Differential Regulation by Calcium Reveals Distinct Signaling Requirements for the Activation of Akt and p70S6k*

Nelly Marmy ConusDagger , Brian A. Hemmings§, and Richard B. Pearson

From the Trescowthick Research Laboratories, Peter MacCallum Cancer Institute, Locked Bag 1, A'Beckett Street, Melbourne, Victoria 3000, Australia and § Friedrich Miescher Institut, P. O. Box 2543, CH-4002, Basel, Switzerland

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Activation of the phosphatidylinositol 3-kinase (PI3K) plays an important role in the mitogenic response of many cell types. Recently, two serine/threonine kinases Akt and p70S6k have been identified as physiological targets of PI3K. Observations that expression of activated forms of Akt led to the activation of p70S6k implied Akt might mediate mitogenic signaling through activation of p70S6k. To clarify the relationship between signaling through these two kinases, we have examined their regulation by various mitogenic stimuli. In this study we have focused on the role of calcium in the regulation of each kinase in Balb/c-3T3 fibroblasts. Depletion of intracellular calcium stores by EGTA pretreatment has no effect on growth factor-induced Akt activation but completely abolishes p70S6k stimulation. Increase of intracellular calcium induced by ionomycin or thapsigargin results in a full activation of p70S6k, whereas little or no activation of Akt is observed. Furthermore, although PI3K in anti-phosphotyrosine immunoprecipitates is only very weakly activated by ionomycin, the calcium-induced stimulation of p70S6k is completely inhibited by the specific PI3K inhibitor wortmannin. We conclude Akt and p70S6k lie on separate signaling pathways. Activation of signaling to Akt is insufficient for the activation of p70S6k, which can be achieved independently of Akt. p70S6k requires a separate calcium-dependent and wortmannin-sensitive process that is likely to be independent of type IA PI3K family members.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Phosphatidylinositol 3-kinase IA (PI3K1 IA) is activated via its docking to activated receptor tyrosine kinases, to src family members, or via direct interaction with activated Ras (1, 2). Due to this mode of activation, PI3K IA can be assayed in anti-phosphotyrosine immunoprecipitates, distinct from the other known PI3K family members types IB, II, and III (3). Activation of PI3K IA has been implicated in the control of cell growth by observations that mutants of the PDGF receptor that cannot bind PI3K are defective in mitogenic signaling and that transformation defective mutants of various oncogenes fail to activate PI3K (1, 4, 5). However, until recently little was known as to how the mitogenic signal is transmitted downstream of PI3K. This situation changed with the identification of the two serine/threonine kinases, p70S6k (6) and Akt (7, 8), as physiologically relevant downstream targets of PI3K IA. PDGF receptor mutants unable to bind PI3K IA fail to activate both p70S6k (6) and Akt (7, 9). In addition, the activation of both kinases by growth factors is inhibited by two different inhibitors of PI3K IA, wortmannin and LY294002 (7, 9-13), strongly arguing in favor of a connection between PI3K IA, Akt, and p70S6k. This connection is further supported by the findings that both p70S6k and Akt are activated following co-transfection with constitutively active PI3K IA (14-16). Furthermore, neutralizing antisera to p70S6k inhibit the mitogenic activity of PI3K IA (17). In addition, Akt is directly activated in vitro by phosphatidylinositol 3,4-bisphosphate, a phospholipid product of PI3K IA, and this activation is dependent on a functional pleckstrin homology domain in Akt (18, 19).

In addition to lying downstream of PI3K IA, both Akt and p70S6k are activated by a similar range of mitogens (EGF, PDGF, fibroblast growth factor, nerve growth factor, insulin, and insulin-like growth factor-1) and phosphatase inhibitors (vanadate and okadaic acid), implying a close connection between the signaling pathways to the two kinases (7-10, 20, 21). Indeed, Akt may mediate mitogenic signaling by activation of p70S6k, since p70S6k is stimulated by active mutants of Akt in co-transfection assays (7, 14, 15). However, there is some indication of differences in their signaling pathways. A kinase inactive mutant of Akt, presumed to act as a dominant negative, failed to inhibit p70S6k stimulation (7), and Akt does not phosphorylate p70S6k in vitro. Whereas inappropriate activation of Akt leads to cellular transformation (22), constitutively active forms of p70S6k are not transforming (23). Moreover, it has recently been shown that Akt but not p70S6k mediates PI3K IA-dependent survival of cerebellar neurons and Rat-1 fibroblasts (24-26).

To clarify the connection between signaling through Akt and p70S6k, we have examined their regulation by other mitogen-stimulated pathways. One obvious candidate for regulating PI3K IA-initiated pathways is the PLCgamma /DAG/1,4,5-inositol triphosphate pathway. Both PI3K IA and PLCgamma are crucial for growth factor-induced mitogenesis (6, 27). In addition, cross-talk between the two is suggested by experiments showing that the lipid products of PI3K IA activate the novel PKC isoforms (28-30). PI3K IA-independent activation of p70S6k via the conventional PKC (cPKC) has been reported (6). Mitogenic stimulation of PLCgamma leads to a rapid and transient increase of intracellular calcium through the second messenger inositol triphosphate (31), as well as the generation of diacyl glycerol (DAG) with subsequent activation of PKC (32). Although the role of this calcium "spike" in mitogenesis is poorly characterized (31, 33, 34), increases in calcium and DAG result in activation of both the conventional and novel PKCs. Although simultaneous activation of PI3K IA and increase of intracellular calcium, via the PLCgamma /DAG/1,4,5-inositol triphosphate pathway, is observed in many cell types, little attention has been given to the interactions between both systems.

