Up-regulation of AMP-activated Protein Kinase in Cancer Cell Lines Is Mediated through c-Src Activation*

We report that the activation level of AMP-dependent protein kinase AMPK is elevated in cancer cell lines as a hallmark of their transformed state. In OVCAR3 and A431 cells, c-Src signals through protein kinase Cα, phospholipase Cγ, and LKB1 to AMPK. AMPK controls internal ribosome entry site (IRES) dependent translation in these cells. We suggest that AMPK activation via PKC might be a general mechanism to regulate IRES-dependent translation in cancer cells.

icillin and 100 mg/ml streptomycin, and all cells were grown in a humidified atmosphere containing 5% CO 2 at 37°C.
siRNA Transfection-A431 and OVCAR3 cells were transfected using DharmaFECT1 according to the manufacturer's protocol. In brief, for a 6-well plate 2 M siRNAs in 1 ϫ siRNA buffer were diluted into serum-free medium and incubated for 5 min at room temperature. 5 l of DharmaFECT1 was diluted with 195 l of serum-free medium, and after 5 min at room temperature the reagent was incubated with 2 M siRNA for 20 min at room temperature. 1.6 ml of DMEM supplemented with 10% FCS was added to the siRNA⅐DharmaFECT1 complex and dropped onto the cells. 96 h after transfection, cells were harvested.
Drug Treatment-At 60 -70% confluence, cells were starved of serum for 2 h. The medium was replaced with medium lacking serum containing PP1, BIS1, rapamycin, AICAR, compound C, or U73122 and the cells were incubated further, as indicated.
Protein Determination-The protein content of cell lysates generated by nondenaturing detergents was quantified using the micro-Bradford assay (13) with the Zor-Selinger modifications (14). Protein determination for denatured lysates was performed using the bound Coomassie Blue method: samples (5 l) and BSA standards (1-10 g) were spotted onto numbered 3MM filter paper strips (0.5 ϫ 1.5 cm; Whatman). Filter papers were stained with Coomassie Brilliant Blue G-250 (2.5 g/liter in 40% methanol, 10% acetic acid; Bio-Rad), and unbound dye was washed with destaining solution (20% methanol, 7% acetic acid). Bound Coomassie was eluted by incubating the filters in 1 ml of 3% SDS at 37°C for 1 h with shaking in a 24-well plate. 200-l samples were transferred to a 96-well plate, and the absorbance of the eluted solution was measured at 595 nm in an ELISA reader.
Western Blot Analysis-Cells were washed with PBS (50 mM NaH 2 PO, 50 mM Na 2 HPO 4 , 0.77 M NaCl), and denatured cell lysates were prepared by scraping the cells in the presence of Laemmli sample buffer (40% glycerol, 0.2 M Tris, pH 6.8, 20% ␤-mercaptoethanol, 12% SDS, and bromphenol blue) and boiling the samples for 10 min. Aliquots of cell extracts containing the same amounts of protein were resolved by SDS-PAGE and electroblotted onto nitrocellulose membranes (Sartorius). The membranes were blocked with TBS-T (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, and 0.2% Tween 20) containing 5% low fat milk, incubated with primary antibodies overnight at 4°C, and then with HRP-conjugated secondary antibodies (Jackson Immunoresearch) for 45 min at room temperature. After each incubation the membranes were thoroughly washed with TBS-T (four times for 3 min). Immunoreactive bands were visualized using enhanced chemiluminescence (ECL). For densitometric analysis, several exposures were made, and only subsaturation exposures were analyzed. Densitometry of immunoblots was performed with ImageJ 1.61 (National Institutes of Health) or EZQuant software.
DNA Transfection and Drug Treatment-A431 and OVCAR3 cells were transfected with the bicistronic reporter construct (1 g of DNA/well). 48 h after transfection the cells were treated for 4 h with AICAR or compound C (CC). After 52 h, cell lysates were prepared and assayed for firefly luciferase and Renilla luciferase activity. The bicistronic translation reporter plasmids (pEF-FFL-IRES-SPL and pEF-SPL-IRES-FFL) were kindly provided by Prof. R. Fukunaga (Osaka, Japan) (15). The control bicistronic reporter plasmid (pRF) was kindly provided by Prof. A.E. Willis (Nottingham, UK) (16). The bicistronic reporter plasmid containing the 5Ј-UTR of HIF-1␣ and the pStemRhifF plasmid, which contains a hairpin before the SPL cistron, were kindly provided by Prof. G. J. Goodall (Adelaide, Australia) (17).
