Extracellular Zinc Activates p70 S6 Kinase through the Phosphatidylinositol 3-Kinase Signaling Pathway*

We have studied a possible role of extracellular zinc ion in the activation of p70S6k, which plays an important role in the progression of cells from the G 1 to S phase of the cell cycle. Treatment of Swiss 3T3 cells with zinc sulfate led to the activation and phosphorylation of p70S6k in a dose-dependent manner. The activation of p70S6k by zinc treatment was biphasic, the early phase being at 30 min followed by the late phase at 120 min. The zinc-induced activation of p70S6k was partially inhibited by down-regulation of phorbol 12-myristate 13-acetate-responsive protein kinase C (PKC) by chronic treatment with phorbol 12-myristate 13-acetate, but this

p70 S6 kinase (p70S6k) 1 was originally recognized as the kinase that regulates the multiple phosphorylation of the 40 S ribosomal protein S6 in vivo (1)(2)(3)(4). Physiological roles of the kinase have been sought using various molecular and pharmacological methods for the past decade. Most worth noting, the inhibition of agonist-induced p70S6k activation in vivo by ei-ther microinjecting neutralizing antibodies (5) or by treatment with the immunosuppressant rapamycin to the cell severely impairs the progression of the cell cycle through the G 1 phase (6 -8). This strongly supports that p70S6k plays important roles during cell growth in the G 1 to S cell cycle transition. Further emphasizing the importance of p70S6k at a molecular level is that the kinase is involved in the selective translational regulation of a unique family of mRNAs (9), presumably by mediating the multiple phosphorylation of 40 S ribosomal protein S6. These mRNAs encode for components of the translational apparatus, including ribosomal proteins and translational elongation factors whose increased expression is essential for cell growth and proliferation (10).
Recently, Thomas' group (11) has shown that a mutant fly that has lost the p70S6k ortholog displays delayed growth and reduced cell and body size compared with those of the wild type fly. These genetic studies in Drosophila melanogaster clearly demonstrate that p70S6k and its downstream targets are not only involved in growth at the cellular level but also affect the development and growth of organs and the organism as a whole.
Although numerous agonists such as growth factors, cytokines, phorbol esters, calcium, inhibitors of protein synthesis, and hormones can activate p70S6k, still little is known about the direct regulators of p70S6k (1)(2)(3)(4). Many studies utilizing either point mutational analysis of platelet-derived growth factor receptor (12), various PI3K mutants (13), or specific inhibitors for PI3K such as wortmannin and LY294002 (12,14) have shown that PI3K is an upstream regulator of p70S6k. In addition, recent progresses in the understanding of the PI3K signaling have led to the discovery of two upstream regulators of p70S6k. The pleckstrin homology domain containing protein kinase phosphoinositide-dependent protein kinase 1 (PDK1) has been identified as the kinase responsible for phosphorylating threonine 229 in the activation loop of p70S6k (15). Interestingly, PDK1 phosphorylates and activates another pleckstrin homology domain containing protein kinase, Akt, as well (16,17). Because p70S6k and Akt share PDK1 as a common upstream regulator, it appears that the PI3K signaling pathway is branched at the level of PDK1 in vivo. However, overexpression of constitutively active or dominantly negative forms of Akt also regulates p70S6k accordingly in vivo (18,19), suggesting that the PI3K signaling pathway is not a typical kinase cascade as exemplified in the mitogen-activated protein kinase pathways.
In addition to these complex mechanisms, another signaling molecule is also involved in the regulation of p70S6k. The immunosuppressant rapamycin strongly inhibits p70S6k in vivo, which is a consequence of the inhibition of the activity of mammalian target of rapamycin (mTOR/FRAP/RAFT) (20 -22). Although there are strong evidences implicating mTOR as an upstream regulator for p70S6k in vivo (6 -8, 23), the exact molecular mechanism by which mTOR regulates p70S6k still remains elusive. In conclusion, such complexity in the regulation of p70S6k is likely due to its activation mechanism, which requires multiple hierarchical phosphorylations by several different kinases.
Zinc is an important trace element in biological systems. It is redox inert and has important roles in modulating the structural and catalytic activities of many cellular proteins. There has been circumstantial evidence suggesting that zinc might be involved in several neurological dysfunctions and other diseases (24,25). For example, zinc interacts with ␤-amyloid and its precursor protein, which are believed to be involved in the pathogenesis of degenerative processes in the brain, particularly in Alzheimer's disease (26). On the other hand, recent experimental evidences support that zinc is involved in cell growth and death in general in vivo (24,25). For example, zinc potentiates the mitogenic signaling of insulin (27) and activates extracellular signal-regulated kinase-mitogen-activated protein kinase 1 and 2 (28). In addition, tyrosine phosphorylations of epidermal growth factor (EGF) receptor are induced by zinc in human epithelial cell lines (29). These findings prompted us to examine the effects of zinc on p70S6k and the PI3K signaling pathway.
