Signal Pathways Involved in Activation of p70 S6K and Phosphorylation of 4E-BP1 following Exposure of Multiple Myeloma Tumor Cells to Interleukin-6*

Interleukin-6 (IL-6) is a prominent tumor growth factor for malignant multiple myeloma cells. In addition to its known activation of the Janus tyrosine kinase-STAT and RAS-MEK-ERK pathways, recent work suggests that IL-6 can also activate the phosphatidylinositol 3-ki-nase (PI3-K)/AKT kinase pathway in myeloma cells. Because activation of the PI3-K/AKT as well as RAS-MEK-ERK pathways may result in downstream stimulation of the p70 S6K (p70) and phosphorylation of the 4E-BP1 translational repressor, we assessed these potential molecular targets in IL-6-treated myeloma cells. IL-6 rapidly activated p70 kinase activity and p70 phosphorylation. Activation was inhibited by wortmannin, rapamycin, and the ERK inhibitors PD98059 and UO126, as well as by a dominant negative mutant of AKT. The concurrent requirements for both ERK and PI3-K/AKT appeared to be a result of their ability to phosphorylate p70 on different residues. In contrast, IL-6-induced phosphorylation of 4E-BP1 was inhibited by rapamycin, wortmannin, and dominant negative AKT but ERK inhibitors had no effect, indicating ERK function was dis-pensable. In keeping with these data, a dominant active AKT mutant was sufficient to induce 4E-BP1

addition, several studies support the importance of IL-6 as a tumor-promoting cytokine in vivo. 1) Serum and bone marrow IL-6 levels are elevated in myeloma patients, and levels correlate with disease activity (3); 2) monoclonal anti-IL-6 antibodies can induce anti-tumor responses (4); 3) IL-6 expression in mice is an absolute requirement for development of murine myeloma (5). Because the predominant effect of IL-6 in nontransformed B-lineage cells is that of inducing differentiation (6), a process that normally results in apoptosis of the terminally differentiated plasma cell, there must be inherent differences in IL-6 signaling in malignant plasma cells that result in continued cell growth and viability. These differences could be therapeutically exploited as they may offer a "therapeutic window." In addition, identification of the signal pathways that mediate IL-6-induced MM cell growth may also provide insight into the critical pathways of IL-6-independent growth. Thus, studies on IL-6-dependent signaling in myeloma cells are important.
The IL-6 receptor consists of a ligand-binding molecule (gp80) and gp130, the signal-transducing molecule. Following IL-6 binding to gp80 and subsequent gp130 oligomerization, the receptor-associated Janus tyrosine kinase kinases become activated and phosphorylate tyrosine residues on gp130. These phosphorylated residues express docking sites for downstream substrates that bind via their SH2 domains. This process leads to activation of two dominant pathways. One is the STAT family of transcription factors, and gp130 signaling in MM cells specifically results in activation of STAT3 and STAT1. A second distinct pathway is activated via adaptor proteins, which also bind to gp130 and induce downstream activation of p21 RAS (7). RAS, in turn, activates several additional effector cascades, one of which consists of RAF/MEK/ERK. Some evidence implicates this latter pathway in proliferation of MM cells because IL-6-induced activation of the pathway correlates with induction of proliferation (8,9) and ERK antisense oligonucleotides prevent proliferation (8). A role for STAT3 activation in expansion of malignant clones may also exist. STAT3 function in the U266 myeloma cell line is critical for protecting against Fas-induced apoptosis (10). Furthermore, gp130-dependent activation of STAT3 protects against apoptosis that occurs during cell cycle transit in other cell types (11).