Luciferase Assay-Cells were transfected with the appropriate reporter plasmids, as above. At the time indicated, cells were harvested, and protein extracts were prepared by lysis with Reporter Lysis Buffer. FFL and SPL activities were assayed with a dual-luciferase kit using a Luminoskan Ascent (Thermo Labsystem) luminometer. For the bicistronic constructs containing the 5Ј-UTR of HIF-1␣, FFL activity was normalized to SPL activity, to correct for transfection efficiencies.
Coomassie Gels-Gels were stained with GelCode blue stain reagent (Pierce), according to the manufacturer's instructions. The amount of purified protein was estimated using a BSA standard curve, which was loaded on the same gel.
In Vitro Kinase Assay-Increasing amounts of GST-LKB1 (10, 20, 40, and 80 ng) were incubated for 30 min at 30°C with 60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl 2 , 3 mM MnCl 2 , 3 M sodium orthovanadate, 1.2 mM DTT, 20 M ATP, and 1 Ci of [␥-32 P]ATP in the presence or absence of active GST-PKC␣ (20 ng/reaction). The reaction was terminated by the addition of SDS sample buffer and the proteins resolved by SDS-PAGE. Proteins were transferred to a nitrocellulose membrane, and the membrane was subjected to autoradiography. Afterward, membranes were blocked with 5% low fat milk in TBST and reacted with anti-LKB1, anti-phospho-LKB1-S428 and anti-PKC␣.
Immunoprecipitation-For co-immunoprecipitation, the cells were lysed by scraping at 4°C with lysis buffer containing 50 mM HEPES, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 150 mM NaCl, 1 mM sodium orthovanadate, 10 mM ␤-glycerolphosphate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 10 g/ml soybean trypsin inhibitor, 10 g/ml leupeptin, 1 g/ml aprotinin, 313 g/ml benzamidine, and 0.2 mM AEBSF. Lysates were centrifuged at 19,000 ϫ g for 10 min. Lysates comprising 0.5-1 mg of protein were used to immunoprecipitate c-Src and PLC␥1 from A431 and OVCAR3 cells. Antibodies against c-Src (mAb 327 or sc-208; Santa Cruz) or PLC␥1 (2822 from Cell Signaling or M156 from Abcam) were coupled to protein G-Sepharose (mAb 327) or A-Sepharose (sc-208, 2822 and M156) (Amersham Biosciences). 60 l of protein A or G was coupled to 250 l of ␣-c-Src (mAb 327) antibody (in medium) or to 2 g of ␣-c-Src (sc-208) antibody by incubating them for 2 h at 4°C. 60 l of protein A was coupled to 1:50 diluted ␣-PLC␥ (2822) antibody or 20 l of ␣-PLC␥ (M156) antibody by incubating them for 2 h at 4°C. After coupling, the beads were washed five times with PBS to remove excess antibody. Lysates were incubated with coupled beads overnight at 4°C on a rotating platform. Immunocomplexes were washed once with lysis buffer and five times with PBS, sample buffer was added to the beads, and the samples were boiled for 10 min before SDS-PAGE separation.

Activity of AMPK Is Elevated in Cancer Cell
Lines-To characterize AMPK regulation in transformed cells, we examined AMPK activity in a set of cancer cell lines, including A431, OVCAR3, HeLa, HT-29, MCF7, SKOV-3, T-24, and PC3 cells. We compared these cells with primary keratinocytes, which represent nontransformed cells of epithelial origin. The levels of the activated (phosphorylated) form of AMPK (Thr-172), as well as the phosphorylation of its direct substrate, ACC (Ser-79), were elevated in A431, OVCAR3, HeLa, T-24, MCF7, and PC-3 cells, compared with K cells, but not in SKOV-3 and HT-29 cells ( Fig. 1 and supplemental Fig. S1). In this study we conducted more detailed experiments on two of the cell lines: OVCAR3 and A431.
