Activation of Phosphatidylinositol 3-Kinase through Glycoprotein 130 Induces Protein Kinase B and p70 S6 Kinase Phosphorylation in Cardiac Myocytes*

Phosphatidylinositol (PI) 3-kinase is known to be activated by cytokine stimulation through different types of receptors to transduce intracellular responses. We have previously reported that leukemia inhibitory factor (LIF) induces the activation of Janus kinase signal transducer and activator of transcription (JAK-STAT) and mitogen-activated protein (MAP) kinase pathways through glycoprotein (gp) 130 in cardiac myocytes. How-ever, whether PI 3-kinase is involved in regulation of gp130 signaling and the activation mechanisms by which it associates with other tyrosine-phosphorylated proteins remain unknown. We found that LIF induced the activation of PI 3-kinase in cardiac myocytes. More-over, JAK1 binds to PI 3-kinase, and LIF stimulation increases the PI 3-kinase activity in JAK1 immunoprecipitates. Activation of MAP kinase and protein kinase B by LIF was attenuated by wortmannin. LIF-induced p70 S6 kinase activation, protein synthesis, and c -fos mRNA expression were inhibited by wortmannin and rapamycin. Both inhibitors failed to appreciably affect the phosphorylation of STAT3. In conclusion, PI 3-kinase is activated with LIF in cardiac myocytes, and JAK1 is found to associate with this enzyme. PI 3-kinase provides a crucial link between gp130, MAP kinase, protein kinase B, and p70 S6 kinase in cardiac myocytes. associated to the P-81 phospho- cellulose paper was counted. Northern Blot Analysis— Isolation of total cellular RNA was per- formed by the acid guanidinium thiocyanate/phenol/chloroform method (30). Northern blot was performed as described previously (24). A la- beled c -fos DNA fragment (484 base pairs) was used as a probe. The relative amounts of specific mRNA were visualized by autoradiography. The hybridization signals of mRNA were normalized to those of human b -actin mRNA. Statistics— Statistical analysis was performed by using Student’s t test as appropriate. Significance was accepted at p , 0.05.

A large number of studies have shown that cytokines share signaling pathways involving activation of protein tyrosine kinases which are required for subsequent cellular responses (1)(2)(3). Recent reports revealed that interleukin-6 related cytokines including interleukin-6, leukemia inhibitory factor (LIF), 1 ciliary neurotrophic factor, oncostatin M, and cardiotrophin-1 activate Janus kinase (JAK)/Tyk family kinases by the formation of homodimers or heterodimers of gp130 (4 -7). gp130 functions as a common cytokine signal transducer for the interleukin-6 family, and is reported to lead to cardiac hypertrophy. These signals might be essential in the physiologic regulation of myocardium by these cytokines (8,9). LIF is also a member of multifunctional cytokines, and induces one to one heterodimerization between the LIF receptor and gp130 (10,11). We have recently demonstrated that JAK signal transducer and activator of transcription (STAT) and mitogen-activated protein (MAP) kinase pathways are present at downstream of gp130 in cardiac myocytes and are rapidly activated by LIF both in vivo and in vitro (12).
Activation of cell proliferation by several different cytokines has been shown to correlate with the intracellular activation of common protein kinase cascades. This may be explained by the functional pleiotropy and redundancy of cytokine receptor systems (13). It is very intriguing that growth factors (e.g. plateletderived growth factor and angiotensin II) and cytokines work through the JAK/Tyk and MAP kinases and lead to the activation of the same or different sets of signal transduction pathways.
In the present study, we investigated the role of PI 3-kinase in LIF-induced hypertrophic and cytoprotective signals in cardiac myocytes. A specific inhibitor of PI 3-kinase, wortmannin, inhibited the LIF-induced activation of MAP kinase, protein kinase B (PKB), p70 S6 kinase, protein synthesis, and c-fos mRNA expression in cardiac myocytes. In addition, we demonstrated for the first time that JAK1 protein kinase may be the protein associated with PI 3-kinase after LIF stimulation, although wortmannin did not affect the phosphorylation of STAT3. Therefore, our study suggests that maximal stimulation of the protein kinase cascade by LIF requires the activation of PI 3-kinase, which may be an important mediator of LIF-induced signal transduction in cardiac myocytes.