In prior work, we identified an additional signal cascade, the phosphatidylinositol 3-kinase (PI3-K)/AKT kinase pathway, that appears important in IL-6-induced myeloma cell growth. PI3-K/AKT is frequently activated in myeloma plasma cells, and inhibiting either PI3-K activity (12) or AKT activity (13) curtails IL-6-dependent and -independent tumor cell expansion. The PI3-K/AKT pathway can promote tumor growth by protecting against apoptosis or enhancing proliferation. Although survival responses may be the result of phosphorylation of one or more of many different substrates (14 -17), stimula-tion of proliferation is thought to be uniquely the result of downstream signaling through the mammalian target of rapamycin (mTOR), which results in phosphorylation and activation of the p70 S6K (p70) and phosphorylation of the 4E-BP1 translational repressor. p70 activation results in increased phosphorylation of the 40 S ribosomal S6 protein and 4E-BP1 phosphorylation disrupts its interaction with the eIF-4E initiation factor, allowing eIF-4E to participate in assembly of a translation initiation complex. These events lead to translational up-regulation of the proteins needed for cell cycle transit. Our prior results indicated that the PI3-K/AKT pathway was more important in MM cell proliferative rather than survival responses (18). Thus, in the current study, we focused on its possible downstream activation of p70 and phosphorylation of 4E-BP1 in IL-6-treated MM cells. Because the RAS/RAF/MEK/ ERK pathway can also mediate p70 (19) or 4E-BP1 phosphorylation (20), and is potentially activated by IL-6 in MM cells, we also tested the role of this cascade in p70/4E-BP1 phosphorylation.

EXPERIMENTAL PROCEDURES
Cell Lines and Reagents-The MM cell lines AF-10 and 8226 were kind gifts from Drs. J. Epstein (University of Arkansas, Little Rock, AR) and James Berenson (UCLA, Los Angeles, CA). The lines were maintained as previously described (2,12). Recombinant IL-6 and insulinlike growth factor-1 (IGF-1) were purchased from R&D Systems (Minneapolis, MN). Phosphospecific antibodies were purchased from New England Biolabs. All other reagents were purchased from Sigma unless otherwise described.
Expression Constructs-The N terminus AKT construct HA-AKT (PH), which includes the PH domain but lacks the kinase domain and the HA-E40K construct, which contains a point mutation in its PH domain, have been previously described (21,22). A recombinant adenovirus that encodes HA-AKT(PH) or HA-E40K was generated by homologous recombination in bacteria. The HA-tagged constructs were first cloned into pAdTrack-CMV, which also contains the green fluorescent protein gene. The resultant construct was linearized and transformed with the supercoiled adenoviral vector, pAdEasy-1 into Escherichia coli and recombinants selected in kanamycin and screened by restriction endonuclease digestion. The recombinant adenovirus was then transfected into 293 cells. Control pAdTrack-CMV recombinant virus was generated in identical fashion but without transgene insertion.
Transfection-Myeloma cells were transduced with adenovirus at varying m.o.i. values for 2 h. Virus was then washed away, and cells were resuspended in media with low serum concentration (1%) to minimize proliferation. At 24 and 48 h, enhanced green fluorescent protein (EGFP) fluorescence demonstrated that Ͼ85% of cells were successfully transfected when using m.o.i. values between 10 and 100.
AKT Kinase Assay-The AKT in vitro kinase assay utilized a nonradioactive kit purchased from New England Biolabs. AKT was immunoprecipitated from cell extracts and incubated with GSK-3 fusion protein in the presence of ATP and kinase buffer. AKT-dependent GSK-3 phosphorylation was detected by immunoblotting with a phosphospecific GSK-3 antibody.
p70 in Vitro Kinase Assay-As previously described (23), p70 S6K was immunoprecipitated from myeloma cells with C18 antibody (Santa Cruz). p70 was then mixed with S6 substrate peptide (Upstate Biotechnology, Inc.) in kinase buffer in the presence of 200 Ci/ml [␥-32 P]ATP. The reaction was stopped by addition of 20 l of 250 mM EDTA, followed by boiling for 5 min. The reaction mixture was then transferred to phosphocellulose columns (Pierce), which were centrifuged to separate free 32 P from S6-labeled 32 P. The amount of labeled S6 peptide adhered to the columns was then assayed by liquid scintillation.
4E-BP-1 Phosphorylation Assay-Phosphorylation of 4E-BP1 was detected by differential migration in SDS-PAGE. As previously described (24), protein lysates were first boiled and then incubated on ice. After centrifugation, supernatant protein was precipitated with 15% trichloroacetic acid for 1 h, followed by two washes with diethyl ether. The pellet was dissolved in Laemmli buffer and electrophoresed in 15% SDS-polyacrylamide gel. Membranes were blotted with anti-4E-BP1 antibody from Santa Cruz Chemicals.
Cell Growth Assay-MM cells were cultured at 2 ϫ 10 5 cells/ml in 6or 12-well plates. Some groups were pretreated with rapamycin or CCI-779 for 2 h before adding IL-6 at 100 units/ml. After 48 h, cells were harvested and viable recovery determined by trypan blue staining.