AMPK Is Regulated by c-Src in OVCAR3 and A431 Cellsc-Src is known to be activated in OVCAR3 and A431 cells (19,20). Therefore, we decided to investigate whether c-Src regulates AMPK activity in these cell lines. Inhibition of c-Src by its inhibitor, PP1 (21), led to reduced activation of AMPK (as indicated by P-AMPK␣) and reduced phosphorylation of its direct substrate, ACC, in both OVCAR3 and A431 cells (Fig. 2, A and B). Furthermore, siRNA-mediated depletion of c-Src using siRNA also resulted in reduced activity of AMPK in both cell lines (Fig. 2, C and D).
c-Src Regulates AMPK through PKC␣-Because previous studies demonstrated that the activation of AMPK is PKC-dependent (22,23), we tested whether PKC mediates AMPK activation by c-Src. Interestingly, the inhibition of PKC by BIS1 significantly attenuated AMPK activity (supplemental Fig. S2, A and B). Nevertheless, PP1 treatment had no effect on the phosphorylation of PKC (Thr-410/Thr-403) (supplemental Fig. S2, C and D). Therefore, PKC is not the intermediate involved in the activation of AMPK by c-Src in these cells. However, the finding that BIS1 treatment led to AMPK inhibition (supplemental Fig. S2, A and B) implied that other PKC isoforms may be involved in the regulation of AMPK by c-Src.
A connection between c-Src and PKC␣ had been reported previously in breast cancer cells (24,25). We therefore examined the effect of Src inhibition on PKC␣ in our cells. Treatment of A431 and OVCAR3 cells with the Src inhibitor, PP1, led to a reduction in the phosphorylated (activated) form of PKC␣ (Thr-638) (18), in both A431 and OVCAR3 cells (supplemental Fig. S3, A and B). Furthermore, siRNA against c-Src also led to a reduction in PKC␣ activation (supplemental Fig. S3, C and D). These results confirm that c-Src regulates PKC␣ in these cell lines.
The phorbol ester, TPA, is known to activate PKC. Treatment of OVCAR3 and A431 cells with TPA resulted in increased phosphorylation of PKC␣ (Thr-638), as well as of AMPK␣ (Thr-172) and ACC (Ser-79) (Fig. 3, A and B). Furthermore, siRNA against PKC␣ led to reduced activity of AMPK in A431 and OVCAR3 cells (Fig. 3, C and D).
Although the up-regulation of PKC␣ by c-Src has been shown previously, the mechanism of this activation remains unknown (25). Recently, it was found that c-Src can be coimmunoprecipitated with PKC␣ from breast cancer cell lines FIGURE 1. AMPK is more active in A431 and OVCAR3 cells than in primary keratinocytes. Phosphorylation of AMPK on Thr-172 indicates constitutive activation in OVCAR3 and A431 cells. P-AMPK␣ and P-ACC levels were normalized to GAPDH levels. The graph shows the calculated averages Ϯ S.D. from three independent experiments. In the cell lines, the direct substrate of AMPK, ACC, was also highly phosphorylated on Ser-79 (P-ACC). (25). Therefore, we tested whether such an association occurs in OVCAR3 and A431 cells. c-Src was immunoprecipitated with two different c-Src-specific antibodies, but Western blot analysis with c-Src and PKC␣ antibodies failed to show an interaction between PKC␣ and c-Src (data not shown). Thus, c-Src regulates PKC␣ in OVCAR3 and A431 cells, in an indirect manner.
c-Src Does Not Regulate PKC␣ through PDK1-The initial step in the activation of PKC can be executed by PDK1 (26,27). Furthermore, it has been reported that the phosphorylation of PDK1 by c-Src is required for PDK1 activation (28,29). Therefore, we explored whether PDK1 mediates PKC␣ activation by c-Src in A431 and OVCAR3 cells. Depletion of PDK1 by siRNA in these cells had a minor effect, if any, on the activity of PKC␣ (supplemental Fig. S4). Therefore, we conclude that PDK1 is not a major player in transmitting the signal from Src to PKC␣.
c-Src Regulates PKC␣ through PLC␥-Regulation of PLC␥1 by c-Src has been reported previously (30 -32), and regulation of PKC isoforms by PLC␥1 has long been known (for review, see Ref. 33). Therefore, we examined whether Src signaled to PKC␣ via PLC␥. Following immunoprecipitation of c-Src from A431 and OVCAR3 lysates, we observed a weak association between c-Src and PLC␥1 (Fig. 4, A and B). The reciprocal co-immunoprecipitation with anti-PLC␥1 antibody confirmed these results (Fig. 4, C and D). Interestingly, an antibody against an  APRIL 29, 2011 • VOLUME 286 • NUMBER 17 epitope in the N terminus of PLC␥1 precipitated c-Src (Fig. 4, C  and D), whereas an antibody against an epitope in the C terminus failed (supplemental Fig. S5).