EXPERIMENTAL PROCEDURES
Reagents-Recombinant mouse LIF, antibody to PI 3-kinase, PKB, anti-phosphotyrosine (4G10), Crosstide (substrate peptide for PKB), and p70 S6 kinase kits were purchased from Upstate Biotechnology Inc., Lake Placid, NY. Antibodies to JAK1, p70 S6 kinase, and STAT3 were from Santa Cruz Biotechnology Inc, Santa Cruz, CA. The MAP kinase assay kit, [␥-32 P]ATP, protein A/G-Sepharose, and enhanced chemiluminescence (ECL) detection system were purchased from Amersham International plc., Oakville, Ontario, Canada. [ 3 H]Leucine was obtained from ICN. Wortmannin, rapamycin, and phorbol 12-myristate 13-acetate were purchased from Sigma. Wortmannin was dissolved in dimethyl sulfoxide to 1 mM stocks, stored at Ϫ20°C, and diluted in medium immediately before use. Rapamycin was dissolved as stock solution in ethanol. PD098059 were purchased from New England Biolabs, Inc., Beverly, MA. Phosphatidylinositol was obtained from Avanti Polar Lipids Inc., Alabaster, AL. Medium-199 (M-199; Flow Laboratories, Inc.) and newborn bovine serum (Life Technologies, Inc.) were used for cell culture. All other chemicals were reagents of molecular biology grade and were obtained from standard commercial sources.
Cell Culture and Stimulation-Primary cultures of neonatal rat cardiac myocytes were prepared from the ventricles of 1-day-old Sprague-Dawley rats (Nippon Dobutsu, Japan) as described previously (24). After an enzymatic dissociation, the cells were preplated for 1 h to selectively enrich for cardiac myocytes. The resultant suspension of myocytes were plated onto 35-or 60-mm culture dishes at a density of 1 ϫ 10 5 cells/cm 2 and cultured in M-199 supplemented with 10% newborn bovine serum and 0.1 mM bromodeoxyuridine. The medium was changed to M-199 and 10% newborn bovine serum 24 h after seeding. We obtained Ͼ90% myocytes in cultures prepared with this procedure. Cells were incubated in a humidified atmosphere of 5% CO 2 , 95% air at 37°C and were generally used for the experiments 4 days after seeding. The medium was changed to M-199 24 h before the experiments.
Protein Synthesis Measurements-Cardiac myocytes seeded in 24well plates were stimulated with LIF (10 3 units/ml) in serum-free medium containing 0.5 Ci/ml [ 3 H]leucine. After 24 h, the cells were washed three times with phosphate-buffered saline and fixed for 30 min with cold 5% trichloroacetic acid to precipitate protein. The cells were then washed once with trichloroacetic acid and three times in tap water. The radioactivity incorporated into the trichloroacetic acid-insoluble fraction was measured by liquid scintillation counting after solubilization in 0.1 M NaOH. The results were expressed as counts per minute per dish.
PI 3-Kinase Assay-Reactions and lipid extraction were essentially performed according to the method of Whitman et al. (25) and Hayashi et al. (26). The immunoprecipitates were subjected to the assay in a 50-l reaction mixture (20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 10 mM MgCl 2 , 0.5 mM EGTA, 120 M adenosine, 50 M ATP). Phosphatidylinositol sonicated in chloroform was added to the reaction at a final concentration of 0.2 mg/ml and vortexed before incubation at 30°C for 10 min. Wortmannin was added to the pellet in various concentrations and samples were preincubated at 30°C for 30 min. The reaction was initiated by the addition of 10 Ci of [␥-32 P]ATP. After 10 min of incubation at 30°C, the reactions were stopped by adding 100 l of 1 M HCl. Phospholipids were immediately extracted with 200 l of CHCl 3 / MeOH (1:1, v/v). Equal volume aliquots from the bottom organic phase were spotted onto thin layer chromatography (TLC) Silica Gel 60 plates (Merck), developing solvent for 2 h, and visualized by autoradiography.
In Vitro Assays for MAP Kinase Activity-Assays of MAP kinase activity were performed as described previously (27). The cells were lysed at 4°C in 0.2 ml of lysis buffer containing 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EGTA, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and 10 g/ml aprotinin. Insoluble material was removed by centrifugation at 15,000 rpm for 30 min. The concentrations of total protein in the supernatant were measured using protein assay system (Bio-Rad). Total cell lysates (1 g of protein/reaction) were incubated in 0.7 mg/ml synthetic peptide substrate containing the phosphorylation sequence PLS/TP in a final volume of 30 l of kinase buffer (10 mM MgCl 2 , 50 M ATP). The reactions were initiated by adding 2 Ci of [␥-32 P]ATP to the mixture. After a 30-min incubation at 30°C, the reactions were terminated by the addition of 10 l of stop solution. Aliquots of the supernatants (30 l) were spotted onto phosphocellulose paper (Whatman International Ltd.), washed twice with 60 mM H 3 PO 4 for 2 min, and twice with distilled water for 2 min, dried, and counted by the Cerenkov technique.