In this study, we examine the role of calcium in the regulation of the PI3K IA/Akt/p70S6k pathway(s) in Balb/c-3T3 fibroblasts to clarify the interconnection between signaling through these kinases. We show that Akt activation is independent of calcium, whereas p70S6k stimulation is totally dependent on calcium. Increases of intracellular calcium levels give little or no activation of Akt and PI3K IA but fully activate p70S6k, and this calcium-induced stimulation is completely blocked by low levels of wortmannin. Thus, the activation of the signaling pathway to Akt is insufficient for activation of p70S6k, which also requires a separate, calcium-dependent process. Furthermore, stimulation of p70S6k by calcium is likely to involve a new wortmannin-sensitive pathway, independent of PI3K IA.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cell Culture and Preparation of Cell Extracts-- Balb/c-3T3 fibroblasts were grown at 37 °C in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Confluent cells were made quiescent by culturing for 24 h in Dulbecco's modified Eagle's medium containing no fetal calf serum. Serum-starved cells were rinsed twice with a Hepes buffer solution containing 120 mM NaCl and 20 mM Hepes, pH 7.4. Cells were then treated at 37 °C with various stimuli diluted in Hepes buffer: 5 nM EGF (Auspep) for 5 min, 50 ng/ml PDGF (Auspep) for 5 min, 1 µM ionomycin for 5 min, 5 µM thapsigargin for 5 min, 0.1 mM pervanadate prepared with 0.2 mM H2O2 (35) for 10 min, 5 µM or 100 nM PMA for 10 min. Alternatively, cells were pretreated for 20 min at 37 °C in Hepes buffer containing 2 mM EGTA, 2 mM BAPTA, 100 nM wortmannin, 5 nM rapamycin, 5 µM the PKC inhibitor Ro-31-8220, 5 µM the inactive analogue Ro-31-6045, or 5 µM calphostin C before stimulation. In some experiments, the cells were pretreated for 24 h with 5 µM PMA. The controls were carried out by incubating the cells for the corresponding period in Hepes buffer containing dimethyl sulfoxide (Me2SO) instead of the agent. Following stimulation, the cells were rinsed twice with ice-cold phosphate-buffered saline (PBS) and lysed in a buffer containing 50 mM Tris, pH 7.5, 120 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 50 mM NaF, 40 mM beta -glycerophosphate, 0.1 mM sodium vanadate, 1 mM benzamidine, 0.5 mM phenylmethylsulfonyl fluoride. Cell extracts were collected with a plastic scraper, homogenized, and cleared by centrifugation at 4 °C for 20 min at 12,000 × g. Protein concentration was measured by the method of Bradford (Bio-Rad), with bovine serum albumin as standard. Aliquots of the supernatant were frozen in liquid nitrogen and stored at -70 °C.

Immunoprecipitation of Akt and in Vitro Akt Kinase Assay-- Akt was immunoprecipitated by incubating 200 µg of protein/assay of cell extract with protein A-Sepharose pre-coupled to an antibody directed to the C-terminal 16 residues of Akt (36) overnight at 4 °C on a roller. The beads were then washed twice at 4 °C with the lysis buffer and once with a kinase assay buffer containing 50 mM Tris, pH 7.5, 1 mM dithiothreitol, 1 mM benzamidine, and 0.5 mM phenylmethylsulfonyl fluoride. Akt kinase activity was assayed using the Crosstide as substrate (37) by incubating the immunoprecipitated Akt for 20 min at 30 °C on a shaking plate in 25 µl of kinase assay buffer containing 100 µM Crosstide, 1 µM protein kinase inhibitor peptide, 10 mM MgCl2, and 50 µM ATP (plus 0.25 µCi of [gamma -32P]ATP). Following a brief centrifugation, the reaction was terminated by spotting the supernatant on P81 phosphocellulose paper. Unincorporated [gamma -32P]ATP was eliminated by three 5-min washes in 75 mM orthophosphoric acid, and phosphorylated Crosstide bound to the paper was counted. Akt kinase assays were conducted in duplicate. The results are expressed in units of Akt activity per mg of protein lysate. One unit of activity results in the transfer of 1 pmol of 32Pi into Crosstide per min under the assay condition listed above.

In Vitro p70S6k and PI3K Assays-- p70S6k activity was assayed as described previously (38) using 40 S ribosome as substrate. The results are expressed in units of p70S6k activity per mg of protein lysate. One unit of activity results in the transfer of 1 pmol of 32Pi into S6 protein per min.

For measurement of PI3K activity, cell lysates (500 µg of protein) were incubated with 2 µg of a mixture of anti-phosphotyrosine antibodies (PY-7E1, PY-1B2, and PY20, Zymed Laboratories) overnight at 4 °C, under constant mixing. The PI3K assays were then performed essentially as described previously (4) in a volume of 100 µl containing 20 mM Hepes, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 50 µM ATP (plus 0.8 µCi of [gamma -32P]ATP), 125 µg/ml phosphatidylinositol, and 62.5 µg/ml phosphatidylserine dispersed by sonication. The samples were incubated for 20 min at 25 °C, and the reactions were stopped by the addition of 100 µl of 1 M HCl. Phospholipids were extracted and separated for 2 h by thin layer chromatography in a chloroform/methanol/glacial acetic acid/H2O (46/41/5/8) developing solvent prepared freshly. The 32P-phospholipids were quantified on a PhosphorImager using ImageQuant software (Molecular Dynamics).