c-Src Activates AMPK in Cancer Cell Lines
In light of these results, we speculated that PLC␥1 mediates the activation of PKC␣ by c-Src. Inhibition of c-Src by PP1 or its depletion by siRNA led to a reduction in the phosphorylation of PLC␥1 at Tyr-783 (supplemental Fig. S6). Phosphorylation is one of the key mechanisms that regulate the activity of PLC␥1, and phosphorylation at Tyr-783 was reported to activate PLC␥1 (34). We also note that in SrcNIH cells, which are NIH 3T3 cells transformed with active Src (Y529F) (12), the levels of phosphorylated PLC␥1 were higher than in the parental NIH 3T3 cell line. Moreover, PP1 strongly inhibited the phosphorylation of PLC␥1 at Tyr-783, in SrcNIH cells (supplemental Fig. S7).
We next inhibited PLC␥ using U73122 (35,36). This led to a decrease in the phosphorylation of PKC␣ (Fig. 4E). Thus, the signal from c-Src to AMPK is transmitted via PLC␥ and PKC␣.
Activation of AMPK by c-Src Is Mediated through PKC␣ and LKB1-Bioinformatics analysis suggested that AMPK␣ might be a direct substrate of PKC␣. However, co-immunoprecipitation failed to detect an interaction between AMPK␣ and PKC␣

c-Src Activates AMPK in Cancer Cell Lines
in OVCAR3 or A431 cells (data not shown). Previously, it had been shown that PKC activates AMPK through the serine/ threonine kinase, LKB1. Following the phosphorylation of LKB1 at Ser-428, LKB1 is translocated from the nucleus to the cytosol, where it associates with AMPK and activates it (22,23). Therefore, we examined whether LKB1 also mediates the activation of AMPK by PKC␣. We found that the inhibition of PKC by BIS1 caused a reduction in the phosphorylation of LKB1 at Ser-428 (Fig. 5, A and B), whereas the activation of PKC␣ by TPA led to increased phosphorylation of LKB at Ser-428 (Fig. 5, C  and D). Furthermore, the depletion of PKC␣ by siRNA led to a marked reduction in the phosphorylation of LKB1. The inhibition of c-Src by PP1 or the depletion of c-Src by siRNA led to a reduction in the phosphorylation of LKB1 (supplemental Fig. S8).
To examine whether PKC␣ directly phosphorylates LKB1, we performed an in vitro kinase assay using purified GST-LKB1 and active GST-PKC␣, in the presence of MgATP and [␥-32 P]ATP and then separated the reaction mixes by SDS-PAGE and transferred them to nitrocellulose. Autoradiography of the blot showed that in the absence of active PKC␣, GST-LKB1 was not significantly phosphorylated. However, in the presence of active PKC␣, [␥-32 P]ATP was incorporated into GST-LKB1, and the degree of incorporated [␥-32 P]ATP was proportional to the amount of GST-LKB1 in the assay. Furthermore, Western blot analysis using anti-phospho-LKB1 antibody showed an increase in Ser-428 phosphorylation of LKB1 (supplemental Fig. S9A). Thus, PKC␣ activates LKB1 in OVCAR3 and A431 cells.
Inhibition of LKB1 using siRNA led to reduced phosphorylation of AMPK and ACC, but not of PKC␣, indicating that LKB1 transmits the signal from PKC␣ to AMPK (supplemental Fig. S8E). Inhibition of Src using PP1 led to decreased phosphorylation of PLC␥, PKC␣, LKB1, AMPK, and ACC (supplemental Fig. S9B). In light of these results, we conclude that c-Src regulates AMPK via PLC␥, PKC␣, and LKB1 in OVCAR3 and A431 cells.
Involvement of AMPK in the Regulation of IRES-dependent Translation-AMPK plays a central role in the regulation of protein synthesis. It inhibits mTOR via the phosphorylation and activation of tuberous sclerosis complex 2 (TSC2). TSC2 is a subunit of the TSC1/TSC2 (hamartin/tuberin) complex which negatively regulates mTOR signaling. Thus, AMPK mediates the inhibition of cap-dependent translation (11,37).
