation may increase the rate of phosphorylation of Bad(Ser-112) by increasing accessibility of Bad(Ser-112) to p90RSK2/3, or by increasing the af®nity of Bad for p90RSK2/3. Equally, Bad(Ser-155) phosphorylation could reduce the rate of dephosphorylation of phosphoBad(Ser-112) through decreased phosphatase activity, or reduced accessibility of phosphorylated Ser-112 to a phosphatase. Interplay between Bad phosphorylation sites has previously been shown for Bad(Ser-136) and Bad(Ser-155).24 Thus, phosphorylation of Bad(Ser-136) and binding to 14-3-3 proteins promotes phosphorylation of Bad(Ser-155) in Bad bound to Bcl-XL.24 Our data indicate that there is also a co-operative effect between the Bad (Ser-155) and Bad(Ser-112) phosphorylation sites.

One factor in protein phosphorylation is the consensus sequence around the kinase substrate residue. The subsequent discussion will relate only to sequences actually found in rat Bad, because substitution of Lys for Arg or substitution of Thr for Ser is allowed. For Bad(Ser-112), the sequence in both rat Bada and Badb with Ser-112 emboldened and underlined is Arg-Ser-Arg-His-Ser-Ser.58 The consensus sequence phosphorylated by p90RSK (and a number of other kinases such as PKB59) is Arg-Xaa-Arg-Xaa-Xaa-Ser where Xaa is any amino acid.60 The consensus sequence phosphorylated by PKA is Arg-Arg-Xaa-Ser, though the sequence Arg-Xaa-Xaa-Ser will suf®ce.61 Thus p90RSK sites could theoretically be phosphorylated by PKA and it could be argued that Bad(Ser-112) is being phosphorylated by PKA in our experiments, especially since the PE-stimulated phosphorylation of Bad(Ser-112) is sensitive to inhibition by propranolol [Fig.5(a)]. However, in practice, it is rare for a p90RSK consensus sequence to be phosphorylated by PKA, and the fact that prazosin [Fig. 5(a)], ERK1/2 cascade inhibitors [Fig. 6(b)] and PKC inhibitors [Fig. 6(b)] inhibit the phosphorylation could not be explained by this scenario. Similarly, for Bad(Ser-155), the sequence in both rat Bada and Badb with Ser-155 underlined is Arg-Arg-Met-Ser.58 Since p90RSK will tolerate the sequence Arg-Arg-Xaa-Ser,60 it could be argued that Bad(Ser-155) could be being phosphorylated by p90RSK. However, although the PE-stimulated phosphorylation of Bad(Ser-155) is inhibited by H89 (an inhibitor of p90RSK as well as PKA)44 [Fig. 6(a)], there is no reason why it should be inhibited by propranolol unless PKA is the kinase directly involved, or unless activation of, PKA leads to activation of p90RSKs which then phosphorylate Bad(Ser-155). To our knowledge there is no evidence of the latter possibility. Whether cAMP is pro-apoptotic or anti-apoptotic in cardiac myocytes is controversial. Whilst the preponderance of published data favours the former view,62,63 others have shown opposite effects.64 However, in such experiments, badrenergic agonists have been used predominantly while relatively few experiments have been carried out with cell membrane-permeating cAMP analogs. Our data suggests that cAMP protects against apoptosis in the cardiac myocyte (Fig. 1). Given that b-adrenergic regulation is so important in regulating contractility in these cells, it seems to us inherently unlikely from a teleological standpoint that physiological levels of b-adrenergic agonists would promote cardiac myocyte apoptosis.

Phosphorylation of Bad in Cardiac Myocytes

In summary, we have shown that PE promotes phosphorylation of pro-apoptotic Bad on Ser-112 and Ser-155 though ERK1/2- and PKA-dependent pathways in neonatal cardiac myocytes. Work in other cells has conclusively shown that these phosphorylations reduce the pro-apoptotic activity of Bad. We suggest that at least part of the cytoprotective effect of PE against 2-DOG-induced apoptosis may result from phosphorylation of Bad on these sites. There is a signi®cant amount of evidence that the ERK1/2 cascade participates in the compensated hypertrophic response in the heart.65 Although this may involve modulation of transcription and translation primarily, we suggest that the potentially anti-apoptotic effects of ERK1/2 mediated through Bad phosphorylation may also participate in the hypertrophic process.