Immunoprecipitation and p70 S6 Kinase Assay-The cells were lysed on ice in 0.5 ml of lysis buffer containing 10 mM potassium phosphate, pH 7.4, 1 mM EDTA, 5 mM EGTA, 10 mM MgCl 2 , 50 mM ␤-glycerophosphate, 0.5% Triton X-100, 1 mM sodium orthovanadate, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 10 g/ml aprotinin. Immunoprecipitations for STAT3 were performed as described previously (12). After 20 min, lysates were centrifuged for 30 min at 15,000 rpm, and the postnuclear lysates were immunoprecipitated with 0.3 g/ml p70 S6 kinase antiserum for 2 h before addition of 30 l of protein A-Sepharose for 1 h. Immunoprecipitates were washed twice with lysis buffer and twice with kinase assay buffer (20 mM MOPS, pH 7.2, 25 mM ␤-glycerophosphate, 5 mM EDTA, 1 mM sodium orthovanadate, and 1 mM dithiothreitol). The immunoprecipitates were resuspended in 30 l of kinase buffer containing 20 M S6 kinase substrate peptide (AKRRRLSSLRA) and 2 M protein kinase inhibitor peptide. The kinase reaction was initiated by the addition of 40 M ATP, 10 mM MgCl 2 mixture and 10 Ci of [␥-32 P]ATP mixture. After a 10-min incubation at 30°C, 20 l of the reaction was spotted onto P-81 phosphocellulose paper and washed twice in 0.75% phosphoric acid and twice in acetone. The dried papers were placed in 5 ml of scintillant and counted by the Cerenkov technique.
PKB Kinase Assay-PKB kinase assay was performed according to the method previously described by Cross et al. (28,29) with modification. Cells were treated as for p70 S6 kinase assay except that lysis buffer (50 mM Tris-HCl, pH 7.5, 0.1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 50 mM sodium fluoride, 10 mM ␤-glycerophosphate, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 0.1% 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and 10 g/ml aprotinin) was used to lyse cells. To immunoprecipitate endogenous PKB, 1 g of anti-PKB antibody coupled to protein G-Sepharose beads were used. The immunoprecipitates were washed three times in lysis buffer containing 0.5 M NaCl, twice in washing buffer (50 mM Tris-HCl, pH 7.5, 0.03% (w/v) Brij-35, 0.1 mM EGTA, and 0.1% 2-mercaptoethanol), and once in kinase assay buffer. The immunoprecipitates were resuspended in 30 l of kinase assay, buffer containing 30 M Crosstide, and 17 M protein kinase A inhibitor peptide. The kinase reaction was processed as for p70 S6 kinase assays, and the radioactivity associated to the P-81 phosphocellulose paper was counted.
Northern Blot Analysis-Isolation of total cellular RNA was performed by the acid guanidinium thiocyanate/phenol/chloroform method (30). Northern blot was performed as described previously (24). A labeled c-fos DNA fragment (484 base pairs) was used as a probe. The relative amounts of specific mRNA were visualized by autoradiography. The hybridization signals of mRNA were normalized to those of human ␤-actin mRNA.
Statistics-Statistical analysis was performed by using Student's t test as appropriate. Significance was accepted at p Ͻ 0.05.

LIF Induces PI 3-Kinase Activation in Cardiac Myocytes-
Several cell line studies indicated that PI 3-kinase plays an important role in the cellular responses to various cytokines (15)(16)(17)(18)(19)(20). To investigate the involvement of PI 3-kinase in gp130 signaling, we examined PI 3-kinase activity in cultured cardiac myocytes after LIF (10 3 units/ml) stimulation. We found that treatment of cardiac myocytes with LIF caused a substantial increase in the PI 3-kinase activity that immunoprecipitated with either anti-phosphotyrosine or anti-PI 3-kinase antibody (Fig. 1A, upper panel). The kinase activity presented a rapid increase at 10 min and declined at 60 min in both immunoprecipitates. Subsequent Western blot analysis with anti-PI 3-kinase antibody were performed to verify the same amount of p85 in all samples (Fig. 1A, middle panel). Quantitation of PI 3-kinase activity obtained by densitometric analysis of autoradiograms were summarized in the lower panel. To determine whether PI 3-kinase is a critical functional component of LIFinitiated signaling, we used the PI 3-kinase specific inhibitor, wortmannin, in this system to confirm the specificity of this activity to PI 3-kinase (31). After 10 min of LIF stimulation, cardiac myocytes were immunoprecipitated by anti-PI 3-kinase antibody and wortmannin was added to the pellet at the concentrations indicated in Fig. 1B. Wortmannin reduced the LIFinduced activation of PI 3-kinase to 50% of the maximum at a concentration of 1 nM and completely abrogated the activity at a concentration of 100 nM.