Here, we show that zinc potently activates p70S6k in a biphasic manner. This activation is completely inhibitable by rapamycin, wortmannin, and LY294002 and is independent from PKC and extracellular calcium levels. In addition, coexpression of dominantly interfering alleles of Akt and PDK1 strongly blocks the activation of p70S6k by zinc. Furthermore, zinc can activate the lipid kinase activity of PI3K. Thus, we conclude that zinc activates p70S6k via the PI3K signaling pathway in vivo.

EXPERIMENTAL PROCEDURES
Cell Culture and Transfection-COS and Swiss 3T3 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Life Technologies, Inc.) at 37°C in a humidified atmosphere with 5% CO 2 . Transient transfection in COS cells were performed at 60% confluency by a DEAE-dextran method as described in the manufacturer's manual (Promega).
Protein Kinase Assay-The endogenous p70S6k in Swiss3T3 cells was immunoprecipitated by anti-p70S6k polyclonal antibody coupled to protein A-Sepharose (Amersham Pharmacia Biotech). Transiently transfected HA-tagged Akt and p70S6k or Myc-tagged PDK1 and p70S6k were immunoprecipitated by 12CA5 anti-HA or 9E10 anti-Myc monoclonal antibody coupled to protein G-Sepharose (Amersham Pharmacia Biotech). The samples were washed twice with Buffer A and then twice with Buffer A containing 500 mM NaCl. Finally, the immune complexes of p70S6k and Akt were washed with Buffer C containing 20 mM Hepes (pH 7.2), 10 mM MgCl 2 , 0.1 mg/ml bovine serum albumin, and 3 mM ␤-mercaptoethanol, and Buffer D containing 20 mM Hepes (pH 7.2), 10 mM MgCl 2 , 10 mM MnCl 2 , 1 mM dithiothreitol, and 0.2 mM EGTA, respectively. S6 phosphotransferase activities were assayed in a reaction mixture consisting of 1ϫ Buffer C, 1 g of S6 protein, 20 M ATP, and 5 Ci of [␥-32 P]ATP at 30°C for 20 min. Akt activities were assayed in a reaction mixture consisting of 1ϫ Buffer D, 1 g of histone H2B, 2 g of PKI, 5 M ATP, and 5 Ci of [␥-32 P]ATP at 30°C for 20 min. Protein kinase assays were terminated by addition of SDS sample buffer, and the samples were subjected to SDS-polyacrylamide gel electrophoresis. Phosphorylated proteins were visualized by autoradiography and quantified with a Phosphoimager (BAS1500, Fuji).
PI3K Lipid Kinase Assay-The PI3K protein complex was immunoprecipitated with anti-phosphotyrosine monoclonal antibody (4G10, Upstate Biotechnology) coupled to protein A-Sepharose. The samples were washed twice with 1% Nonidet P-40 and 1 mM sodium orthovanadate in phosphate-buffered saline, twice with washing buffer containing of 100 mM Tris-HCl (pH 7.5), 500 mM LiCl, and 1 mM sodium orthovanadate, and finally twice with ST consisting of 150 mM NaCl and 50 mM Tris-HCl (pH 7.2). Then the samples were resuspended in phosphatidylinositol kinase buffer containing 20 mM Hepes (pH 7.2), 100 mM NaCl, 10 g/ml leupeptin, and 10 g/ml pepstatin. Following addition of a phosphatidylinositol/EGTA mixture consisting of 1 mg/ml phosphoinositide and 2.5 mM EGTA, the samples were incubated at room temperature for 10 min. Then a mixture consisting of 20 mM Hepes (pH 7.4), 5 mM MnCl 2 , 10 M ATP, 20 Ci of [␥-32 P]ATP was added, and the samples were further incubated at 30°C for 15 min. The reactions were stopped by addition of 1 M HCl, and phospholipids were extracted with CHCl 3 . Dried samples were separated by thin layer chromatography. Phosphorylated lipids were visualized by autoradiography.