Appendix In this paper, we have used the amino acid numbering system of murine Bad in order to avoid confusion with other published material. However, the situation is somewhat different for rat Bad for which two splice variants (Bada and Badb) have recently been cloned.58 Rat Bada and Badb are 205 and 220 amino acid residues in length (including the initiator Met residue), respectively,58 whereas murine Bad contains 204 residues.66 Both Bada and Badb transcripts are expressed in rat heart.58 Rat Bada and Badb are identical in their ®rst N-terminal 165 residues but diverge thereafter. Sequence alignment shows that rat Bada and murine Bad show identity in 196 residues, whereas rat Badb and murine Bad are identical in 159 residues. The major difference between rat Bada and murine Bad is that a single Pro residue is inserted in the rat Bada sequence after Gln-89. Since this lies N-terminal to the Bad phosphorylation sites, the residues corresponding to Ser-112, Ser-136, Ser-155 and Ser-170 in murine Bad are Ser-113, Ser-137, Ser-156 and Ser-171, respectively, in rat Bada. The numbering of these residues in rat Badb is the same as in rat Bada, even though there is a sequence divergence between Bada and Badb around Ser-171.

Acknowledgment This work was funded by the British Heart Foundation.

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References 1. CHIEN KR, KNOWLTON KU, ZHU H, CHIEN S. Regulation of cardiac gene expression during myocardial growth and hypertrophy: molecular studies of an adaptive physiologic response. FASEB J 1991; 5: 3037ÿ ÿ3046. 2. SUGDEN PH, CLERK A. Cellular mechanisms of cardiac hypertrophy. J Mol Med 1998; 76: 725ÿ ÿ746. 3. BOGOYEVITCH MA, GLENNON PE, ANDERSSON MB, CLERK A, LAZOU A, MARSHALL CJ, PARKER PJ, SUGDEN PH. Endothelin-1 and ®broblast growth factors stimulate the mitogen-activated protein kinase signaling cascade in cardiac myocytes. The potential role of the cascade in the integration of two signaling pathways leading to myocyte hypertrophy. J Biol Chem 1994; 269: 1110ÿ ÿ1119. 4. CLERK A, BOGOYEVITCH MA, ANDERSSON MB, SUGDEN PH. Differential activation of protein kinase C isoforms by endothelin-1 and phenylephrine, and subsequent stimulation of p42 and p44 mitogenactivated protein kinases in ventricular myocytes cultured from neonatal rat hearts. J Biol Chem 1994; 269: 32 848ÿ ÿ32 857. 5. BOGOYEVITCH MA, KETTERMAN AJ, SUGDEN PH. Cellular stresses activate c-Jun N-terminal protein kinases (JNKs) in ventricular myocytes cultured from neonatal rat hearts. J Biol Chem 1995; 270: 29 710ÿ ÿ29 717. 6. CLERK A, MICHAEL A, SUGDEN PH. Stimulation of the p38 mitogen-activated protein kinase pathway in neonatal rat ventricular myocytes by the G proteincoupled receptor agonists endothelin-1 and phenylephrine: a role in cardiac myocyte hypertrophy? J Cell Biol 1998: 142: 523ÿ ÿ535. 7. NICOL RL, FREY N, PEARSON G, COBB M, RICHARDSON J, OLSON EN. Activated MEK5 induces serial assembly of sarcomeres and eccentric cardiac hypertrophy. EMBO J 2001; 20: 2757ÿ ÿ2767. 8. MARAIS R, WYNNE J, TREISMAN R. The SRF accessory protein Elk-1 contains a growth factor-regulated transcriptional activation domain. Cell 1993; 73: 381ÿ ÿ393. 9. TREISMAN R. Ternary complex factors: growth factor regulated transcriptional activators. Curr Opin Gen Dev 1994; 4: 96ÿ ÿ101. 10. STURGILL TW, RAY LB, ERIKSON E, MALLER JL. Insulinstimulated MAP-2 kinase phosphorylates and activates ribosomal protein S6 kinase II. Nature 1988; 334: 715ÿ ÿ718. 11. FROÈDIN M, GAMMELTOFT S. Role and regulation of 90 kDa ribosomal S6 kinase (RSK) in signal transduction. Mol Cell Endocrinol 1999; 151: 65ÿ ÿ77. 12. LIANG Q, WIESE RJ, BUENO OF, DAI YS, MARKHAM BE, MOLKENTIN JD. The transcription factor GATA4 is activated by extracellular signal-regulated kinase 1- and 2-mediated phosphorylation of serine 105 in cardiomyocytes. Mol Cell Biol 2001; 21: 7460ÿ ÿ7469. 13. MOLKENTIN JD. The zinc ®nger-containing transcription factors GATA-4, -5, and -6. Ubiquitously expressed regulators of tissue-speci®c gene expression. J Biol Chem 2000; 275: 38 949ÿ ÿ38 952. 14. MOLKENTIN JD, LU J-R, ANTOS C, MARKHAM B, RICHARDSON J, ROBBINS J, GRANT SR, OLSON EN.