IL-6 Activates mTOR-dependent p70 S6K Activity in Myeloma Cells
To assess the activation and roles of p70/4E-BP1 phosphorylation in IL-6-stimulated MM cells, we primarily used the AF-10 MM cell line for these studies for the following reasons. 1) Although the line can grow in the absence of IL-6, addition of exogenous IL-6 reproducibly increases proliferation; 2) the two dominant IL-6 signal pathways that are classically stimulated in MM cells, STAT1/STAT3 activation and RAS/RAF/MEK/ ERK activation, are consistently activated in AF-10 cells; and 3) IL-6 rapidly activates PI3-K and AKT in these cells. In addition, AF-10 cells have functional IGF-1 receptors, allowing us to use IGF-1 stimulation as a positive control for p70/4E-BP1 phosphorylation.
Using a p70 in vitro kinase assay, we could demonstrate that IL-6 rapidly activated p70 kinase function ( Fig. 1A) with kinetics and a magnitude that was similar to that achieved with IGF-1. IL-6-stimulated activity developed between 5 and 15 min of incubation and peaked at 15 min (2.5-fold over unstimulated control cells). Activity slightly decreased by 30 min (2.1fold over control) and returned to base line by 60 min (1.2-fold of control; data not shown). The results shown in Fig. 1 were obtained from cells stimulated with 100 units/ml IL-6. This was optimal IL-6-induced stimulation, as experiments using 1000 units/ml did not result in greater kinase stimulation, whereas 1 and 10 units/ml were less effective (data not shown). IGF-1 likewise stimulated kinase activity in AF-10 cells. Pretreatment with the mTOR inhibitor rapamycin completely abrogated the ability of both IL-6 and IGF-1 to activate kinase activity (Fig. 1A).
IL-6-induced p70 kinase activity correlated with IL-6-induced p70 phosphorylation. Utilizing phosphospecific p70 antibodies and IGF-1 treatment as a positive control, we could show that IL-6 induced phosphorylation at serine 411, threonine 421/serine 424, and threonine 389 (Fig. 1B). These are critical residues, phosphorylation of which results in optimal p70 kinase activity. Clear induction of phosphorylation was seen by 15 min of incubation. As shown, pretreatment with rapamycin prevented phosphorylation of all these residues. Thus, as for p70 kinase activity, mTOR regulates IL-6-dependent p70 phosphorylation.

IL-6-induced p70 S6K Activity Is Dependent upon Both PI3-K/AKT and MEK/ERK Pathways
IL-6 is capable of activating the PI3-K/AKT pathway (12) as well as the RAS/RAF/MEK/ERK (8,9) pathway in MM cells. To specifically test activation of these pathways in the AF-10 MM cell line, we exposed cells to IL-6 (100 units/ml) for varying durations and performed Western blot for the phosphorylated, activated form of AKT or p42/p44 ERK. In five separate experiments, exposure to IL-6 consistently induced phosphorylation of ERK and AKT in AF-10 cells. In Fig. 2A, results of two of these experiments are shown. In the left panel, p42 ERK demonstrates an increase in phosphorylation by 5 min, which increases to maximal levels by 30 min, and p44 ERK also demonstrates maximal phosphorylation by 30 min. In the second experiment shown (right panel), maximal phosphorylation is again demonstrated by 30 min of IL-6 exposure, although the major effect (in this experiment) was on p44 ERK (3.9-fold increase by densitometry) as compared with p42 (only 2.1-fold increase). Similarly, IL-6 markedly induced AKT phosphorylation at 15 and 30 min (one representative experiment shown in Fig. 2B).
In these experiments, we also tested the effectiveness and specificity of wortmannin, a PI3-K inhibitor, and UO126 and PD98059, two unrelated MEK/ERK inhibitors. Preliminary experiments identified the range of concentrations that effectively inhibit either AKT or ERK phosphorylation. We then tested effective lower concentrations of the inhibitors to test the specificity of inhibition. At 0.1 M, wortmannin (W) completely abrogated IL-6-dependent AKT phosphorylation ( Fig. 2B) but had no effect on ERK phosphorylation ( Fig. 2A, right panel). In contrast, UO126 (UO) at 12.5 M, and PD98059 (PD) at 50 M, completely inhibited IL-6-dependent ERK phosphorylation ( Fig. 2A, left panel) but had no effect on AKT phosphorylation (Fig. 2B).