Immunoblotting-Cell lysates were boiled in 1ϫ SDS sample buffer for 5 min. The samples were subjected to SDS-polyacrylamide gel electrophoresis, and separated proteins were electrically transferred to nitrocellulose membranes (Schleicher & Schuell). Membranes were incubated for 15 min in blocking solution (Tris-buffered saline containing 0.1% Tween-20 (TBS-T), 2% bovine serum albumin, and 0.02% sodium azide) and further incubated with the appropriate primary antibodies for 1 h. The membranes were then washed with TBS-T and incubated for 30 min with either anti-mouse or anti-rabbit secondary antibody conjugated to horseradish peroxidase. Bound antibodies were detected with enhanced chemiluminescence (Amersham Pharmacia Biotech).

Dose-and Time-dependent Activation of p70S6k by Zinc-To
examine the effects of zinc on p70S6k, quiescent Swiss 3T3 cells were treated with ZnSO 4 for various doses and time periods, and the S6 phosphotransferase activities of p70S6k of their cell lysates were examined by immune complex kinase assays. As shown in Fig. 1A, the protein kinase activities of p70S6k were slightly induced by up to 50 M ZnSO 4 but strongly activated by 100 M ZnSO 4 to levels comparable with EGF stimulation (Fig. 1A, top and middle panels). In the immunoblot analyses of the same lysates used for the kinase assay, we could detect the slow migrating species of p70S6k in the 100 M ZnSO 4 -treated sample (Fig. 1A, bottom panel). As previously reported, such retarded species of p70S6k are also seen following growth factor stimulation and represent the highly phosphorylated and activated forms of the kinase (6).
Next, we examined the activation time course of p70S6k by zinc. Quiescent cells were treated with 100 M ZnSO 4 for various time periods. Unexpectedly, we reproducibly observed that zinc stimulation induces p70S6k activity in a biphasic manner (Fig. 1B). The earlier phase appears between 0 and 60 min and peaked at 30 min, followed by the late phase peaking at 120 min. These time-dependent activities of p70S6k were again tightly co-related with the phosphorylation of the kinase (Fig.  1B, bottom panel).
To confirm that the zinc sulfate-mediated activation of p70S6k is indeed induced specifically by zinc divalent ion, we examined the effects of other similar salts on the S6 kinase activity of p70S6k. As shown in Fig. 2, only ZnSO 4 and ZnCl 2 , but not MgSO 4 , MgCl 2 , NaCl, or CaCl 2 , induced the activities of p70S6k. This result unequivocally demonstrates that zinc ion is specifically responsible for activation of p70S6k.
Protein Kinase C-and Calcium-independent Activation of p70S6k by Zinc-The PMA-dependent activation of p70S6k requires PMA-responsive PKC (30). This was demonstrated by rechallenging PMA to cells with PKC down-regulated by chronic pre-exposure (20 h) to PMA. To test the role of PKC in the biphasically induced activation of p70S6k by zinc, we stim-ulated the PKC-down-regulated cells with zinc for 30 and 120 min, each time point representing the early and the late phase of the activation kinetics of p70S6k. Interestingly, PKC downregulation partially (ϳ30%) inhibited the activation of p70S6k by 30 min of zinc treatment but did not affect the activation of p70S6k by 120 min of stimulation (Fig. 3A). These results support the possibility that PMA-responsive PKCs might be partially involved, but only in the early activation mechanism of the zinc-induced activation and phosphorylation of p70S6k. To further study the involvement of PKC, we treated cells with Go6976, a specific calcium-dependent PKC inhibitor, followed by treatment with zinc sulfate. As expected, this drug partially inhibited the p70S6k activation induced by 30 min zinc treatment but did not reduce the kinase activity induced by 120 min treatment (Fig. 3B). These results support that the overall activation and phosphorylation of p70S6k by zinc are mainly regulated by PMA-responsive PKC-independent mechanisms.
Others have previously reported that calcium channels such as NMDA receptor and AMPA receptor mediate zinc ion influx in addition to calcium ion influx in neuron cells and that the rise in zinc and calcium ion levels induce a variety of physiological responses (24,25). In addition, several reports support the possible involvement of calcium in the regulation of p70S6k (31,32). Therefore, we examined whether calcium plays a role in the activation of p70S6k by zinc. The activation and phosphorylation of p70S6k from Swiss 3T3 cells cultured in a calcium-free Dulbecco's modified Eagle's medium (obtained from Life Technologies, Inc.) were strongly induced by zinc (Fig. 4,  fourth column). As a control, the addition of calcium to the cell did not significantly stimulate the S6 kinase activities of p70S6k, despite slightly increasing phosphorylation of the kinase. In addition, when we applied calcium and zinc together, calcium rather slightly inhibited the zinc-mediated activation of p70S6k at higher doses (Fig. 4). These data indicate that extracellular calcium is not involved in the zinc-induced activation of p70S6k.