762

15.

16.

17.

18.

19.

20. 21. 22. 23. 24.

25.

26.

27.

28.

29.

D. M. Valks et al.

A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell 1998; 93: 215ÿ ÿ228. DE WINDT LJ, LIM HW, TAIGEN T, WENCKER D, CONDORELLI G, DORN GW II, KITSIS RN, MOLKENTIN JD. Calcineurin-mediated hypertrophy protects cardiomyocytes from apoptosis in vitro and in vivo: an apoptosis-independent model of dilated heart failure. Circ Res 2000; 86: 255-263. YUE TL, WANG C, GU JL, MA XL, KUMAR S, LEE JC, FEUERSTEIN GZ, THOMAS H, MALEEFF B, OHLSTEIN EH. Inhibition of extracellular signal-regulated kinase enhances ischemia/reoxygenation-induced apoptosis in cultured cardiac myocytes and exaggerates reperfusion injury in isolated perfused heart. Circ Res 2000; 86: 692ÿ ÿ699. WANG Y, HUANG S, SAH VP, ROSS J, BROWN JH, HAN J, CHIEN KR. Cardiac muscle cell hypertrophy and apoptosis induced by distinct members of the p38 mitogen-activated protein kinase family. J Biol Chem 1998; 273: 2161ÿ ÿ2168. MA XL, KUMAR S, GAO F, LOUDEN CS, LOPEZ BL, CHRISTOPHER TA, WANG C, LEE JC, FEUERSTEIN GZ, YUE TL. Inhibition of p38 mitogen-activated protein kinase decreases cardiomyocyte apoptosis and improves cardiac function after myocardial ischemia and reperfusion. Circulation 1999; 99: 1685ÿ ÿ1691. ANDREKA P, ZANG J, DOUGHERTY C, SLEPAK TI, WEBSTER KA, BISHOPRIC NH. Cytoprotection by Jun kinase during nitric oxide-induced cardiac myocyte apoptosis. Circ Res 2001; 88: 305ÿ ÿ312. GROSS A, MCDONNELL JM, KORSMEYER SJ. BCL-2 family members and the mitochondria in apoptosis. Genes Dev 1999; 13: 1899ÿ ÿ1911. STRASSER A, O'CONNOR L, DIXIT VM. Apoptosis signaling. Annu Rev Biochem 2000; 69: 217ÿ ÿ245. DATTA SR, BRUNET A, GREENBERG ME. Cellular survival: a play in three Akts. Genes Dev 1999; 13: 2905ÿ ÿ2927. VANHAESEBROECK B, ALESSI DR. The PI3K-PDK1 connection: more than just a road to PKB. Biochem J 2000; 346: 561ÿ ÿ576. DATTA SR, KATSOV A, HU L, PETROS A, FESIK A, YAFFE MB, GREENBERG ME. 14-3-3 proteins and survival kinases cooperate to inactivate BAD by BH3 domain phosphorylation. Mol Cell 2000; 6: 41ÿ ÿ51. LIZCANO JM, MORRICE N, COHEN P. Regulation of BAD by cAMP-dependent protein kinase is mediated via phosphorylation of a novel site, Ser155. Biochem J 2000; 349: 547ÿ ÿ557. TAN Y, DEMETER MR, RUAN H, COMB MJ. BAD Ser-155 phosphorylation regulates J BAD:Bcl-XL interaction and cell survival. J Biol Chem 2000; 275: 25 865ÿ ÿ25 869. VIRDEE K, PARONE PA, TOLKOVSKY AM. Phosphorylation of the pro-apoptotic protein BAD on serine 155, a novel site, contributes to cell survival. Curr Biol 2000; 10: 1151ÿ ÿ1154. ZHOU X-M, LIU Y, PAYNE G, LUTZ RJ, CHITTENDEN T. Growth factors inactivate the cell death promoter, BAD, by phosphorylation of its BH3 domain on serine 155. J Biol Chem 2000; 275: 25 046ÿ ÿ25 051. DRAMSI S, SCHEID MP, MAITI A, HOJABRPOUR P, CHEN X, SCHUBERT K, GOODLETT DR, AEBERSOLD R, DURONIO V. Identi®cation of a novel phosphorylation site,