Immunoblotting-- Cell lysates were boiled in Laemmli sample buffer for 3 min. Cell lysates, containing 10 µg of total protein for the determination of Akt and 40 µg for p70S6k, were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) on 10% slab gels, and proteins were transferred to polyvinylidene difluoride membranes. Membranes were blocked for 30 min in PBS containing 0.1% Tween 20 (PBS-T) and 5% (w/v) dry skim milk powder and incubated overnight with anti-p70S6k (39) or anti-Akt (36) antisera. The membranes were then washed with PBS-T and incubated for 2 h with an anti-rabbit secondary antibody conjugated to horseradish peroxidase. Bound antibodies were detected with the enhanced chemiluminescence (ECL) system (Amersham Corp.).

Intracellular Calcium Measurements-- Serum-starved Balb/c-3T3 cells were harvested by trypsinization and suspended at a density of 106 cells/ml in Dulbecco's modified Eagle's medium containing 0.1% (w/v) bovine serum albumin. The cells were loaded with 2 µM fura-2/AM for 30 min at 37 °C in the same medium. Cells were washed once and resuspended in Hepes buffer at the same density. For fluorimetric measurements, 3 × 106 fura-2-loaded cells were placed in a cuvette, and fluorescence was monitored using a dual excitation wavelength spectrofluorimeter (SPEX Industries Inc., Edison, NJ). Cells were excited with light alternating between 340 and 380 nm, and fluorescence emitted was measured at 505 nm. The results of excitation (F340 and F380) were averaged over 1-s intervals. Autofluorescence at each wavelength, which represented less than 10% of the signal from loaded cells, was subtracted before calculation of the ratio F340/F380. This ratio is proportional to change in intracellular calcium (40), and the results are expressed as change in the ratio from base line.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Differential Calcium Requirements for EGF-dependent Activation of Akt and p70S6k-- To study the relationship between the activation of Akt and p70S6k, we have examined their relative regulation by EGF in Balb/c-3T3 fibroblasts. Western blot analysis of Akt and p70S6k revealed the characteristic bandshifting associated with the activation of these kinases by treatment of the cells with EGF (Fig. 1A). When the cells were incubated with sufficient EGTA to deplete the intracellular calcium stores, the EGF-induced electrophoretic mobility decrease of Akt was still observed, whereas p70S6k remained in its basal state (Fig. 1A). Measurement of intracellular calcium levels confirmed that the intracellular calcium stores were almost totally depleted after treatment with EGTA under the conditions stated above (Table I). EGF treatment did not increase intracellular calcium levels significantly, indicating a basal level of calcium is required for the activation of p70S6k. Relative calcium levels were measured as described under "Experimental Procedures." Pretreatment with the PI3K inhibitor wortmannin inhibited the bandshifting of both kinases, whereas the immunosuppressant rapamycin specifically inhibited p70S6k (Fig. 1A).


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Fig. 1.   Differential calcium sensitivity of Akt and p70S6k activation by EGF. A, Western blot analysis of Akt and p70S6k. Balb/c-3T3 fibroblasts were serum-starved for 24 h before incubation for 10 min with Hepes buffer (20 mM Hepes, pH 7.4, 120 mM NaCl) containing vehicle (Me2SO or medium; Con) or 5 nM EGF (EGF). To chelate intracellular calcium cells were preincubated for 20 min with 2 mM EGTA before stimulation with EGF (+EGTA). Alternatively, cells were preincubated for 20 min in presence of 100 nM wortmannin (+Wort) or 5 nM rapamycin (+Rapa) before EGF stimulation. Similar results were obtained in at least two additional experiments. B, Akt immunoprecipitation assay. Akt activity was quantified for its ability to phosphorylate the Crosstide as described under "Experimental Procedures." The values represent the mean of at least four different cell preparations ± S.D. One unit (unit/mg) of Akt activity results in the transfer of 1 pmol of 32P into Crosstide per min. C, p70S6k assay. p70S6k activity was measured using 40 S ribosomes as substrate (see "Experimental Procedures"). One unit/mg of activity results in the transfer of 1 pmol of 32P into protein S6 per min. The values represent the mean of at least four different cell preparations assayed in duplicate and expressed ± S.D.

                              
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Table I
Effect of EGF, PDGF, and thapsigargin in the presence or absence of EGTA, BAPTA, or wortmannin on relative intracellular calcium levels
Serum-starved Balb/c-3T3 cells were loaded with 2 µM fura-2/AM as described under "Experimental Procedures" and treated for 5 min at 37 °C with 5 nM EGF, 50 ng/ml PDGF, or 5 µM thapsigargin. Alternatively, cells were pretreated for 20 min at 37 °C in Hepes buffer containing 2 mM EGTA, 2 mM BAPTA, or 100 nM wortmannin. The results are expressed as change in ratio of fura-2 fluorescence from base-line (Control) and are expressed as the mean of at least two different cell preparations assayed in duplicate ± S.D.

These results indicate a differential dependence of Akt and p70S6k on calcium for activation and were confirmed by direct kinase assays. The EGF-induced activation of Akt was independent of calcium (Fig. 1B), whereas the activation of p70S6k by EGF was dependent on the presence of calcium (Fig. 1C).

Differential Calcium Requirements for PDGF- and Pervanadate-induced Activation of Akt and p70S6k-- To characterize further the calcium requirement for Akt and p70S6k activation, we tested two other stimuli known to potently activate these kinases, PDGF and the tyrosine phosphatase inhibitor pervanadate. Unlike EGF, PDGF induced substantial increases in intracellular calcium levels, and these levels remained below base line if the cells were pretreated with either EGTA or BAPTA (Table I). Pervanadate and PDGF markedly decreased the electrophoretic mobility of Akt and p70S6k (Fig. 2A). p70S6k bandshifting was abolished by EGTA treatment, whereas Akt was unaffected. These results were quantified by direct kinase assays (Fig. 2, B and C). EGF, PDGF, and pervanadate induced a 5- to 6-fold activation of Akt activity and an 8- to 9-fold increase of p70S6k activity. The stimulation of Akt activity by all agents was not affected by calcium chelation (Fig. 2B), whereas this treatment completely inhibited p70S6k activation by PDGF (Fig. 2C). The stimulation of p70S6k activity by pervanadate was only partially blocked (43% inhibition) following EGTA preincubation (Fig. 2C), indicating the existence of a calcium-independent pathway capable of stimulating p70S6k.