We next used these inhibitors at the same effective and specific concentrations to test effects on p70S6 kinase activity. p70 and 4E-BP1 in Myeloma Cells fected into AF-10 cells, and anti-HA immunoblots as well as AKT in vitro kinase assays in IL-6-treated cells demonstrated expression of the truncated PH construct (Fig. 3B) and inhibition of IL-6-induced activation of endogenous AKT (Fig. 3C). p70 in vitro kinase assays also demonstrated a significant inhibition (p Ͻ 0.05) of IL-6-induced p70 kinase activity in PH-transfected cells (Fig. 3A). As shown, EGFP control-transfected cells still were capable of increasing their p70 activity to Ͼ2-fold increase over non-IL-6-treated cells, whereas dominant negative PH-transfected AF-10 cells only could increase activity to 1.2-fold over control when exposed to IL-6. Thus, the PI3-K/AKT pathway and the MEK/ERK pathway both regulate IL-6-dependent p70 kinase activity. As inhibition of each distinct pathway alone could abrogate p70 activity, the results suggest that the two pathways are additive or synergistic rather than overlapping.

Differential Phosphorylation of Specific p70 Residues in IL-6-stimulated MM Cells
Phosphorylation of p70 on Thr 421 /Ser 424 -The p70 kinase is activated by hierarchical phosphorylation at multiple sites (25). Initial phosphorylation at serine 411, threonine 421, and serine 424 in the auto-inhibitory domain relieves pseudosubstrate suppression. Activation also requires subsequent PI3-Kdependent phosphorylation at threonine 389 in the adjacent linker domain. These events then synergize to allow phosphorylation at threonine 229 in the catalytic domain. Phosphorylation at Thr 229 then results in optimal kinase activity. Thus, one possible explanation for a concurrent requirement of MEK/ ERK and PI3-K/AKT in p70 activation was that each pathway was necessary for differential phosphorylation steps on different residues of p70 to allow full activation. We, again, used the phosphospecific p70 antibodies to test this hypothesis. Initial p70 phosphorylation on Thr 421 /Ser 424 in the auto-inhibitory domain was activated by IL-6 after 15 and 30 min of incubation and returned toward base line at 60 min (Fig. 4, A and B). Wortmannin, used in concentrations that abrogated AKT activation, had no effect on Thr 421 /Ser 424 phosphorylation (Fig.  4A). In contrast, the MEK/ERK inhibitors PD98059 and UO126 both inhibited Thr 421 /Ser 424 phosphorylation induced by IL-6 (Fig. 4B). These data support the hypothesis that IL-6-induced phosphorylation of p70 on Thr 421 /Ser 424 is dependent upon signaling through RAS/RAF/MEK/ERK. Further evidence that the PI3-K/AKT pathway was not critical for IL-6-dependent Thr 421 /Ser 424 phosphorylation was obtained with use of the PH dominant negative-expressing adenovirus. As shown in Fig.  4C, there was no effect on IL-6-dependent Thr 421 /Ser 424 phosphorylation when AF-10 cells were transfected with the PH construct. Thus, although PI3-K/AKT function was critical for p70 kinase activity (Fig. 3) and, as will be shown below, for phosphorylation at other p70 residues, it was not required for Thr 421 /Ser 424 phosphorylation.
Phosphorylation of p70 on Serine 411-In contrast to the above results on phosphorylation of Thr 421 /Ser 424 , phosphorylation of Ser 411 was regulated by both MEK/ERK and PI3-K/ AKT pathways. As shown in Fig. 5A, IL-6-induced Ser 411 phosphorylation was clearly inhibited by pretreatment with PD98059, UO126, and wortmannin. In addition, IL-6-treated, PH-transfected AF-10 cells were incapable of Ser 411 phosphorylation compared with EGFP control (C)-transfected cells (Fig.  5B). These data indicate independent regulation of Ser 411 phosphorylation by both pathways.