30.

31.

32.

33.

34.

35.

36. 37.

38.

39.

40.

41.

42.

Ser170, as a regulator of Bad pro-apoptotic activity. J Biol Chem 2002; 277: 6399ÿ ÿ6405. ZHA J, HARADA H, YANG E, JOCKEL J, KORSMEYER SJ. Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14ÿ628. 3-3 not BCL-Xl. Cell 1996; 87: 619ÿ DATTA SR, DUDEK H, TAO X, MASTERS S, FU H, GOTOH Y, GREENBERG ME. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 1997; 91: 231ÿ ÿ241. DEL PESO L, GONZALEZ-GARCIA M, PAGE C, HERRERA R, NUNEZ G. Interleukin-3-induced phosphorylation of Bad through the protein kinase Akt. Science 1997; 278: 687ÿ ÿ689. HARADA H, ANDERSEN JS, MANN M, TERADA N, KORSMEYER SJ. p70S6 kinase signals cell survival as well as growth, inactivating the pro-apoptotic molecule BAD. Proc Natl Acad Sci USA 2001; 98: 9666ÿ ÿ9670. BONNI A, BRUNET A, WEST AE, DATTA SR, TAKASU MA, GREENBERG ME. Cell survival promoted by Ras-MAPK signaling pathway by transcription-dependent and independent mechanisms. Science 1999; 286: 1358ÿ ÿ1362. BERTOLOTTO C, MAULON L, FILIPPA N, BAIER G, AUBERGER P. Protein kinase Cy and e promote Tcell survival by a Rsk-dependent phosphorylation and inactivation of BAD. J Biol Chem 2000; 275: 37 246ÿ ÿ37 250. SHIMAMURA A, BALLIF BA, RICHARDS SA, BLENIS J. Rsk1 mediates a MEK-MAP kinase survival signal. Curr Biol 2000; 10: 127ÿ ÿ135. TAN Y, RUAN H, DEMETER MR, COMB MJ. p90RSK blocks Bad-mediated cell death via a protein kinase C-dependent pathway. J Biol Chem 1999; 274: 34 859ÿ ÿ34 867. BOGOYEVITCH MA, ANDERSSON MB, GILLESPIE-BROWN J, CLERK A, GLENNON PE, FULLER SJ, SUGDEN PH. Adrenergic receptor stimulation of the mitogenactivated protein kinase cascade and cardiac hypertrophy. Biochem J 1996; 314: 115ÿ ÿ121. IWAKI K, SUKHATME VP, SHUBEITA HE, CHIEN KR. a- and b-adrenergic stimulation induces distinct patterns of immediate early gene expression in neonatal rat myocardial cells, fos/jun expression is associated with sarcomere assembly; Egr-1 induction is primarily an a1-mediated response. J Biol Chem 1990; 265: 13 809ÿ ÿ13 817. COOK SA, SUGDEN PH, CLERK A. Regulation of Bcl-2 family proteins during development and in response to oxidative stress in cardiac myocytes: association with changes in mitochondrial membrane potential. Circ Res 1999; 85: 940ÿ ÿ949. CHILOECHES A, PATERSON HF, MARAIS RM, CLERK A, MARSHALL CJ, SUGDEN PH. Regulation of Ras.GTP loading and Ras-Raf association in neonatal rat ventricular myocytes by G protein-coupled receptor agonists and phorbol esters. Activation of the ERK cascade by phorbol esters is mediated by Ras. J Biol Chem 1999; 274: 19 762ÿ ÿ19 770. IWAI-KANAI E, HASEGAWA K, ARAKI M, KAKITA T, MORIMOTO T, SASAYAMA S. a- and b-Adrenergic pathways differentially regulate cell type-speci®c apoptosis in rat cardiac myocytes. Circulation 1999; 100: 305ÿ ÿ311.