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Fig. 2.   Calcium requirement for PDGF- and pervanadate-induced stimulation of Akt and p70S6k. A, Western blot analysis of Akt and p70S6k. Balb/c-3T3 fibroblasts were serum-starved for 24 h before incubation for 10 min with Hepes buffer containing vehicle (Me2SO or medium; Con) or 0.1 mM pervanadate (Per), 5 nM EGF (EGF), or 50 ng/ml PDGF (PDGF). Cells were incubated for 20 min in the presence of 2 mM EGTA (+) before stimulation to assess calcium requirement for p70S6k and Akt activation by growth factors and pervanadate. Similar results were obtained in at least two more experiments. B and C, Akt and p70S6k assays were performed as described for Fig. 1. Hatched bars indicate the activities in the absence of EGTA and solid bars in the presence of EGTA. Activities are expressed relative to that of unstimulated cells (Con). The values represent the mean of at least two separate cell preparations ± S.D.

Preincubation with 100 nM wortmannin inhibited the pervanadate-induced stimulation of p70S6k activity by 85%, whereas it completely blocked the activation by EGF and PDGF. A combination of wortmannin and EGTA did not further inhibit p70S6k activation by pervanadate (results not shown). Thus, the wortmannin-independent pathway that can stimulate p70S6k is also calcium-independent.

Differential Effects of Increasing Intracellular Calcium Levels on Akt and p70S6k Activities-- To determine the effects of an increase of intracellular calcium on the activity of both kinases, ionomycin and thapsigargin, a specific inhibitor of the endoplasmic reticulum calcium-ATPases was used (41). Ionomycin and thapsigargin induced similar and substantial increases in intracellular calcium levels, and EGTA or BAPTA pretreatment reduced calcium levels below base line in the presence of thapsigargin (Table I). Incubation in the presence of these agents induced little or no activation of Akt, although it fully activated p70S6k (6-7-fold increase). Preincubation with EGTA abolished the effects of ionomycin and thapsigargin (Fig. 3, A and B). Simultaneous treatment with ionomycin and EGF did not lead to any increase of Akt and p70S6k activation induced by EGF alone (results not shown). Time course and dose-response analysis showed no effect on Akt activity and indicated that the concentration of ionomycin and thapsigargin and the duration of stimulation used were optimal for p70S6k stimulation (results not shown). Thus, the activation of Akt is not required for the calcium stimulation of p70S6k activity in Balb/c-3T3 fibroblasts.


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Fig. 3.   Effect of increasing intracellular calcium levels with ionomycin or thapsigargin on Akt and p70S6k. Serum-starved Balb/c-3T3 cells were incubated for 5 min in Hepes buffer containing vehicle (Me2SO; Control), 1 µM ionomycin (Iono), or 5 µM of thapsigargin (Thaps). Akt (A) and p70S6k (B) activities are expressed relative to those of unstimulated cells (Control). The values represent the mean of four different cell preparations ± S.D. Hatched bars indicate the activities in the absence of EGTA and solid bars in the presence of EGTA.

Role of PI3K in the Calcium-induced Activation of p70S6k-- Due to its pivotal role in the regulation of Akt and p70S6k in response to growth factors, the involvement of PI3K in the stimulation of both kinases by calcium was examined. Cells were stimulated with ionomycin or thapsigargin following 20 min preincubation with 100 nM wortmannin, a dose that gives specific inhibition of PI3K (42). This treatment resulted in Akt and p70S6k activities below control levels (Table II), indicating that PI3K is absolutely required for the stimulation by calcium. The measurement of intracellular calcium levels in fura-2/AM-loaded cells showed that the preincubation with wortmannin had no effect on the increase of calcium induced by PDGF or thapsigargin (Table I).

                              
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Table II
The effect of pretreatment with wortmannin on activation of p70S6k by ionomycin
Balb/c-3T3 fibroblasts were preincubated for 20 min in the presence of 100 nM wortmannin or vehicle (Me2SO) prior to the addition of vehicle (Control) or 5 µM thapsigargin. Akt and p70S6k activities were measured as described in Fig. 1. One unit of activity resulted in the transfer of 1 pmol of 32P into Crosstide or S6 per min, respectively. The values represent the mean of two different cell preparations ± S.D. assayed in duplicate.

To characterize further the involvement of PI3K in the calcium-induced activation of p70S6k, the role of a calcium in the regulation of PI3K activity in anti-phosphotyrosine immunoprecipitates was examined. Ionomycin treatment resulted in weak activation of PI3K (2-fold), approximately 60-fold less than that induced by EGF treatment. Preincubation with EGTA resulted in a 20% inhibition of EGF-induced PI3K activation (Fig. 4). These results indicate the possible existence of a PI3K/Akt-independent, calcium-dependent pathway leading to the activation of p70S6k. However, because the calcium-induced stimulation of p70S6k is completely inhibited by low doses of wortmannin (Table II), it is possible that other PI3K isoforms, not detected in the anti-phosphotyrosine immunoprecipitation, are involved.