LIF Induces Tyrosine Phosphorylation of the p85 Subunit of PI 3-Kinase-We next examined the tyrosine phosphorylation of the p85 subunit of PI 3-kinase after 10 min of LIF stimulation. As shown in Fig. 2 (upper panel), stimulation of cardiac myocytes with LIF markedly increased tyrosine phosphorylation of the p85 subunit of PI 3-kinase. Similar amounts of protein were detected with anti-PI 3-kinase antibodies, showing that equivalent amounts of p85 were immunoprecipitated (Fig. 2, lower panel).
LIF Stimulates p70 S6 Kinase Activity in Cardiac Myocytes-To examine whether p70 S6 kinase is activated by LIF, p70 S6 kinase mobility shift and immunocomplex assays were performed. Fig. 3 (upper panel) shows a time course of p70 S6 kinase activation after LIF stimulation. Treatment of the cells with LIF for 15 min caused a large increase in the phosphorylation of p70 S6 kinase (␣I and ␣II), which was accompanied by the slowest migration in electrophoretic mobility. Western blot analysis with anti-JAK1 antibody was used to normalize the levels of p70 S6 kinase in all samples (Fig. 3, middle panel). To further analyze the changes in the phosphorylation state of p70 S6 kinase, immunocomplex assay was performed with S6 peptide used as a substrate. The kinase assay was performed after immunoprecipitation with an anti-p70 S6 kinase antibody to eliminate other kinases that phosphorylate p70 S6 kinase. We confirmed that LIF activates p70 S6 kinase, which results in a 2.8-fold increase in phosphorylation of the S6 peptide at 15 min (Fig. 3, lower panel).
Wortmannin Inhibits Activation of p70 S6 Kinase Induced by LIF-We next examined the effects of rapamycin on LIF-induced p70 S6 kinase activation. Cardiac myocytes were pretreated with the indicated concentrations of rapamycin for 30

FIG. 1. LIF induces PI 3-kinase activation in cardiac myocytes.
A, upper panel, analysis of PI 3-kinase activity in cardiac myocytes stimulated with LIF. Cardiac myocytes were stimulated with 10 3 units/ml LIF for the time indicated. 1% Nonidet P-40 cell lysates were immunoprecipitated with either anti-phosphotyrosine antibody (left) or anti-p85 subunit of PI 3-kinase antibody (right), and the precipitates were assayed for PI 3-kinase activity. The 32 P-labeled lipid products (PI3-P) were separated by TLC. The origin contains residual [␥-32 P]ATP that was not removed by organic phase, the amount of which is not relevant. Middle panel, the relative amount of p85 subunit of PI 3-kinase were shown by Western blot analysis. The equal volume of divided cell lysates were immunoprecipitated with anti-PI 3-kinase antibody and the immunoprecipitates were separated on 7.5% SDS-PAGE, transferred onto Immobilon-P membrane. Protein bands were visualized by ECL detection system after incubation with anti-PI 3-kinase antibody. Experiments were repeated three times with similar results. Lower panel, PI 3-kinase activity were quantified by densitometric analysis of the autoradiogram of TLC plates derived from the upper panel. Results were expressed as fold of control. Data are mean Ϯ S.E. from three independent experiments. *, p Ͻ 0.05 versus 0 min. B, inhibition of in vitro PI 3-kinase assay by wortmannin. Cardiac myocytes were stimulated with 10 3 units/ml LIF for 10 min and the cell lysates were immunoprecipitated with anti-PI 3-kinase antibody. The samples were preincubated with wortmannin at 30°C for 30 min before the reaction. PI 3-kinase assay was performed in the absence or presence of various concentrations of wortmannin as indicated. Experiments were repeated three times with similar results.
FIG. 2. LIF induces tyrosine phosphorylation of the p85 subunit of PI 3-kinase. Cardiac myocytes were stimulated with 10 3 units/ml LIF for 10 min, and the cell lysates were immunoprecipitated with anti-phosphotyrosine (upper panel) or anti-PI 3-kinase antibodies (lower panel). The immunoprecipitates were separated on 7.5% SDS-PAGE and transferred onto Immobilon-P membrane. Protein bands were visualized by ECL detection system after incubation with anti-PI 3-kinase antibody. Molecular mass protein markers indicate 97 and 66 kDa. Experiments were repeated three times with similar results. min, and p70 S6 kinase mobility shift assays were performed after 15 min of LIF stimulation. As shown in Fig. 4A (upper panel), p70 S6 kinase was rapidly dephosphorylated in the presence of 3 ng/ml rapamycin, and the inhibitory effects occurred in a dose-dependent manner. Several studies using the selective inhibitor wortmannin indicated that PI 3-kinase acts upstream of p70 S6 kinase in a signaling cascade induced by a number of tyrosine kinase receptors (18,32,33). To support our hypothesis that PI 3-kinase is coupled to the p70 S6 kinase signaling cascade in cardiac myocytes, we used wortmannin in the assay. Pretreatment of cardiac myocytes with 100 nM wortmannin for 15 min led to complete inactivation of p70 S6 kinase (Fig. 4A, upper panel, lane 3). This inhibitory effect of wortmannin occurred in a concentration-dependent manner (Fig.  4B, upper panel, lanes 4 -6). Subsequent Western blot analysis with anti-JAK1 antibody were performed to verify the same amount of p70 S6 kinase in all samples (Fig. 4, A and B, middle  panel). These results correlated well with inactivation of kinase activities (Fig. 4, A and B, lower panel).