Phosphorylation of Bad in Cardiac Myocytes 43. ZHU H, MCELWEE-WITMER S, PERRONE M, CLARK KL, ZILBERSTEIN A. Phenylephrine protects neonatal rat cardiomyocytes from hypoxia and serum deprivation-induced apoptosis. Cell Death Differ 2001; 7: 773ÿ ÿ784. 44. DAVIES SP, REDDY H, CAIVANO M, COHEN P. Speci®city and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 2000; 351: 95ÿ ÿ105. 45. PHAM FH, COLE SM, CLERK A. Regulation of cardiac myocyte protein synthesis through phosphatidylinositol 3 0 kinase and protein kinase B. Adv Enzyme Regul 2001; 41: 73ÿ ÿ86. 46. ALESSI DR. The protein kinase C inhibitor Ro 318220 and GF 109203X are equally potent inhibitors of MAPKAPK kinase-1b (Rsk-2) and p70 S6 kinase. FEBS Lett 1997; 402: 121ÿ ÿ123. 47. PHAM FH, SUGDEN PH, CLERK A. Regulation of protein kinase B and 4E-BP1 by oxidative stress in cardiac myocytes. Circ Res 2000; 86: 1252ÿ ÿ 1258. 48. KAMAKURA S, MORIGUCHI T, NISHIDA E. Activation of the protein kinase ERK5/BMK1 by receptor tyrosine kinases. Identi®cation and characterization of a signaling pathway to the nucleus. J Biol Chem 1999; 274: 26 563ÿ ÿ26 571. 49. MODY N, LEITCH J, ARMSTRONG C, DIXON J, COHEN P. Effects of MAP kinase cascade inhibitors on the MKK5/ERK5 pathway. FEBS Lett 2001; 502: 21ÿ ÿ24. 50. HAUNSTETTER A, IZUMO S. Towards antiapoptosis as a new treatment modality. Circ Res 2000; 86: 371ÿ ÿ376. 51. KANG PM, IZUMO S. Apoptosis and heart failure: a critical review of the literature. Circ Res 2000; 86: 1107ÿ ÿ1113. 52. OH H, FUJIO Y, KUNISADA K, HIROTA H, MATSUI H, KISHIMOTO T, YAMAUCHI-TAKIHARA K. Activation of phosphatidylinositol 3-kinase through glycoprotein 130 induces protein kinase B and p70 S6 kinase phosphorylation in cardiac myocytes. J Biol Chem 1998; 273: 9703ÿ ÿ9710. 53. KUWAHARA K, SAITO Y, KISHIMOTO I, MIYAMOTO Y, HARADA M, OGAWA E, HAMANAKA I, KAJIYAMA N, TAKAHASHI N, IZUMI T, KAWAKAMI R, NAKAO K. Cardiotrophin-1 phosphorylates akt and BAD, and prolongs cell survival via a P13K-dependent pathway in cardiac myocytes. J Mol Cell Cardiol 2000; 32: 1385ÿ ÿ1394. 54. NEGORO S, OH H, TONE E, KUNISADA K, FUJIO Y, WALSH K, KISHIMOTO T, YAMAUCHI-TAKIHARA K. Glyocoprotein 130 regulates cardiac myocyte survival in doxirubicin-induced apoptosis through phosphatidylinositol 3-kinase/Akt phosphorylation and Bcl-xL/caspase 3 interaction. Circulation 2000; 103: 555ÿ ÿ561.

