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Keywords:

  • adaptor protein;
  • apoptotic resistance;
  • autophagy;
  • nutrient deprivation;
  • p66Shc

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Macroautophagy or autophagy is a lysosome-dependent process in which enzymatic degradation and recycling of cytosolic components occur in stressful contexts. The mechanisms underlying the signaling from starvation to the regulation of autophagy are not fully understood. We previously showed that the Src family member p66Shc (focal adhesion-associated 66 kDa isoform of the Src homology and collagen) promotes anoikis and suppresses tumor metastasis via k-Ras-dependent control of proliferation and survival. However, the role of p66Shc in low-nutrient-induced autophagy-related pathways remains elusive. In this work, human lung adenocarcinoma A549 cells were used to further investigate the biological effects of p66Shc on autophagy and apoptotic resistance. Here, we show that deficiency of p66Shc mitigates the low-nutrient-induced autophagy process in the levels of microtubule-associated protein 1A light chain protein 3B (LC3B) conversion, in the number of autophagic vacuoles and in p62/sequestosome 1 protein degradation. However, autophagy-related protein Beclin 1 was not significantly changed during low-nutrient treatment. Furthermore, we found that prolonged phosphorylation of extracellular signaling-regulated kinase (Erk)1/2, but not phosphorylation of Akt is significantly sustained when p66Shc expression is inhibited by shRNA. In addition, cleavage of caspase 7 and poly(ADP-ribose) polymerase, but not caspase 6 and 9 are retarded with this effect compared to the shRNA control cells. Together, these findings suggest the possibility that p66Shc plays a pivotal role in coordinately regulating autophagy process and apoptotic resistance in A549 cells under nutrient-limited conditions.


Abbreviations
3-MA

3-methyladenine

Erk

extracellular signaling-regulated kinase

LC3B

microtubule-associated protein 1A light chain protein 3B

mTOR

mammalian target of rapamycin

PARP

poly(ADP-ribose) polymerase

RFP

red fluorescent protein

shRNA

short hairpin RNA

TSC1

tuberous sclerosis protein 1

ULK

uncoordinated family member (unc)-51-like kinase

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Macroautophagy or autophagy is a lysosome-dependent process in which enzymatic degradation and recycling of cytosolic components occur in stressful contexts including nutrient deprivation [1]. During nutrient deprivation, mammalian target of rapamycin (mTOR) and AMPK have been well characterized as critical signaling pathways regulating prosurvival; however, excessive autophagy also leads to cell death, a process named autophagic cell death or type II apoptosis [2, 3]. Factors involved in the induction and regulation of autophagy are: potent tumor suppressors, including Beclin 1, autophagy-related 4, phosphatase and tensin homolog, tuberous sclerosis protein 1 (TSC1), TSC2, liver kinase B1, UV radiation-resistance-associated gene, as well as the Bcl-2 family proteins [4, 5]. However, autophagy in different cancer cells has different effects at different stages of tumorigenesis and progression. Thus, a somewhat more antithetic relationship to the other alternative pathways of cell death which are obviously tightly regulated exists in cancer cells under stressful conditions such as low nutrient levels.

The mammal adaptor proteins ShcA (p66Shc, p52Shc and p46Shc) share a C-terminal Src homology 2 domain, a central collagen-homologous domain and an N-terminal phosphotyrosine-binding domain and have N-termini of different lengths [6, 7]. Despite their high structural similarity, p66Shc and p52Shc/p46Shc are involved in multiple signaling mechanisms to support diversity in cell homeostasis [8]. Of the two ShcA transcripts and three isoforms, p66Shc functions as a critical regulator of longevity in mice [9]. We have previously shown that p66Shc is also a focal adhesion-associated protein, mediates anoikis through a RhoA-dependent mechanosensory test, and thus plays an important role in preventing solid cancer metastasis [10, 11]. Here, we explore the function of downregulated p66Shc in a nutrient-deprivation condition, characterize the downstream signaling, apoptotic and autophagic pathways caused by silencing p66Shc in lung cancer A549 cells, and identify the delicate regulation of apoptotic resistance and autophagic survival pathways sustained by p66Shc in nutrient-poor conditions.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

p66Shc is induced by nutrient deprivation and enhances cell death

It is well known that nutrient deprivation induces autophagy in many types of cultured cells. To define a feasible period of autophagy induced by nutrient deprivation, A549 (human lung adenocarcinoma) cells were incubated in basal medium with 0.1% fetal bovine serum and amino acid deprivation for up to 3 days. We observed that the p66Shc expression, but not the other isoforms of p52Shc and p46Shc, was enhanced in a timely fashion by nutrient deprivation at the protein (Fig. 1A,B) and mRNA levels (Fig. 1C). Next, we conducted a p66Shc knockdown assay using lentiviral transduction as well as control shRNA against fire fly luciferase. We found that cell death was increased by nutrient deprivation in A549 cells, whereas p66Shc knockdown by either small hairpin (sh)RNAs (1) or (2) mitigated this adverse effect (Fig. 1D), implying that inhibition of p66Shc expression blocks nutrient-deprivation-induced cell death.