PI3K-and mTOR-dependent Signaling Pathways Mediate the Activation of p70S6k by Zinc-Our immunoblot analyses clearly show that the activation of p70S6k by zinc is mediated by phosphorylations, as with activation by other agonists. As described in the introduction, p70S6k is regulated by the PI3K signaling pathway and the mTOR-dependent pathway in vivo. Therefore, we examined the effects of rapamycin, a specific inhibitor of mTOR, or wortmannin and LY294002, two structurally unrelated PI3K inhibitors, on the activation and phosphorylation of p70S6k by zinc. As shown in Fig. 5, the zincinduced activation and phosphorylation of p70S6k were strongly inhibited by these drugs. These results strongly suggest that the zinc-mediated activation and phosphorylation of p70S6k is dependent on mTOR and PI3K in vivo.
Zinc Activates Akt in a Biphasic Manner-To understand how the PI3K pathway mediates the zinc-mediated activation of p70S6k, we first examined the zinc-induced activation of Akt in Swiss 3T3 cells. Akt plays a pivotal role in the PI3K pathway and is activated by phosphorylations at threonine 308 in the activation loop and serine 473 within the C-terminal domain (33). These phosphorylations are tightly correlated with the activities of Akt in vivo (33). As shown in Fig. 6, quiescent Swiss 3T3 cells were stimulated with 100 M of zinc sulfate for various time intervals as indicated, and the Akt activities were determined by immunoblot analyses with serine 473 phosphospecific Akt antibody. Surprisingly, zinc induced Akt serine 473 phosphorylation in a biphasic manner (Fig. 6), just like p70S6k shown in Fig. 1B. The early phase appears as early as 1 min and peaked at 5 min, followed by the late phase peaking at 60 min (Fig. 6). This activation pattern of Akt is very similar to that induced by growth factors such as EGF, platelet-derived growth factor, and insulin (34).
Zinc Activates p70S6k via the PI3K Signaling Pathway in Vivo-Because we found Akt to be activated by zinc, we next examined whether other components of the PI3K pathway are affected by zinc in the activation of p70S6k. Recent studies showed that PDK1 (15) and Akt (18,19) relay the growth factor-induced activation signals from PI3K to p70S6k either directly or indirectly. To confirm their involvement in the zincmediated activation of p70S6k in vivo, we co-expressed a dominant negative form of Akt or PDK1, both kinase-dead (KD) mutants, with p70S6k in COS cells. Zinc strongly induced p70S6k activity in COS cells, supporting that the zinc-mediated activation of p70S6k is not a cell type-specific phenomenon (Fig. 7). As expected, co-expression of wild type (wt) Akt (Fig.  7A) or PDK1 (Fig. 7B) with p70S6k slightly augmented the activation of p70S6k by zinc. However, co-expression of dominant negative Akt (Fig. 7A) or dominant negative PDK1 (Fig.   7B) blocked the zinc-induced activation of p70S6k down to the unstimulated control level. These results strongly suggest that PDK1 and Akt play major roles as upstream regulators in the zinc-mediated activation of p70S6k.
Next, we examined whether zinc directly regulates the activities of PDK1 and Akt. As shown in Fig. 8, addition of zinc ion failed to affect the phosphotransferase activity of immunoprecipitated PDK1 and Akt in our experimental conditions (Fig. 8, A and B, respectively), which suggests that extracellular zinc regulates component(s) further upstream of PDK1 in the PI3K signaling pathway in vivo. Therefore, we examined whether the lipid kinase activity of PI3K was stimulated by exogenous zinc. Indeed, zinc strongly stimulated the lipid kinase activity of PI3K by 7-fold, comparable with EGF stimulation, and this activation was inhibited by wortmannin (Fig.  9). DISCUSSION Zinc is present in nearly all body tissues, especially in the thyroid, pancreas, brain, and reproductive organs (25). This mineral is involved in the body's enzymatic reactions, protein synthesis, and carbohydrate metabolism, etc. In addition, zinc is essential for cell growth and is required for healing and maintaining healthy tissues. It has been understood that these important physiological roles of zinc might stem from the cofactor-like roles of the divalent ion in the cell. However, a recent output of research, mainly focusing on zinc-mediated neurotoxicity, has implied that zinc itself may be directly involved in various cell signalings (24). However, no systematic research has been conducted on how zinc is involved in the regulation of cell growth and survival at the molecular level. Here, we first demonstrate the involvement of zinc in the cell signaling activity of p70S6k through the PI3K signaling pathway. Because the PI3K pathway has important roles in regulating cell growth, apoptosis, and development, our present results not only provide a novel mechanism in the regulation of the p70S6k and the PI3K pathway but also instate zinc as a major player in cell signal transduction.