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Fig. 4.   Effect of intracellular calcium levels on PI3K activity. Balb/c-3T3 fibroblasts were preincubated for 20 min with vehicle (Me2SO) or 2 mM EGTA prior to addition of vehicle (Con), 1 µM ionomycin (Iono), or 5 nM EGF. PI3K activity of 500 µg of protein lysates was assayed as described under "Experimental Procedures." Results, expressed as PhosphorImager units, represent the mean ± S.D. of two cell preparations. A control where 100 nM of wortmannin (Wort) was added in vitro to EGF-stimulated extract was carried out.

Role of PKC in Akt and p70S6k Stimulation-- To examine the differential effects of calcium on Akt and p70S6k activities, we focused on the calcium-regulated PKC (cPKC). Cells were incubated in the presence of PMA for 24 h, to down-regulate PKC, and then stimulated with various mitogens. PKC down-regulation had no effect on Akt activation by any of the stimuli tested (results not shown), whereas a partial inhibition of the stimulation of p70S6k activity induced by pervanadate (40%), EGF (41%), PDGF (47%), and ionomycin (45%) was observed (Fig. 5A). Re-addition of PMA to cells in which PKC was down-regulated gave no activation of p70S6k suggesting that the conditions used were sufficient to down-regulate PKC (Fig. 5A). To characterize further the involvement of PKC in the Akt and p70S6k pathway, cells were exposed to 5 µM PMA for 10 min. As shown previously (7, 9), PMA treatment did not stimulate Akt kinase activity (results not shown) but activated p70S6k activity 5-fold (Fig. 5B). Following EGTA exposure, the stimulation of p70S6k by PMA was inhibited by approximately 60%, although after pretreatment with wortmannin (100 nM) this inhibition was increased to 75% (Fig. 5B). At more relevant PMA levels (100 nM), this inhibition was almost complete (90%, results not shown). Combination of EGTA and wortmannin completely inhibited the stimulation of p70S6k activity by PMA.


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Fig. 5.   Effect of the incubation with PMA on p70S6k activity in Balb/c-3T3 fibroblasts. A, serum-starved Balb/c-3T3 cells were incubated for 24 h in medium containing vehicle (hatched bars) or 5 µM PMA (solid bars) prior to stimulation with 0.1 mM pervanadate (Per), 5 nM EGF (EGF), 50 ng/ml PDGF (PDGF), 5 µM ionomycin (Iono), or 5 µM PMA (PMA). p70S6k activity, assayed as described in Fig. 1, is expressed relative to that of unstimulated cells (Con). The values represent the mean of two separate cell preparations ± S.D. B, serum-starved Balb/c-3T3 cells were preincubated for 20 min with 2 mM EGTA, 100 nM wortmannin, 5 µM specific PKC inhibitor Ro-31-8220, or with a combination of 100 nM wortmannin and 2 mM EGTA prior to addition of vehicle (Me2SO; Control) or 5 µM PMA for 10 min. p70S6k activity, assayed as described in Fig. 1, is expressed relative to that of unstimulated cells (Control), and the values represent the mean of two different cell preparations ± S.D.

Because of the uncertainty of the specificity of the PMA response, especially at 5 µM, these studies were extended using the specific PKC inhibitor Ro-31-8220 (43). Preincubation of the cells with Ro-31-8220 completely inhibited the stimulation of p70S6k by PMA (Fig. 5B) and by EGF and ionomycin (results not shown). However, the inactive analogue Ro-31-6045 also completely inhibited the EGF and ionomycin-induced activation of p70S6k, suggesting that these compounds have a direct inhibitory effect on p70S6k, as was shown in vitro (44). The structurally unrelated PKC inhibitor, calphostin C (5 µM, under room light), did not inhibit the activation of p70S6k by either EGF or PDGF (results not shown).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Activation of PI3K has been implicated in the control of cell growth and motility, cellular transformation, secretion, and apoptosis; however, little is known as to how the mitogenic signal is transmitted downstream of PI3K (9-11). Recently, the two serine/threonine kinases Akt and p70S6k have been identified as physiologically relevant downstream elements of this pathway. A number of reports have indicated that these kinases lie on a common signaling pathway (7, 14, 15). Transient transfection of constitutively active Akt in Rat-1 cells is sufficient to activate p70S6k (7), leading to the hypothesis that Akt lies upstream of p70S6k in a PI3K-dependent signal transduction pathway. To dissect the connection(s) between mitogenic signaling through Akt and p70S6k pathways, we have measured their relative endogenous activities in response to a range of stimuli known to induce activation of these kinases. In this study we have concentrated on the activation states of the endogenous kinases to avoid potential artifacts associated with overexpression. For example, overexpression of p70S6k in 293 cells results in hyperphosphorylation of sites minimally labeled in endogenous kinase (45, 46) and a marked decrease in the specific activity of the kinase.2 In particular, we have focused on the role of calcium in regulating each kinase in both the absence and presence of mitogenic stimuli.

In this study we show that EGF, PDGF, and pervanadate all stimulate Akt and p70S6k in Balb/c-3T3 fibroblasts. While the activation of both kinases by these agents has been reported (7, 9, 20, 21), the role of calcium in their regulation was unknown. Treatment of Balb/c-3T3 fibroblasts with EGTA under conditions that deplete both intra- and extracellular calcium completely inhibits p70S6k activation by EGF and PDGF, whereas it has no effect on Akt activation. Conversely, increasing the calcium levels in Balb/c-3T3 fibroblasts by ionomycin or thapsigargin induced a full activation of p70S6k although little or no stimulation of Akt activity was observed. Thus, activation of Akt is insufficient to activate p70S6k in the absence of calcium, whereas activation of p70S6k induced by calcium does not require Akt activity. The two kinases appear to lie on distinct pathways with a separate calcium-dependent pathway being required for p70S6k activity. Calcium may play dual roles in the regulation of p70S6k. Basal levels of calcium are required for EGF and probably other growth factors to induce activation of the kinase, although increases in intracellular calcium levels are sufficient to activate the enzyme.