LIF Increases the Protein Synthesis in Cardiac Myocytes Which Is Inhibited by Wortmannin and Rapamycin-We showed that LIF treatment caused a 2.1-fold increase in [ 3 H]leucine incorporation over 24 h (Fig. 5, lane 4). We next tested the effects of wortmannin and rapamycin on protein synthesis. As shown in Fig. 5, wortmannin (100 nM) and rapamycin (3 ng/ml) do not simply suppress the basal level of [ 3 H]leucine incorporation (lanes 2 and 3, respectively) but specifically inhibit LIF-stimulated protein synthesis. Preincubation with wortmannin for 15 min completely inhibited LIFstimulated increase in [ 3 H]leucine incorporation (Fig. 5, lane  5). To determine whether the increase in protein synthesis was mediated via p70 S6 kinase, cardiac myocytes were preincubated with rapamycin for 30 min before LIF stimulation. Rapamycin significantly suppressed LIF-induced [ 3 H]leucine incorporation (Fig. 5, lane 6).
LIF Induced MAP Kinase Activation Is Inhibited by Wortmannin but Not by Rapamycin and PKC Down-regulation-To investigate the involvement of PI 3-kinase in LIF induced MAP kinase activation in cardiac myocytes, we examined the effects of wortmannin on MAP kinase activity. MAP kinase activity was not affected by wortmannin nor rapamycin in the vehicletreated cells (Fig. 6, lanes 2 and 3). It was significantly augmented 5 min after LIF stimulation (Fig. 6, lane 4). When cardiac myocytes were pretreated with wortmannin at a dose of 100 nM for 15 min, activation of MAP kinase induced by LIF was inhibited by 50% as compared with the kinase activity without pretreatment (Fig. 6, lane 5). We next examined whether rapamycin and PKC down-regulation would influence the activation of MAP kinase induced by LIF. The cells were pretreated with 3 ng/ml rapamycin for 30 min and 5 M phorbol 12-myristate 13-acetate for 24 h, respectively, before LIF stimulation. Activation of MAP kinase achieved with LIF was affected neither by rapamycin nor phorbol 12-myristate 13-acetate pretreatment (Fig. 6, lanes 6 and 7).
JAK Protein Kinase Associates with PI 3-Kinase following LIF Stimulation-We have previously reported that LIF induces tyrosine phosphorylation of JAK1 and JAK2 within 15 min in cardiac myocytes (12). Therefore, we examined the possible relationship between JAK protein kinase and PI 3-kinase. Cell lysates from cardiac myocytes were immunoprecipitated with antibody to JAK1 and analyzed by immunoblotting with an antibody to the p85 subunit of PI 3-kinase. We showed that PI 3-kinase was co-immunoprecipitated, but there was no significant increase in the amount of PI 3-kinase protein after 15 min LIF stimulation (Fig. 7A). These results indicate that these associations do not directly enhance the kinase activity induced by LIF. The specificity of the antibody used for immunoblotting was verified by preincubation of the antibody with an excess amount of peptide antigen, which eliminated the signal (Fig. 7A, preabsorbed). The immunoblots were reprobed with anti-phosphotyrosine and anti-JAK1 antibodies. As shown in Fig. 7B, tyrosine phosphorylation of JAK1 immunoprecipitates was only observed after LIF stimulation (upper panels). We also confirmed that the same amount of proteins were immunoprecipitated in each sample (Fig. 7B, lower panels). To investigate whether the PI 3-kinase which co-immunoprecipitated with JAK1 remains active, anti-JAK1 immunoprecipitates were analyzed for PI 3-kinase activity. We observed that LIF stimulation induced increased PI 3-kinase activity associated with JAK1, which completely inhibited by wortmannin (Fig. 7C).