Previously, we showed that in the course of cell transformation a switch from cap to IRES-dependent translation occurred. This switch corresponded with the progressive activation of AMPK during transformation (4). Here, we examined whether AMPK can divert translation toward IRES-dependent translation in OVCAR3 and A431 cells. To this end we used a luciferase reporter system that utilizes reciprocal bicistronic plasmids from which both cap and IRES-dependent translation can be measured (15). In both cell lines, the activation of AMPK by AICAR (11) led to an increase in IRES-dependent translation (supplemental Fig. S10, A, B, E, and F) and a reduction in capdependent translation (supplemental Fig. S10, C, D, G, and H). Correspondingly, the addition of the AMPK inhibitor, compound C (38), led to a decline in the IRES fraction. Interestingly, compound C treatment also reduced cap-dependent translation (supplemental Fig. S10, C, D, G, and H). To verify that we were indeed measuring IRES-dependent translation from the HIF 5Ј-UTR, and not read-through translation, we used the  APRIL 29, 2011 • VOLUME 286 • NUMBER 17 pStemRHifF plasmid (Fig. S10I), in which cap-dependent translation is strongly inhibited. The ratio of firefly to Renilla luciferase activity was even higher from pStemRhifF than from pRhifF, indicating that cap-dependent translation of the Renilla luciferase was repressed and the firefly luciferase was read from the IRES site in the 5Ј-UTR (17).

c-Src Activates AMPK in Cancer Cell Lines
Hif-1␣ is directly involved in cell survival and was reported to undergo protein translation by both cap and IRES-dependent mechanisms (17). It has been reported that c-Src regulates the synthesis of Hif-1␣ protein (39). Furthermore, it has been documented that AMPK regulates Hif-1␣ via a signaling pathway that is independent of the PI3-kinase/PKB and MAPK path-

c-Src Activates AMPK in Cancer Cell Lines
ways (40). In view of these results, we hypothesized that through AMPK, c-Src might regulate the IRES-dependent protein synthesis of Hif-1␣ in A431 and OVCAR3 cells.
The depletion of c-Src or of PKC␣ using siRNA, as well as PP1 treatment, led to a reduction in the level of Hif-1␣ in OVCAR3 cells (Fig. 6, A and B). We note that Hif-1␣ could not be detected on Western blots from A431 cells.
Rapamycin, a selective inhibitor of mTOR, and hence of caprelated translation, had no effect on the accumulation of Hif-1␣ in OVCAR3 cells (Fig. 6B). Furthermore, the activation of AMPK by AICAR treatment led to increased accumulation of Hif-1␣ (Fig. 6C). These findings were corroborated by the demonstration that IRES-dependent translation from the 5Ј-UTRs of HIF1␣ increased under AICAR treatment, whereas compound C treatment led to reduced translation from the 5Ј-UTR of HIF1␣ (Fig. 6D).
Regulation of AMPK by c-Src Is Cell Type-specific-The regulation of AMPK by c-Src was seen in a number of cell lines, including L-HF1, BP-HF1 (4), A431, and OVCAR3 cells. However, in HeLa and MCF7 cells, the inhibition of c-Src by PP1 had no effect on AMPK activity (supplemental Fig. S11, A and B), suggesting that the c-Src 3 PKC␣ 3 AMPK module is cell type-(or tissue)-specific. Even in these cells, however, the inhibition of PKC by BIS1 treatment led to a reduction in the phosphorylation of AMPK and ACC (supplemental Fig. S11, C and  D), suggesting that this part of the signaling module may be widespread but that the upstream elements and PKC isotypes may vary.

DISCUSSION
In this study, we delineate a signaling pathway from the oncoprotein c-Src to AMPK. AMPK, a Ser/Thr protein kinase, serves as an energy sensor in all eukaryotic cells (7,8). We observed an increase in the activation of AMPK in a number of cancer cell lines, including OVCAR3 and A431 cells ( Fig. 1 and  supplemental Fig. S1). The increased activity of AMPK in A431 and OVCAR3 cells is c-Src-dependent, as inhibition of c-Src resulted in decreased activity of AMPK (Fig. 2). In primary keratinocytes, we saw no evidence of a connection between c-Src and AMPK (supplemental Fig. S12).
A signaling pathway leading to the activation of AMPK␣ by c-Src has not been previously established in cancer cells. Here, we have found that in OVCAR3 and A431 cells, c-Src regulates AMPK␣ through PKC␣, PLC␥, and LKB1. Although PKC can be activated by PDK1, and PDK1 has been reported to be regulated by c-Src (28), we were unable to find evidence for PDK1dependent activity of PKC␣ in OVCAR3 and A431 cells (supplemental Fig. S4). However, PDK1 was not completely abolished by the siRNA in this assay, and the remaining protein might have been sufficient to induce PKC␣ activation.