Due to the central role of PI3K in the regulation of both enzymes, we examined its role in this differential calcium effect. Calcium levels sufficient to fully activate p70S6k following ionomycin or thapsigargin treatment gave little or no activation of PI3K in anti-phosphotyrosine immunoprecipitates (PI3K IA). Similarly, calcium chelation only slightly inhibited PI3K IA activation by EGF but totally inhibited p70S6k. Thus, the PI3K IA activity profile mirrors that of Akt but not that of p70S6k. The calcium-induced activation of p70S6k appears to be independent of PI3K IA. Paradoxically, activation of p70S6k by calcium was totally inhibited by wortmannin, despite the lack of the calcium-induced activation of either PI3K IA or Akt.

Why is the activation of p70S6k by calcium totally inhibited by wortmannin? Wortmannin may be inhibiting an alternative target to PI3K as it has been shown to inhibit other enzymes such as myosin light chain kinase (47), cPLA2 (48), and phosphatidylinositol 4-kinase (49). However, with the exception of cPLA2, inhibition of these enzymes was observed at much higher levels of wortmannin than used in the present study. A more likely explanation is that a wortmannin-sensitive PI3K isoform distinct from the type IA isoforms might be implicated in p70S6k activation by calcium (Fig. 6). Wortmannin inhibition of MAPK activation by platelet-activating factor (PAF) through a mechanism independent of the conventional p85/p110 heterodimeric PI3K has already been shown in a macrophage cell line (50). A human PI3K highly homologous to the yeast Vps34p kinase has been identified which has a substrate specificity restricted to phosphatidylinositol (51) and might therefore be a good candidate for regulating p70S6k activity independently of Akt pathway which requires phosphatidylinositol 3,4-bisphosphate for activation. Interestingly, PI3K from Arabidopsis thaliana (AtVPS34), which has 61% homology to the yeast VPS34, contains an N-terminal calcium-dependent lipid-binding domain possibly required for phosphatidylinositol binding whose phosphorylation may recruit additional proteins to the membrane (52).


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Fig. 6.   Hypothetical pathways involved in the differential activation of Akt and p70S6k. According to this model the two kinases lie on separate pathways with the activation of p70S6k requiring a calcium-dependent process that is partially independent of PKC and involves activation of a PI3K isoform distinct from the type IA PI3K family members.

What is the calcium-dependent intermediate required for activation of p70S6k? It has been suggested that the calcium-dependent activation of p70S6k in GN4 rat liver epithelial cells is mediated by tyrosine kinase/proline-rich tyrosine kinase 2 (CADTK/PYK2) (53). However, although the activation of p70S6k correlated with the increased expression and activation of CADTK in GN4 cells, TPA did not stimulate p70S6k activity in these cells although it did stimulate CADTK. Similar to our results, wortmannin completely inhibited the activation of p70S6k by calcium; however, wortmannin had no effect on the calcium-dependent activation of CADTK suggesting that PI3K is downstream of CADTK or unrelated to the activation of CADTK. The most likely candidate for a calcium-dependent intermediate to p70S6k is a member of the conventional PKC family (cPKC; alpha , beta , gamma ) given that the PLCgamma /PKC pathway is activated simultaneously to the PI3K pathway and these PKC isoforms are calcium-dependent. In this study we showed that activation of cPKCs by PMA results in strong activation of p70S6k, independent of Akt. This observation is consistent with cPKCs mediating the differential effects of calcium on the two kinases. However, down-regulation of cPKCs with PMA gave only a 45% inhibition of the ionomycin-induced activation of p70S6k. This is similar to the inhibition observed for growth factor- and pervanadate-induced activation and indicates that a significant proportion of the calcium signal is independent of cPKC. Ionomycin stimulation of p70S6k activity was totally inhibited by wortmannin. Thus, ionomycin stimulation of p70S6k is likely to involve a third, wortmannin-sensitive pathway, independent of the PI3K/Akt pathway and partially independent of cPKC. Unlike the stimulation of p70S6k by growth factors and calcium, the PMA-induced activation was only partially inhibited by EGTA indicating phorbol esters can initiate calcium-independent pathways not activated by EGF and PDGF (Fig. 6).

The use of the PKC inhibitor Ro-31-8220 and its inactive analogue Ro-31-6045 to characterize further the involvement of PKC in p70S6k pathway revealed the lack of specificity of these compounds which both strongly inhibited any stimulation of p70S6k. This result will nevertheless be of great practical interest if Ro-31-6045 proves to be a specific inhibitor of p70S6k. An alternative PKC inhibitor that is structurally unrelated to Ro-31-8220, calphostin C, did not inhibit the activation of p70S6k by either EGF or PDGF, strengthening our conclusion that a calcium-dependent, PKC-independent pathway exists leading to the activation of p70S6k.

In this study we find that the activation of the signaling pathway to Akt is insufficient for the activation of p70S6k, which can be achieved independently of Akt. Thus, the two kinases are likely to lie on separate pathways with the activation of p70S6k requiring a separate calcium-dependent process that is partially independent of PKC and may involve activation of a PI3K isoform distinct from the type IA PI3K family members. Differential regulation of Akt and p70S6k by calcium might reflect their differential involvement in intracellular events such as apoptosis, differentiation, and mitogenesis.