Effect of Constitutively Expressed Activated AKT
Although the above experiments utilizing the PH AKT dominant negative confirm an AKT-dependent pathway for IL-6induced p70 kinase activation and phosphorylation, the results also suggest an interaction between AKT and the MEK/ERK pathway is required for p70 activity. To further support the notion that AKT must interact with MEK/ERK-dependent events, and, by itself, is insufficient to activate p70, we transfected a dominant active form of AKT, E40K (22), which possesses enhanced kinase activity caused by a point mutation resulting in an increased affinity of the PH domain for second messenger phospholipids (22). AF-10 cells were transfected with the adenovirus expressing E40K or control virus, and, 24 h later, cells were harvested and assayed. As with the PH dominant negative AKT-expressing adenovirus (above), the transfection efficiency was again very high (Ͼ85% at m.o.i. 10). The transfected AKT was constitutively phosphorylated as shown by immunoblot assay (Fig. 7B). In addition, the E40K was functional as a kinase inducing BAD phosphorylation and GSK-3 phosphorylation. However, MM cells transfected with FIG. 4. Phosphorylation of p70 on Thr 421 /Ser 424 . In A, AF-10 cells were pretreated with or without wortmannin and then stimulated with IL-6 for 0, 15, 30, or 60 min (min). Extracts were then immunoblotted with total p70 or phosphospecific p70 (p70-P) that specifically recognize phosphorylated Thr 421 /Ser 424 . In B, cells were treated similarly except that pretreatment was with or without PD98059 or UO126. In C, cells were transfected with control or dominant negative PH AKT (PH) and, 24 h later, stimulated with or without IL-6 for 15 min.   (Fig. 7B, right panel). There was little phosphorylation of Thr 421 /Ser 424 or Thr 389 induced by transfection of E40K.

Studies on 8226 MM Cells
To demonstrate that the above results were not peculiar to AF-10 MM cells, we also studied a second MM cell line, 8226, in a limited number of experiments. The 8226 cell line expresses functional IL-6 receptors and is protected against apoptosis by this cytokine (26). Several signal transduction pathways are modulated by IL-6 in 8226 cells, and, most importantly, AKT activation is induced (12). In addition, the 8226 MM line expresses a mutated ras oncogene and constitutively activated ERK1 and ERK2 (26). Initial in vitro kinase assays demon-strated that IL-6 activated p70 kinase activity in 8226 cells by 15 and 30 min of incubation (Fig. 8A). At 15 min the increase was at 1.7-fold of control (untreated 8226 cells) and, at 30 min, it was 2.2-fold of control. Wortmannin (0.1 M), PD98059 (50 M), and rapamycin (100 nM) all significantly (p Ͻ 0.05) inhibited activation of the kinase in 8226 cells. As the transfection efficiency with adenoviral vectors was as high in 8226 cells (Ͼ90% at m.o.i. 10) as it was in AF-10 MM cells, we could test the effect of the dominant negative PH AKT. As shown in Fig.  8B, PH-transfected 8226 cells were incapable of activating p70 kinase activity when exposed to IL-6 (p Ͻ 0.05). These data are similar to the above results with the AF-10 MM cell line in demonstrating a requirement for both MEK/ERK function as well as PI3-K/AKT activity in IL-6 induction of p70 activity.
A similar pattern of results was seen when phosphorylation of p70 on Thr 389 was studied. As shown in Fig. 8C, wortmannin, rapamycin, and PD98059 all curtailed the ability of IL-6 to induce Thr 389 phosphorylation on p70 in 8226 cells.

In Vitro Phosphorylation of p70
The above data in both AF-10 and 8226 MM cells indicated a requirement for the MEK/ERK pathway in p70 phosphorylation on Thr 389 , a key residue required for optimal induction of kinase activity. To determine whether ERK could directly phosphorylate this residue, we immunoprecipitated p70 from resting AF-10 cells (cultured in the absence of serum) and incubated it in vitro with activated ERK1 or ERK2 (purchased from Upstate Biotechnology, Inc.) for 10 min at 30°C. Phosphorylation of p70 was then assayed by immunoblot with phosphospecific antibodies. As shown in Fig. 9, activated ERK2 and, to a lesser extent, ERK1 were capable of in vitro phosphorylation of Ser 411 and Thr 421 /Ser 424 when added at 10 ng/l reaction. When 10-fold less activated ERK1 and ERK2 was added, little substrate phosphorylation was seen (data not shown). In contrast, activated ERKs could not phosphorylate Thr 389 on the immunoprecipitated p70 (Fig. 9). These data indicate ERK is incapable of directly phosphorylating Thr 389 and suggest that the MEK/ERK requirements for Thr 389 phosphorylation are the result of phosphorylation at the other residues, which allow access of Thr 389 to different kinases.