763

55. WU W, LEE WL, WU YY, CHEN D, LIU TJ, JANG A, SHARMA PM, WANG PH. Expression of constitutively active phosphatidylinositol 3-kinase inhibits activation of caspase 3 and apoptosis in cardiac muscle cells. J Biol Chem 2000; 275: 40113ÿ ÿ40119. 56. BOLUYT MO, ZHENG J-S, YOUNES A, LONG X, O'NEILL L, SILVERMAN H, LAKATTA EG, CROW MT. Rapamycin inhibits a1-adrenergic receptor-stimulated cardiac myocyte hypertrophy but not activation of hypertrophy-associated genes. Evidence for involvement of p70 S6 kinase. Circ Res 1997; 81: 176ÿ ÿ186. 57. SCHLUÈTER KD, SIMM A, SCHAFER M, TAIMOR G, PIPER HM. Early response kinase and PI 3-kinase activation in adult cardiomyocytes and their role in hypertrophy. Am J Physiol 1999; 276: H1655ÿ ÿH1663. 58. HAMNEÂR S, ARUMAÈE U, LI-YING Y, SUN Y-F, SAARMA M, LINDHOLM D. Functional characterization of two splice variants of rat Bad and their interaction with Bcl-w in sympathetic neurons. Mol Cell Neurosci 2001; 17: 97ÿ ÿ106. 59. OBATA T, YAFFE MB, LEPARC GG, PIRO ET, MAEGAWA H, KASHIWAGI A, KIKKAWA R, CANTLEY LC. Peptide and protein library screening de®nes optimal substrate motifs for AKT/PKB. J Biol Chem 2000; 275: 36 108ÿ ÿ36 115. 60. LEIGHTON IA, DALBY KN, CAUDWELL FB, COHEN PT, COHEN P. Comparison of the speci®cities of p70 S6 kinase and MAPKAP kinase-1 identi®es a relatively speci®c substrate for p70 S6 kinase: the N-terminal kinase domain of MAPKAP kinase-1 is essential for peptide phosphorylation. FEBS Lett 1995; 375: 289ÿ ÿ293. 61. SONGYANG Z, BLECHNER S, HOAGLAND N, HOEKSTRA MF, PIWNICA-WORMS H, CANTLEY LC. Use of an orientated peptide library to determine the optimal substrates of protein kinases. Curr Biol 1994; 4: 973ÿ ÿ982. 62. HASEGAWA K, IWAI-KANAI E, SASAYAMA S. Neurohormonal regulation of myocardial cell apoptosis during the development of heart failure. J Cell Physiol 2001; 186: 11ÿ ÿ18. 63. SINGH K, XIAO L, REMONDINO A, SAWYER DB, COLUCCI WS. Adrenergic regulation of cardiac myocyte apoptosis. J Cell Physiol 2001; 189: 257ÿ ÿ265. 64. HENAFF M, HATEM SN, MERCADIER JJ. Low catecholamine concentrations protect adult rat ventricular myocytes against apoptosis through cAMPdependent extracellular signal-regulated kinase activation. Mol Pharmacol 2000; 58: 1546ÿ ÿ1553. 65. SUGDEN PH. Signalling pathways in cardiac myocyte hypertrophy. Ann Med 2001; 33: 611ÿ ÿ622. 66. YANG E, ZHA J, JOCKEL J, BOISE LH, THOMSON CB, KORSMEYER SJ. Bad, a heterodimeric partner for BelXL and Bcl-2, displaces Bax and promotes cell death. Cell 1995; 80: 285ÿ ÿ291.