image

Figure 1. p66Shc is induced by nutrient stress and enhances cell death. (A) Lung cancer A549 cells were treated with limited nutrient of 0.1% fetal bovine serum and amino acids deprivation for indicated time. At the end of treatment, cell lysate was collected and subject to western blotting analysis of Shc isoform expression, with β-actin loading control. (B) p66Shc protein levels shown in (A) were determined by densitometric analysis of the protein bands on western blots and normalized to β-actin value at 0 d of treatment (n = 4). (C) qRT-PCR analysis of relative mRNA levels of p66Shc as treated in (A). The mRNA intensities were normalized to that of β-actin and expressed by fold of control cells (day 0). (D) p66Shc knockdown with shRNA (1) and (2) decreases cell death by nutrient stress. (Left) Cell death assay in p66Shc knockdown cells by nutrient deprivation for 24 h (n = 4). (Right) Western blotting demonstrates p66Shc knockdown effect by shRNA (1) and (2) as well as control against fire fly luciferase (control). Data are mean ± SEM of three independent replicates. *P < 0.05, **P < 0.01, ***P < 0.001.

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Deficiency of p66Shc mitigates autophagy process by nutrient deprivation

To investigate the effect of reduced p66Shc expression on nutrient-deprivation-induced autophagy, A549 cells expressing two shRNAs against p66Shc were generated. Because of a green fluorescent protein cassette present in the shRNA lentiviral vector, we examined red fluorescent protein (RFP)–LC3B transfection in these cells and assessed autophagosome induction using fluorescence confocal microscopy. LC3B, one of the microtubule-associated proteins, becomes cleaved and lipidated with the phospholipid, phosphatidylethanolamine and recruited to the autophagosomal membrane during autophagosome formation [1, 12]. In A549 cells maintained in 10% fetal bovine serum, RFP–LC3B displayed a sustained level of autophagosome formation (Fig. 2A, upper). Following nutrient deprivation for 24 h, RFP–LC3B puncta increased in both empty vector and p66Shc-knockdown A549 cells (Fig. 2A, lower); however, silencing p66Shc showed the formation of less punctate in RFP–LC3B transfected cells compared to empty control cells (Fig. 2A, lower and Fig. 2B).

image

Figure 2. Silencing p66Shc mitigates chronic nutrient starvation-induced autophagy. (A) Representative confocal images of A549 cells transiently expressing RFP–LC3B maintained as described in 'Experimental procedures'. Scale bars, 5 μm. (B) Quantification of RFP–LC3B fluorescent intensity in puncta-positive cells shown in (A). The arbitrary unit of punctae was determined as the mean ± SEM from 50 cells for each condition (*< 0.05, **< 0.01). (C) A549 cells were treated as indicated and cell lysates were western blotted with anti-(LC3B) and anti-(p62) IgG, with β-actin as a loading control. (D, E) Plot depicts densitometric analysis of LC3B-II, -I and p62 band intensity, normalized to β-actin levels and expressed as fold change from untreated control. (F) Western blotting against LC3B and β-actin of 0.1 μm bafilomycin A1-treated or untreated control and p66Shc-shRNA knockdown A549 cells. (G) The LC3B-II/I ratio was determined by densitometric analysis of the protein bands on western blots. The results were expressed as mean ± SEM of three determinations.

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Because the change in autophagic flux shows a dynamic formation of autophagosome, we tested the LC3B-I to -II conversion and p62 degradation by western blotting in p66Shc knockdown A549 cells. As shown in Fig. 2C–E, knockdown of p66Shc decreased LC3B-I to -II conversion, but not p62 degradation compared with shRNA controls, indicating that p66Shc facilitates autophagy induced by nutrient deprivation. However, silencing p66Shc did not completely inhibit LC3B-I to -II conversion in A549 cells with nutrient deprivation after 3 days (Fig. 2C,D). More importantly, the elevated level of LC3B-II in the p66Shc knockdown cells was not further increased in the presence of late autophagy inhibitor, bafilomycin A1 (Fig. 2F,G), indicating that p66Shc is necessary for the augmentation of autophagosome formation rather than reduced autophagosome–lysosome fusion. Collectively, these results suggest a regulatory role of p66Shc in autophagy process induced by nutrient deprivation.