IL-6 Induction of 4E-BP1 Phosphorylation
To investigate the second potential downstream target of the PI3-K/AKT/mTOR pathway, 4E-BP1, we used immunoblot assays that allowed discrimination of three forms of 4E-BP1, depending upon their state of phosphorylation. 4E-BP1 was resolved into as many as three separate bands, designated ␣, ␤, and ␥ in order of increasing electrophoretic mobility. These forms arise from differences in the phosphorylation state with an increased phosphorylation causing a decrease in mobility. Thus, ␤ and ␥ represent the more phosphorylated forms of 4E-BP1. As shown in Fig. 10A, IL-6 treatment of AF-10 cells resulted in hyperphosphorylation of 4E-BP1, as shown by an increase in the relative proportion of the more highly phosphorylated ␥ and ␤ forms of 4E-BP1 (left panel). The effect of IL-6 was equal or even more impressive than IGF in these experiments. In the presence of the MEK/ERK inhibitor, PD98059, used in the same concentration that inhibited p70 kinase activity (50 M), the IL-6-induced hyperphosphorylation of 4E-BP1 was unaffected. However, both wortmannin and rapamycin prevented 4E-BP1 phosphorylation (Fig. 10A). As shown, there was minimal detection of the ␥ and ␤ forms of 4E-BP1 in these inhibitor-treated cells. To test the role of AKT in IL-6induced phosphorylation of 4E-BP1, we transfected AF-10 cells with the same control (con) and dominant negative PH AKTexpressing adenoviral vectors as described above. As shown in   In control-transfected cells exposed to IL-6 (con), a relative decrease in the hypophosphorylated ␣ form and corresponding increase in the hyperphosphorylated ␥ form was demonstrated, indicating IL-6 induction of 4E-BP1 phosphorylation. In contrast, IL-6 cannot induce phosphorylation of 4E-BP1 in PH-transfected AF-10 cells (Fig. 10B) and, in fact, the relative amount of the hyperphosphorylated ␥ form of 4E-BP1 even decreased somewhat when these latter cells were exposed to IL-6. Thus, these data indicate that AKT function as well as PI3-K and mTOR is required for IL-6-induced phosphorylation of 4E-BP1 in AF-10 MM cells and that the MEK/ERK pathway is dispensable. To further support this notion, we transfected AF-10 cells with the E40K active AKT construct and 24 h later tested 4E-BP1 phosphorylation. As shown in Fig. 10C, there were again three differentially phosphorylated forms of 4E-BP1 in both control-transfected (C) and E40K-transfected (E) cells. However, E40K transfection resulted in a significantly greater relative expression of a hyperphosphorylated ␥ 4E-BP1 form and a corresponding decrease in the ␣ hypophosphoryl-ated form. This indicates that AKT was required and sufficient for IL-6-induced 4E-BP1 phosphorylation.

Inhibition of mTOR Curtails IL-6-induced MM Cell Growth
To test the biological effects of mTOR signaling in IL-6treated MM cells, we used the mTOR inhibitors rapamycin and its newly developed analogue, CCI-779 (27). In preliminary experiments, we demonstrated effective inhibition of IL-6-induced p70 activity and phosphorylation as well as 4E-BP1 phosphorylation was afforded by CCI-779, used in comparable concentrations to rapamycin. AF-10 cells were then treated with increasing concentrations of rapamycin or CCI-779 for 2 h and then stimulated with IL-6. Viable cell recovery was enumerated 48 h later. As shown in Fig. 11, rapamycin or CCI-779, used at the highest concentration, had no effect on growth of AF-10 cells in the absence of IL-6. This was expected as untreated cells demonstrate little, if any, activation of p70 kinase activity or p70/4E-BP1 phosphorylation. In contrast, both drugs effectively abrogated the ability of IL-6 to stimulate MM cell growth in a concentration-dependent fashion. DISCUSSION The results of this study demonstrate that concurrent stimulation of the MEK/ERK and PI3-K/AKT cascades is required for activation of the p70 kinase in IL-6-treated myeloma cells. A similar interaction between the two upstream pathways was required for p70 phosphorylation on Thr 389 , a key residue required for kinase activation. In contrast, although the PI3-K/ AKT pathway was required for IL-6-dependent 4E-BP1 phosphorylation, the MEK/ERK pathway was dispensable. Both IL-6-dependent p70 activation/phosphorylation and 4E-BP1 phosphorylation was inhibited by rapamycin, and this drug also inhibited IL-6-dependent cell growth.