Several groups have proposed that zinc can enter cells through calcium channels along with calcium (35,36), and Kiss and co-workers (37) reported that extracellular calciuminduced stimulation of DNA synthesis and p70S6k activity in NIH 3T3 was dependent on zinc, leading us to first suspect that zinc regulates p70S6k through calcium-and PKC-dependent mechanisms. However, our results clearly showed that an increase in extracellular zinc strongly stimulates p70S6k in a manner that is independent of both PKC and extracellular calcium (Figs. 3 and 4). These interesting effects of zinc on Quiescent Swiss 3T3 cells were pretreated with (ϩ) or without (Ϫ) wortmannin (Wort) and further treated with zinc (100 M) or EGF (50 ng/ml) for 2 min. Cell lysates were prepared as described under "Experimental Procedures," and anti-phosphotyrosine antibody was added to the cleared cell lysates. Immune complexes were subjected to lipid kinase assays for PI3K, and phospholipids were extracted and separated by thin layer chromatography. The quantitated 32 P incorporation into phosphatidylinositol 3-phosphate (PIP) (bottom panel) was shown as a bar graph (top panel) that represents the means of three independent experiments Ϯ S.D. p70S6k are very similar to those seen following growth factormediated cell stimulation (30).
A decade ago, Thomas' group (38) showed that the EGFstimulated p70S6k activities exhibit biphasic activation kinetics; the early phase of activation appears at 10 -15 min, followed by the late phase at between 30 -60 min, which is sensitive to PKC down-regulation. Our data demonstrated that zinc also induces p70S6k and Akt in a biphasic manner (Figs. 1B and 6). The early peak of p70S6k activity appears at 30 min, and the late peak appears at 120 min following zinc stimulation. Interestingly, the p70S6k activities detected during the early peak is partially (about 30%) sensitive to down-regulation of PKC and to a specific PKC inhibitor, but the late peak is completely insensitive to PKC down-regulation (Fig. 3). This interesting pattern of PKC dependence of the zinc-induced p70S6k activity is highly consistent with our previous results from the platelet-derived growth factor-dependent activation of p70S6k (12); p70S6k is regulated by the PKC-dependent pathway and the PI3K-dependent pathway in a 3:7 ratio.
In this paper, we confirmed that zinc stimulates p70S6k activity through the PI3K-dependent pathway using specific inhibitors of PI3K, wortmannin, and LY294002 (Fig. 5) and a dominant interfering allele of Akt and PDK1 (Fig. 7). Moreover, we demonstrated that zinc stimulates the lipid kinase activity of PI3K (Fig. 9). However, the imminent question of how zinc activates PI3K remains. In Fig. 9, we showed that the PI3K activities associated with the anti-phosphotyrosine immunoprecipitates were raised significantly following in vivo zinc stimulation. This result suggests that tyrosine kinases are involved in the activation of the PI3K signaling pathway by zinc. May and Contoreggi (39) have found that zinc exerted the insulin-like effects by generating hydrogen peroxide in isolated rat adipocytes. Intracellular reactive oxygen species induces the tyrosine phosphorylation of numerous cytosolic proteins and activation of growth factor receptor tyrosine kinases such as EGF receptor (40). Furthermore, oxidative stress activates PI3K and causes the accumulation of phosphatidylinositol 3, 4-bisphosphate, which recruits Akt to the plasma membrane and activates it (41). From these results, we can postulate a model that zinc induces the generation of reactive oxygen species such as hydrogen peroxide in the cell and consequently activates growth factor receptor tyrosine kinases to stimulate the PI3K signaling pathway.
Alternatively, zinc may activate nonreceptor tyrosine kinases associated with PI3K. For example, a recent study implicated that focal adhesion kinase, a nonreceptor tyrosine kinase, functions as the upstream mediator of PI3K activation in T98 glioblastoma cells (42). In addition, focal adhesion kinase and Src family tyrosine kinases were tyrosine-phosphorylated and induced to associate with PI3K in response to hydrogen peroxide treatment (42,43,44). Thus, it is possible that these nonreceptor tyrosine kinases may also be involved in the zinc-mediated activation of PI3K.
Up to now, the molecular mechanisms behind the role of zinc in cell growth and metabolism have been unknown. The present study suggests that zinc exerts its physiological functions through the PI3K pathway. Further studies on how zinc activates PI3K and on the precise mechanisms through which the PI3K/p70S6k signaling pathway acts to modulate the response to zinc, are needed to fully understand this newly discovered role of zinc in the cell.