Induction of STAT3 Phosphorylation Does Not Require PI 3-Kinase and MAP Kinase Kinase (MEK) Activation in Cardiac
Myocytes-We next examined the involvement of PI 3-kinase, p70 S6 kinase, MAP kinase in the downstream signaling events of JAKs by studying the status of STAT3 activation under the conditions of wortmannin, rapamycin, and MEK inhibitor PD098059 pretreatment. As shown in Fig. 8A, LIF induced tyrosine phosphorylation of STAT3 within 5 min in cardiac myocytes. But interestingly, neither wortmannin nor rapamycin affected the LIF-induced tyrosine phosphorylation STAT3. In addition, there was a discernible reduction in the electrophoretic mobility of STAT3␣, which is phosphorylated on serine after 10 min LIF stimulation, and then declined to basal level at 60 min (Fig. 8B,  lanes 2 and 3). But this serine phosphorylated activity was not inhibited by wortmannin, rapamycin, and PD098059 (Fig. 8B,  lanes 4 -7). Hence, inhibition of the PI 3-kinase, p70 S6 kinase, and MEK pathways do not prevent the LIF-induced serine phosphorylation of STAT3 in cardiac myocytes.
Effects of Wortmannin and Rapamycin on c-fos mRNA Expression-A recent report demonstrated that LIF, cardiotrophin-1, and phenylephrine caused c-fos mRNA expression in cardiac myocytes (34). To further investigate the selectivity of wortmannin and rapamycin action, we evaluated the effects of these inhibitors on c-fos mRNA expression in cardiac myocytes. As shown in Fig. 9, LIF-induced c-fos mRNA expression was partially inhibited by wortmannin and rapamycin. The inhibitory effects of these inhibitors on c-fos mRNA expression supports the hypothesis that PI 3-kinase and p70 S6 kinase are involved in the signaling cascade leading to cardiac myocyte hypertrophy.
LIF Induced PKB Activation Is Sensitive to Wortmannin but Not to Rapamycin-The PKB encodes a serine-threonine protein kinase which is activated by several growth factor-generated signals that are transduced via PI 3-kinase (35)(36)(37). As shown in Fig. 10A, LIF induced a rapid activation of PKB at 5 min in cardiac myocytes and the kinase activity reached maximal at 15 min. We then analyzed whether PKB activation might be a part of PI 3-kinase and/or p70 S6 kinase-dependent signaling pathways. The effect of the wortmannin and rapamycin on the PKB activation was examined (Fig. 10B). LIF stimulation increased PKB kinase activity over five times and it was completely inhibited by wortmannin but not by rapamycin (Fig. 10B, lanes 5 and 6). Thus, LIF regulation of PKB employs PI 3-kinase but not p70 S6 kinase in cardiac myocytes. DISCUSSION In this study, we demonstrated that treatment of cardiac myocytes with LIF resulted in the activation of PI 3-kinase followed by the activation of MAP kinase, PKB, and p70 S6 kinase. In addition, we demonstrated the possibility that JAK protein kinases associate with PI 3-kinase, and a PI 3-kinasemediated pathway is involved in LIF-induced hypertrophic and cytoprotective signals in cardiac myocytes.
We have shown that LIF caused a substantial increase in the production of PI 3-kinase products in a time-dependent man-

FIG. 6. LIF-induced MAP kinase activation is inhibited by wortmannin but not by rapamycin and PKC down-regulation.
Cardiac myocytes were stimulated with 10 3 units/ml LIF for 5 min in the absence or presence of the indicated inhibitors as described in ner, and it was inhibited by wortmannin in a concentrationdependent manner (Fig. 1). Furthermore, the specificity of this kinase assay was performed by including adenosine in kinase buffer as an inhibitor of PI 4-kinase. The differences of PI 3-kinase activities between the immunoprecipitates with antiphosphotyrosine and anti-PI 3-kinase antibody depend on the precipitability of each antibody (26). The findings that PI 3-kinase activity were observed in control immunoprecipitates correlated well with the evidence that PI 3-kinase products is constitutively produced in the absence of growth factor stimulation (20,38). LIF-induced increase in PI 3-kinase activity detected in anti-PI 3-kinase immunoprecipitates may be due to, at least in part, increased tyrosine phosphorylation of PI 3-kinase itself (Fig. 2). A recent report has demonstrated that the ␣ 1 -adrenergic receptor phosphorylates the p85 subunit of purified PI 3-kinase on tyrosine residues in vascular smooth muscle cells (19).
Although the direct interaction between p70 S6 kinase and PI 3-kinase is poorly understood, several studies using wortmannin indicate that PI 3-kinase acts upstream of p70 S6 kinase (18,32,33). Recent studies have demonstrated that PI 3-kinase provides a signal necessary for the activation of p70 S6 kinase, which directs the site-specific phosphorylation of Thr-252 in the p70 catalytic domain (39). Our results demonstrate that LIF, like other growth factors, also activates p70 S6 kinase in cardiac myocytes, and this activity is completely inhibited by wortmannin and rapamycin.