Our results confirm and extend a previous study, which documented the ability of gp130-generated signals to activate the p70 kinase in leukemia inhibitory factor (LIF)-treated cardiac myocytes (28). LIF induced p70 activation, which was sensitive to wortmannin and rapamycin, implicating PI3-K and mTOR. However, because wortmannin also inhibited LIF-dependent activation of mitogen-activated protein kinases in these cells, it was not clear whether ERK mitogen-activated protein kinases or other PI3-K targets such as PDK1 or AKT were upstream activators of p70. In contrast, wortmannin has no effect on IL-6-dependent phosphorylation of ERKs in our myeloma cells. Thus, the ability of wortmannin to inhibit p70 activation/phosphorylation in myeloma cells was independent of any effects on ERK. In addition, use of a dominant negative construct confirmed a role for AKT downstream of PI3-K in IL-6-dependent p70 activation. However, two unrelated MEK/ FIG. 9. In vitro phosphorylation of p70. p70 S6K was immunoprecipitated from resting AF-10 myeloma cells and mixed with activated ERK2, ERK1 (at 10 ng/l in the reaction mixture) or no kinase (C) in kinase buffer. Phosphorylation of p70 substrate was then assessed by immunoblot using phosphospecific antibodies for Ser 411 , Thr 421 /Ser 424 , or Thr 389 .

FIG. 10. Phosphorylation of 4E-BP1 in myeloma cells.
In A, AF-10 cells were pretreated with or without (no inhib) PD98059, wortmannin, or rapamycin and then stimulated with or without IGF-1 or IL-6 for 15 min. Lysates then immunoblotted for 4E-BP1. Differentially phosphorylated 4E-BP1 was resolved by migration in SDS-PAGE. In B, AF-10 cells were transfected with control (con) or PH vector and, 24 h later, stimulated with or without IL-6 for 15 min. Lysates then assayed for 4E-BP1 phosphorylation by immunoblot. In C, AF-10 cells were transfected with control (C) or E40K vector (E) and, 24 h later, also assayed for 4E-BP1 phosphorylation. p70 and 4E-BP1 in Myeloma Cells ERK inhibitors, which had no effect on AKT activation, also significantly curtailed IL-6-induced p70 activation. In addition, a constitutively active AKT construct could not, by itself, induce p70 activation. Taken together, these results support independent requirements for both MEK/ERK and PI3-K/AKT pathways in IL-6-induced p70 kinase activation. Concurrent requirements for these two pathways in p70 activation have been previously shown in insulin-stimulated (19) and UV-stimulated (30) cells.
Because the p70 kinase is activated by phosphorylation at several residues, we considered the possibility that the two required upstream activating pathways were responsible for separate phosphorylation events. In keeping with that hypothesis, MEK/ERK inhibitors effectively prevented IL-6-induced p70 phosphorylation at Thr 421 /Ser 424 , whereas wortmannin and the dominant negative AKT had no effect. These data are consistent with prior studies that identified these residues as targets for proline-directed ERK kinases (31). As Thr 421 /Ser 424 phosphorylation in the p70 auto-inhibitory domain is required to relieve pseudosubstrate inhibition and allow subsequent phosphorylation at Thr 389 , this event could explain the requirement for MEK/ERK in optimal p70 activation and Thr 389 phosphorylation. The inability of activated ERK1 or ERK2 to directly phosphorylate Thr 389 in vitro further supports this notion. On the other hand, wortmannin and the dominant negative AKT clearly inhibited Thr 389 phosphorylation, which is consistent with prior studies that indicate a PI3-K-dependent pathway is crucial for Thr 389 phosphorylation.
The results of p70 Ser 411 phosphorylation are not as easy to explain. This residue is also in the C-terminal auto-inhibitory domain, and its phosphorylation would also be important for relieving pseudosubstrate inhibition. Our results suggest both MEK/ERK or PI3-K/AKT pathways may induce Ser 411 phosphorylation. A previous study (30) demonstrated that UV-induced Ser 411 phosphorylation was inhibited by a MEK/ERK inhibitor and dominant negative ERK and Jun kinase constructs. As Jun kinase activity is actually decreased by IL-6 treatment of myeloma cells (26), it is unlikely to be mediating Ser 411 phosphorylation in our cells.