    ACKNOWLEDGEMENTS

We are indebted to Jan Groves for excellent technical assistance, to Dr. Elizabeth Johnson for help with the measurement of intracellular calcium levels, to Dr. Varuni Kanagasundaram for help with the PI3K assays, and to Drs. Catherine Monnot and Matthew O'Connell for critical reading of this manuscript.

    FOOTNOTES

* This work was supported by a Project Grant from the Anti-Cancer Council of Victoria (to R. B. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of a Postdoctoral fellowship from Schweizerische Stiftung für medizinisch-biologische Stipendien.

To whom correspondence should be addressed. Tel.: 61 3 9656 1247; Fax: 61 3 9656 1411; E-mail: rick{at}res.petermac.unimelb.edu.au.

1 The abbreviations used are: PI3K, phosphatidylinositol-3-kinase; PKC, protein kinase C; cPKC, calcium-regulated PKC; PLC, phospholipase C; DAG, diacylglycerol; PMA, phorbol 12-myristate 13-acetate; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; BAPTA, bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid; PBS, phosphate-buffered saline.

2 R. B. Pearson, P. B. Dennis, and G. Thomas, unpublished observations.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

  1. Kapeller, R., and Cantley, L. C. (1994) BioEssays 16, 565-576[Medline] [Order article via Infotrieve]
  2. Rodriguez-Viciana, P., Warne, P. H., Dhand, R., Vanhaesebroeck, B., Gout, I., Fry, M. J., Waterfield, M. D., Downward, J. (1994) Nature 370, 527-532[CrossRef][Medline] [Order article via Infotrieve]
  3. Domin, J., and Waterfield, M. J. (1997) FEBS Lett. 410, 91-95[CrossRef][Medline] [Order article via Infotrieve]
  4. Whitman, M., Kaplan, D. R., Shaffhausen, B., Cantley, L., and Roberts, T. M. (1985) Nature 315, 239-242[Medline] [Order article via Infotrieve]
  5. Carpenter, C. L., and Cantley, L. C. (1996) Curr. Opin. Cell Biol. 8, 153-158[CrossRef][Medline] [Order article via Infotrieve]
  6. Chung, J., Grammar, T. C., Lemon, K. P., Kazlauskas, A., Blenis, J. (1994) Nature 370, 71-75[CrossRef][Medline] [Order article via Infotrieve]
  7. Burgering, B. M. T., and Coffer, P. (1995) Nature 376, 599-602[CrossRef][Medline] [Order article via Infotrieve]
  8. Downward, J. (1995) Nature 376, 553-554[CrossRef][Medline] [Order article via Infotrieve]
  9. Franke, T. F., Yang, S., Chan, T. O., Datta, K., Kazlauskas, A., Morrison, D. K., Kaplan, D. R., Tsichlis, P. N. (1995) Cell 81, 727-736[Medline] [Order article via Infotrieve]
  10. Andjelkovic, M., Jakubowicz, T., Cron, P., Ming, X.-F., Han, J.-W., and Hemmings, B. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5699-5704[Abstract/Free Full Text]
  11. Cheatham, B., Vlahos, C. J., and Cheatham, L. (1994) Mol. Cell. Biol. 14, 4902-4911[Abstract]
  12. Arcaro, A., and Wymann, M. P. (1993) Biochem. J. 296, 297-301[Medline] [Order article via Infotrieve]
  13. Vlahos, C. J., Matter, W. F., Hui, K. Y., Brown, R. F. (1994) J. Biol. Chem. 269, 5241-5248[Abstract/Free Full Text]
  14. Reif, K., Burgering, B. M. T., and Cantrell, D. A. (1997) J. Biol. Chem. 272, 14426-14433[Abstract/Free Full Text]
  15. Kohn, A. D., Takeuchi, F., and Roth, R. A. (1996) J. Biol. Chem. 271, 21920-21926[Abstract/Free Full Text]
  16. Klippel, A., Reinhard, C., Kavanaugh, W. M., Apell, G., Escobedo, M.-A., Williams, L. T. (1996) Mol. Cell. Biol. 16, 4117-4127[Abstract]
  17. McIlroy, J., Chen, D., Wjasow, C., Michaeli, T., and Backer, J. M. (1997) Mol. Cell. Biol. 17, 248-55[Abstract]
  18. Klippel, A., Kavanaugh, W. M., Pot, D., and Williams, L. T. (1997) Mol. Cell. Biol. 17, 338-344[Abstract]
  19. Franke, T. F., Kaplan, D. R., Cantley, L. C., Toker, A. (1997) Science 275, 665-668[Abstract/Free Full Text]
  20. Susa, M., Vulevic, D., Lane, H. A., Thomas, G. (1992) J. Biol. Chem. 267, 6905-6909[Abstract/Free Full Text]
  21. Ming, X.-F., Burgering, B. M. T., Wennstrom, S., Claesson-Welsh, L., Heldin, C-H., Bos, J. L., Kozma, S. C., Thomas, G. (1994) Nature 371, 426-429[CrossRef][Medline] [Order article via Infotrieve]
  22. Ahmed, N. N., Franke, T. F., Bellacosa, A., Datta, K., Gonzalez-Portal, M., Taguchi, J. R., Testa, J. R., Tsichlis, P. N. (1993) Oncogene 8, 1957-1963[Medline] [Order article via Infotrieve]
  23. Mahalingam, M., and Templeton, D. J. (1996) Mol. Cell. Biol. 16, 405-413[Abstract]