The inhibitory effects of wortmannin and rapamycin on LIFinduced protein synthesis demonstrate the functional relevance of PI 3-kinase and p70 S6 kinase activation, and which are consistent with previous reports (40,41). They showed that rapamycin at 10 ng/ml reduced the basal protein content. However, this is not due to a general toxic effect of rapamycin, because the increase in protein content caused by serum stimulation was not affected by rapamycin. As we used lower dose (3 ng/ml) of rapamycin in these experiments and it was also similar to its inhibition of p70 S6 kinase activation, these observations were not due to toxic or nonspecific effects of rapamycin. p70 S6 kinase is reported to be related to cardiac hypertrophy in response to angiotensin II and ␣ 1 -adrenergic receptor (40,41). These findings suggest that PI 3-kinase is FIG. 7. JAK protein kinase associates with PI 3-kinase following LIF stimulation. A, cardiac myocytes were treated with 10 3 units/ml LIF for 15 min and immunoprecipitated by anti-JAK1 antibody. Whole lysates and each immunoprecipitate were blotted with anti-PI 3-kinase antibody or antibody preabsorbed with a peptide antigen for 2 h. Control indicates a basal level of PI 3-kinase association with JAK1 immunoprecipitates. They were not enhanced by LIF stimulation. B, the membrane used above was stripped and first probed with antiphosphotyrosine antibody and then reprobed with the indicated antibodies. Anti-phosphotyrosine blot of JAK1 immunoprecipitates showed that LIF induced tyrosine phosphorylation of JAK1 in cardiac myocytes (upper panel). The amounts of precipitated protein were the same in each sample (lower panel). All blots were representative of three independent experiments. C, lysates from cardiac myocytes were prepared as described in A. Although a basal PI 3-kinase activity was present in JAK1 immunoprecipitates, LIF induced a significantly increased PI 3-kinase activity. The kinase activity was completely inhibited by wortmannin (100 nM). Data were representative of three independent experiments. involved in gp130 mediated cardiac hypertrophy via p70 S6 kinase (Fig. 5).
PI 3-kinase has been reported to act upstream of MAP kinase or p21 ras activation (27,(42)(43)(44)(45)(46)(47). Although, several reports have demonstrated an inhibition of MAP kinase activation by wortmannin, Scheid and Duronio (48) have recently shown that the effect of wortmannin and LY-294002 may be attributed to inhibition at a site upstream of MAP kinase that is distinct from PI 3-kinase (48). Based on a recent report, we support the former hypothesis, that constitutively-activated PI 3-kinase activates Ras, Raf, and MAP kinase and stimulates fos transcription, suggesting that this enzyme lies upstream of Ras (49). Therefore, this study was undertaken to determine whether PI 3-kinase is actually involved in the activation process of MAP kinase in cardiac myocytes. As presented in Fig. 6, PI 3-kinase is required for maximal MAP kinase activation in cardiac myocytes under LIF stimulation.
Schiemann and Nathanson (50) proposed that activation of the MAP kinase cascade by LIF occurs through a bifurcated signaling in 3T3 L1 cells. One pathway appears to be Ca 2ϩ -dependent and probably acts through PKC, while the other is independent of Ca 2ϩ but is sensitive to wortmannin (27). Our data demonstrate that LIF-induced MAP kinase activation in cardiac myocytes was not inhibited following 24 h of administration of phorbol esters to down-regulate PKC. This result indicates that LIF-mediated MAP kinase activation proceeds predominantly through a PKC independent manner, resulting in wortmannin-sensitive fashion in cardiac myocytes (Fig. 6).
We revealed that JAK1 immunoprecipitates made a complex with p85 and participated in the recruitment of p85 subunit to the LIF signaling complex in cardiac myocytes (Fig. 7A). Since JAK1 protein contains the Y-X-X-M sequences, JAK⅐p85 complexes may interact with these sequences and the SH2 domain of p85 (51). We confirmed the tyrosine phosphorylation of JAK protein kinase after LIF stimulation. Activation of PI 3-kinase itself in JAK1 immunoprecipitates also supports our speculations that only activated protein kinase functions as regulators of p85 for signaling events downstream of gp130 (Fig. 7, B and C). Alternatively, JAK protein kinase activation by LIF may lead to the phosphorylation of a secondary protein on tyrosine residues and promote its association with p85 in cardiac myocytes.