The dual requirement for MEK/ERK and PI3-K/AKT pathways in IL-6 induction of p70 kinase activity and Thr 389 phosphorylation was also demonstrated in a second myeloma cell model, the 8226 line. This is of particular interest in that the MEK/ERK pathway is constitutively activated in that line because of an oncogenic ras mutation and IL-6 is incapable of further ERK activation (26). The ability of the MEK/ERK inhibitor PD98059 to prevent IL-6-induced p70 kinase activity and Thr 389 phosphorylation in 8226 cells suggests some MEK/ ERK-mediated p70 phosphorylation must be present, either constitutively or cytokine-induced, to allow for further p70 kinase activation. Because IL-6 does not induce proliferation in 8226 cells, it is also clear that p70 activation, by itself, is not sufficient for a complete proliferative response.
The role of AKT in activation of p70 is controversial. Although some membrane-localized constitutively active forms of AKT can induce p70 activation (32,33), similarly active but non-membrane-localized mutants may not (33). Those results suggest that an additional membrane-localized, p70-activating kinase might be stimulated by the co-localized AKT. In addition, some dominant-interfering AKT constructs significantly inhibit p70 activation (34), whereas others have little effect (33). In contrast, most studies (33,35,36) demonstrate AKT is sufficient for 4E-BP1 phosphorylation. Our results are similar to these previous studies, in that the dominant active E40K AKT we used was ineffective in activating p70 kinase activity or inducing p70 Thr 389 phosphorylation but was capable of inducing 4E-BP1 phosphorylation. However, our dominant-interfering AKT mutant prevented both p70 activation as well as 4E-BP1 phosphorylation. Differences in the dominant negative AKT mutants used, vectors employed and cytokine stimulation (almost all prior studies analyze insulin stimulation) may account for the differences in our results when compared with previous studies (33,37). In this regard, it is certainly possible that the inhibitory effect of our PH dominant negative AKT on p70 activation is the result of inhibition of a parallel pathway, possibly mediated by PDK1. If the PH construct inhibited PDK1 function, this would explain the prevention of p70 kinase activation as p70 phosphorylation at Thr 229 would be abrogated. In addition, recent evidence supports the ability of PDK1 to also phosphorylate p70 on Thr 389 (38), which could also explain the ability of PH to inhibit p70 Thr 389 phosphorylation.
Rapamycin was an effective inhibitor of p70 kinase activation, p70 phosphorylation, and 4E-BP1 phosphorylation, indicating the ability of mTOR to regulate all those events. mTOR can phosphorylate p70 at Thr 389 in vitro (39,40) but more recent studies (41)(42)(43)(44) indicate that mTOR-mediated Thr 389 phosphorylation and p70 activation occurs via an inhibition of protein phosphatase 2A-mediated dephosphorylation. Thus, rapamycin may inhibit Thr 389 phosphorylation by activation of protein phosphatase 2A. The ability of rapamycin to also inhibit IL-6-dependent phosphorylation at Ser 411 and Thr 421 / Ser 424 may also be caused by enhanced activity of this phosphatase because these residues in the auto-inhibitory domain are not substrates for mTOR in vitro (39). The rapamycininduced inhibition of 4E-BP1 phosphorylation may also be the result of activation of a phosphatase or direct inhibition of mTOR kinase activity as mTOR can directly phosphorylate 4E-BP1 under some conditions (41). The inhibition of p70 activation and 4E-BP1 phosphorylation could explain the resulting cell cycle block induced by rapamycin. In addition, more recent work indicates rapamycin can induce cellular apoptosis, possibly because of inhibition of p70 phosphorylation of BAD (45). However, some cell types are resistant to the cytoreductive effects of rapamycin (29) for unclear reasons. To test the cellular effects of inhibiting mTOR function, we used rapamycin and its newly developed analogue, CCI-779. Although these drugs had no effect on the growth of AF-10 MM cells cultured without IL-6, they abrogated the IL-6 proliferative response in a dose-dependent fashion. Their lack of effect on unstimulated cells rules out nonspecific toxicity. Furthermore, the concentration of both drugs that curtailed IL-6-dependent cell growth effectively abrogated IL-6-dependent p70 and 4E-BP1 phosphorylation but had no effect on IL-6-dependent AKT activation (data not shown). As IL-6 is an important growth factor for tumor cells in vivo in myeloma patients, targeting IL-6-mediated activation of p70 and phosphorylation of 4E-BP1 is a promising therapeutic modality.