  24. Dudek, H., Datta, S. R., Franke, T. F., Birnbaum, M. J., Yao, R., Cooper, G. M., Segal, R. A., Kaplan, D. R., Greenberg, M. E. (1997) Science 275, 661-664[Abstract/Free Full Text]
  25. Kauffmann-Zeh, A., Rodriguez-Viciana, P., Ulrich, E., Gilbert, C., Coffer, P., Downward, J., and Evan, G. (1997) Nature 385, 544-548[CrossRef][Medline] [Order article via Infotrieve]
  26. Kulik, G., Klippel, A., and Weber, M. J. (1997) Mol. Cell. Biol. 17, 1595-1606[Abstract]
  27. Valius, M., and Kazlauskas, A. (1993) Cell 73, 321-334[Medline] [Order article via Infotrieve]
  28. Ettinger, S. L., Lauener, R. W., and Duronio, V. (1996) J. Biol. Chem. 271, 14514-14518[Abstract/Free Full Text]
  29. Akimoto, K., Takahashi, R., Moriya, S., Nishioka, N., Takayanagi, J., Kimura, K., Fukui, Y., Osada, S., Mizuno, K., Hirai, S., Kazlaukas, A., and Ohno, S. (1996) EMBO J. 15, 788-798[Abstract]
  30. Moriya, S., Kazlauskas, A., Akimoto, K., Hirai, S., Mizuno, K., Takenawa, T., Fukui, Y., Watanabe, Y., Ozaki, S., and Ohno, S. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 151-155[Abstract/Free Full Text]
  31. Berridge, M. J. (1993) Nature 361, 315-325[CrossRef][Medline] [Order article via Infotrieve]
  32. Nishizuka, Y. (1992) Science 258, 607-614[Medline] [Order article via Infotrieve]
  33. Wahl, M., and Gruenstein, E. (1993) Mol. Cell. Biol. 4, 293-302
  34. Short, A. D., Bian, J., Ghosh, T. K., Waldron, R. T., Rybak, S. L., Gill, D. L. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4986-4990[Abstract]
  35. Posner, B. I., Faure, R., Burgess, J. W., Bevan, A. P., Lachance, D., Zhang-Sun, G., Fantus, I. G., Ng, J. B., Hall, D. A., Lum, B. S., Shaver, A. (1994) J. Biol. Chem. 269, 4596-4604[Abstract/Free Full Text]
  36. Jones, P. F., Jakubowicz, T., Pitossi, F. J., Maurer, F., Hemmings, B. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4171-4175[Abstract]
  37. Cross, D. A. E., Alessi, D. R., Cohen, P., Andjelkovic, M., and Hemmings, B. A. (1995) Nature 378, 785-789[CrossRef][Medline] [Order article via Infotrieve]
  38. Lane, H. A., and Thomas, G. (1991) Methods Enzymol. 200, 269-291
  39. Lane, H. A., Fernandez, A., Lamb, N. J. C., Thomas, G. (1993) Nature 363, 170-173[CrossRef][Medline] [Order article via Infotrieve]
  40. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260, 3440-3450[Abstract]
  41. Thastrup, O., Cullen, P. J., Drobak, B. K., Hanley, M. R., Dawson, A. P. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2466-2470[Abstract]
  42. Wymann, M. P., Bulgarelli-Leva, G., Zvelebil, M. J., Pirola, L., Vanhaesebroeck, B., Waterfield, M. D., Panayotou, G. (1996) Mol. Cell. Biol. 16, 1722-1733[Abstract]
  43. Twomey, B., Muid, R. E., Nixon, J. S., Sedgwick, A. D., Wilkinson, S. E., Dale, M. M. (1990) Biochem. Biophys. Res. Commun. 171, 1087-1092[Medline] [Order article via Infotrieve]
  44. Alessi, D. R. (1997) FEBS Lett. 402, 121-123[CrossRef][Medline] [Order article via Infotrieve]
  45. Ferrari, S., Pearson, R. B., Siegmann, M., Kozma, S. C., Thomas, G. (1993) J. Biol. Chem. 268, 16091-16094[Abstract/Free Full Text]
  46. Pearson, R. B., Dennis, P. B., Han, J.-W., Williamson, N. A., Kozma, S. C., Wettenhall, R. E. H., Thomas, G. (1995) EMBO J. 14, 5279-5287[Abstract]
  47. Nakanishi, S., Kakita, S., Takahashi, I., Kawahara, K., Tsukuda, E., Sano, T., Yamada, K., Yoshida, M., Kase, H., Matsuda, Y., Hashimoto, Y., and Nonomura, Y. (1992) J. Biol. Chem. 267, 2157-2163[Abstract/Free Full Text]
  48. Cross, M. J., Stewart, A., Hodgkin, M. N., Kerr, D. J., Wakelam, M. J. O. (1995) J. Biol. Chem. 270, 25352-25355[Abstract/Free Full Text]
  49. Nakanishi, S., Catt, K. J., and Balla, T. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5317-5321[Abstract]
  50. Ferby, I. M., Waga, I., Hoshino, M., Kume, K., and Shimizu, T. (1996) J. Biol. Chem. 271, 11684-11688[Abstract/Free Full Text]
  51. Volinia, S., Dhand, R., Vanhaesebroeck, B., Mac. Dougall, L. K., Stein, R., Zvelebil, J., Domin, J., Panaretou, C., Waterfield, M. D. (1995) EMBO J. 14, 3339-3348[Abstract]
  52. Welters, P., Takegawa, K., Emr, S. D., Chrispeels, M. J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11398-11402[Abstract/Free Full Text]
  53. Graves, L. M., He, Y., Lambert, J., Hunter, D., Li, X., and Earp, H. S. (1997) J. Biol. Chem. 272, 1920-1928[Abstract/Free Full Text]


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