The precise downstream function of second messengers generated by JAK-p85 association has not yet been established. Pfeffer et al. (52) demonstrated that STAT3 acts as an adapter to couple PI 3-kinase to the type I interferon receptor, and wortmannin reduces the serine kinase activity of STAT3. They showed that a consensus Y-X-X-M p85 binding motif is present in STAT3 at Tyr-656 and is required for p85 interaction with STAT3. In our experiments, STAT3 was not found to co-immunoprecipitate with p85 (data not shown). The effects of wortmannin on STAT3 phosphorylation are consistent with the data obtained by Pfeffer et al. (52) in that wortmannin had little or no effect on tyrosine phosphorylation of STAT3 in Daudi cells (Fig. 8A). It is well known that serine phosphorylation of STAT3␣ is required for maximal STAT3 transcription activity by the MEK/extracellular-regulated protein kinase-dependent pathway (53,54). However, our data showed that neither wortmannin, rapamycin, nor the maximal dose of PD098059 produced a reduction in the slowly migration band of STAT3␣ (Fig. 8B). Interestingly, our data are consistent with the previous findings that interleukin-6 induces extracellular-regulated protein kinase-independent but H-7-sensitive STAT3 serine phosphorylation in HepG2 cells (55). These results suggest that LIF-induces wortmannin-insensitive STAT3 phosphorylation by the MEK-independent pathway in cardiac myocytes.
Several recent studies have shown that different responses occurred in an interleukin-2-dependent T-cell line and vascular smooth muscle cells, where rapamycin was found to selectively block p70 S6 kinase but not c-fos induction with interleukin-2 and angiotensin II stimulation, respectively (56,57). It is well FIG. 9. Effect of wortmannin and rapamycin on c-fos mRNA expression. Cardiac myocytes were stimulated with 10 3 units/ml LIF for 15 min in the absence or presence of wortmannin (100 nM) and rapamycin (3 ng/ml). Total RNA was extracted from the cells and analyzed by Northern blot using 32 P-labeled c-fos cDNA fragment as a probe. Human ␤-actin mRNA were shown below to indicate that equal amount of RNA was loaded in each lane. The extent of hybridization was visualized by autoradiography. Similar results were obtained in three independent experiments.
FIG. 10. LIF-induced PKB activation is sensitive to wortmannin but not to rapamycin. A, cardiac myocytes were incubated in serum-free medium for 24 h and treated with 10 3 units/ml LIF for the times indicated. Cell lysates were immunoprecipitated with anti-PKB antibody preconjugated with protein G-Sepharose, and the kinase activity were determined using [␥-32 P]ATP and Crosstide as a substrate. *, p Ͻ 0.05 versus 0 min. B, cardiac myocytes were incubated with or without wortmannin (100 nM) for 15 min or rapamycin (3 ng/ml) for 30 min, and then stimulated with 10 3 units/ml LIF for 15 min or left untreated. PKB kinase activity was analyzed in immune complex assay using Crosstide as a substrate. Data are mean Ϯ S.E. from three independent experiments each performed in triplicate. *, p Ͻ 0.05 versus LIF (Ϫ) (lane 1); †, p Ͻ 0.05 versus LIF (ϩ) (lane 4). established that p70 S6 kinase regulates translation, but not transcription. These findings indicate that p70 S6 kinase increased protein synthesis without affecting the phenotypic expression. It is possible that induction of c-fos gene expression through p70 S6 kinase by LIF may be regulated by different mechanisms than those after interleukin-2 or angiotensin II stimulation. On the other hand, these data are consistent with a previous report that PI 3-kinase functions upstream of both Ras and Raf in mediating downstream insulin regulation of gene transcription through the c-fos serum response element (58). Consequently, our findings suggest that there is a close correlation between the activation of PI 3-kinase and p70 S6 kinase by LIF and its hypertrophic effect in cardiac myocytes.
We recently reported the evidence that the activation of gp130 transduces cytoprotective signals through bcl-xL in cardiac myocytes (59). PI 3-kinase is also crucial in cell survival (23,60). Our data demonstrated for the first time that PKB was activated as a consequence of increased PI 3-kinase activity in cardiac myocytes stimulated with LIF (Fig. 10B, lane 5). In contrast, a recent study suggested that cAMP stimulated PKB in a wortmannin-insensitive manner, indicating that activation of PKB does not only proceed through PI 3-kinase-dependent signals (61). Our results are consistent with very recent studies that PKB activity was found to correlate with the amount of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate both in vivo and in vitro (62,63). Furthermore, the constitutive active form of PKB is reported to stimulate p70 S6 kinase in T cells (36). Therefore, PI 3-kinase signals are sufficient to activate PKB, which may function as an upstream regulator of p70 S6 kinase and be also a critical mediator of LIF-induced cell protection in cardiac myocytes (64).
In conclusion, we have shown that PI 3-kinase is necessary for LIF-induced full activation of MAP kinase, PKB, and p70 S6 kinase in cardiac myocytes. Activation of PI 3-kinase by LIF is associated with tyrosine phosphorylation of JAK1, and thereby promotes its association with p85. PI 3-kinase pathways contribute to LIF induced c-fos mRNA expression and regulate protein synthesis. These results suggest that PI 3-kinase may play important roles in gp130-mediated hypertrophic and cytoprotective signals in cardiac myocytes.