Another possible connection between c-Src and PKC␣ is through PLC␥. It has long been known that PLC␥ regulates the activity of PKC isoforms (for review, see Ref. 33). Furthermore, a connection between PLC␥ and c-Src was postulated previously (30 -32). Here we have shown, using siRNA techniques and pharmacological tools, that c-Src regulates PLC␥ in A431 and OVCAR3 cells (supplemental Fig. S6). Furthermore, we monitored a weak physical association between c-Src and PLC␥1 (Fig. 4). It is interesting to note that only an antibody that targets an epitope in the N terminus of PLC␥ could precipitate c-Src (Fig. 4, C and D), whereas an antibody against an epitope in the C terminus failed to achieve this (supplemental Fig. S5). The C-epitope antibody may interfere with the interaction of c-Src and PLC␥1. The finding that the SH3 domain of PLC␥1 is in close proximity to the C terminus supports this hypothesis (41).
The existence of a link between c-Src and PLC␥1 is strongly supported by the finding that the activity of PLC␥1 is higher in SrcNIH cells than in the parental NIH 3T3 cell line. Moreover, we found that the Src inhibitor, PP1, reduces Tyr-783 phosphorylation on PLC␥1 in a dose-dependent manner (supplemental Fig. S7). Hence, in OVCAR3 and A431 cells, c-Src signals to PKC␣ via PLC␥.
LKB1 is the major upstream kinase of AMPK (for review, see Refs. 7, 8), therefore we looked for a connection between PKC␣ and LKB1. We found that the phosphorylation of LKB1 by PKC␣ leads to activation of AMPK (Fig. 5). Additionally, in a cell-free assay, PKC␣ directly phosphorylated LKB1 at Ser-428 (supplemental Fig. S9). In light of these results, we conclude that AMPK is regulated by c-Src through PKC␣, PLC␥1, and LKB1 in OVCAR3 and A431 cells.
We speculate that this signaling pathway, in which Src or other tyrosine kinases activate AMPK via PKC, might be common to a number of cell types. It would be interesting to investigate whether this pathway is activated in tumor tissue. It has been recently shown that in endothelial cells AMPK is activated by c-Src, via the PI3-kinase pathway (22,23). Even though c-Src does not regulate AMPK␣ in HeLa and MCF7 cells (supplemental Fig. S11, A and B), we did find reduced activity of AMPK when PKC was inhibited in these cells (supplemental Fig. S11, C and D). We speculate that not only c-Src, but also other tyrosine kinases or receptor tyrosine kinases might be involved in the regulation of AMPK via PKC. Furthermore, not only PKC␣, but also other PKC isoforms, might be involved in the regulation of AMPK. As mentioned above, it has been reported previously that AMPK is PKC-dependent in heart and skeletal muscle (22,23). This dependence was not seen in A431 and OVCAR3 cells, but might be relevant to other cell lines. Further, we note that increased AMPK activity was monitored in HeLa cells compared with primary keratinocytes (supplemental Fig. S1), although it is well known that HeLa cells are LKB1-deficient (42).
AMPK is activated under stress, enabling cells to survive. Cancer cells are subjected to significant stress, due to the accumulation of chromosomal aberrations and damaged proteins, as well as to their rapid rates of proliferation and the consequent metabolic stress. Although LKB1 is generally considered to be a tumor suppressor, the role of AMPK in cancer is unclear. It has been suggested that AMPK is essential for tumors to adapt to metabolic stress (43 and references therein). The activation of AMPK has been reported to decrease cap-dependent translation (11). Here, we have shown that AMPK also plays a role in the regulation of IRES-dependent translation. We found that IRES-dependent translation was up-regulated when AMPK was activated and down-regulated when AMPK was inhibited (supplemental Fig. S10, A, B, E, and F). As expected, following AMPK activation, there was a reduction in cap-dependent translation (supplemental Fig. S10, C, D, G, and H). Surprisingly, AMPK inhibition by compound C also reduced cap-dependent translation (supplemental Fig. S10, C, D, G, and  H), possibly due to a toxic effect of compound C on A431 and OVCAR3 cells. The activation of AMPK led to the increased accumulation of Hif-1␣, whereas rapamycin treatment (which inhibits cap-dependent translation) had no effect on the accumulation of this protein in OVCAR3 cells (Fig. 6). Thus, activation of AMPK by c-Src leads to induced IRES-dependent translation in OVCAR3 cells.
In summary, we have shown that AMPK is regulated by c-Src through PKC␣, PLC␥, and LKB1 in OVCAR3 and A431 cells. Furthermore, our results imply a correlation between AMPK activation and IRES-dependent translation. We suggest that activation of AMPK by c-Src may signal the cell to save energy, to allow cell survival under stress.