Phosphorylation of Erk1/2(Thr202/Tyr204) but not Akt1 (Ser473) are sustained in p66Shc knockdown cells by nutrient deprivation

Erk1/2 are conserved serine/threonine kinases that regulate many cellular programs including proliferation, differentiation and cell death, and have been implicated in autophagy [13]. We tested whether the levels of phosphorylated Erk1/2 in p66Shc knockdown cells were changed during nutrient deprivation. The time course effect (up to 3 days) of nutrient deprivation was investigated in p66Shc knockdown A549 cells. A549 cells showed high basal levels of phosphorylated Erk1/2 (Thr202/Tyr204) and phosphorylated Akt1 (Ser473), which decreased strongly with nutrient deprivation (Fig. 3A–D). However, in p66Shc-deficient A549 cells, levels of phosphorylated Erk1/2 (Thr202/Tyr204), but not Akt1 (Ser473), were actively prolonged by nutrient deprivation (Fig. 3A–D). Moreover, knockdown of p66Shc in A549 cells reduced BrdU incorporation by ~ 40% compared with vector control (Fig. 3E), suggesting a specific effect of p66Shc on proliferation in A549 cells. 3-Methyladenine (3-MA), an inhibitor of early stage of autophagy through inhibiting class III phosphatidylinositol kinase activity, also markedly increased phosphorylation of Erk1/2 after 1 day of nutrient starvation (Fig. 3F). Together, these data showed that silencing p66Shc sustains prolonged activation of Erk1/2, but not Akt1, by nutrient deprivation treatment.

image

Figure 3. p66Shc knockdown sustains phosphorylated Erk1/2(Thr202/Tyr204), but not Akt1(Ser473) and decreases cell proliferationby nutrient starvation. A549 cells were transduced with control or p66Shc shRNA and nutrient starved as indicated time points. (A, B) Western blots were performed for total Erk1/2 and phospho-Erk1/2 (Thr202/Tyr204), respectively. Membrane was stripped and reprobed with β-actin as an equal protein-loading control. Results shown are representative of three independent blots. (C, D) Western blots were performed for total Akt1 and phosphorylated Akt1 (Ser473), respectively. Results shown are representative of three independent blots. (E) p66Shc knockdown decreases BrdU uptake in A549 cells. Data are the mean ± SEM of six determinations. (F) 3-MA (5 mm), an early autophagy inhibitor, was applied to A549 cells for 0 or 1 day after 1 day of nutrient starvation. The immunoblot demonstrates that 3-MA markedly increases the phosphorylation of Erk1/2 as shown in the upper panel. *< 0.01, **< 0.05.

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Deficiency of p66Shc increases apoptosis resistance by nutrient deprivation

Nutrient deprivation is one of the inducers of apoptosis in cultured cells. We further examined the issue of whether caspases pathways could be involved in p66Shc knockdown A549 cells. The nutrient deprivation-induced apoptosis detected by breakdown of DNA fragments and released histones was abrogated by nutrient deprivation (Fig. 1D). Moreover, we also noticed that A549 cells in this work bear an activating mutation in k-Ras (Fig. S1). Notably, nutrient deprivation inhibited cleavage of caspase 7 and poly(ADP-ribose) polymerase (PARP) by knockdown of p66Shc (Fig. 4A,B). However, nutrient deprivation showed no significant effect on cleavage of caspase 6 and 9 despite p66Shc absence (Fig. 4C), indicating that p66Shc-associated apoptosis is mediated by a nutrient-deprivation-independent pathway. These data suggest that p66Shc is a positive regulator of nutrient-deprivation-induced autophagy in A549 cells.

image

Figure 4. p66Shc knockdown increases apoptosis resistance by nutrient starvation in A549 cells. (A) A549 cells were infected with control or p66Shc shRNA, following nutrient starvation for the indicated time. Cleaved caspase 7 and PARP were immunoblotted with β-actin as a loading control. (B) Cleaved caspase 7 and PARP protein levels shown in (A) were determined by densitometric analysis of the protein bands on western blots and normalized to β-actin value at 0 days of treatment. (C) Cleaved caspase 6 and 9 were immunoblotted as measured in (A). Experiments are representative of three independent blots with similar results.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

In this study, we found that a deficiency of p66Shc mitigates the low-nutrient-induced process of autophagy in lung cancer A549 cells through prolonged activation of Erk1/2 (Thr202/Tyr204), but not phosphorylated Akt1 (Ser473). Moreover, sustained autophagy by silencing p66Shc results in decreased proliferation and enhanced apoptotic resistance. Recent work has indicated that feedback between p66Shc and Nrf2, a stress-related transcription factor, increases cancer cell survival, and tumor progression [14]. We propose that finetuned autophagic survival and apoptotic resistance induced by nutrient deprivation are regulated by adaptor protein p66Shc. This scenario is supported by several of our observations.

First, deficiency of p66Shc mitigates, but does not completely inhibit, nutrient-deprivation-induced autophagy (Fig. 2A,C). It has been reported that p66Shc knockout mice gain a prolonged lifespan by decreasing the endogenous reactive oxygen species with a normal phenotype [9]. It is also well known that malignant tumor cells struggle to survive in a metabolically stressed microenvironment such as nutrient deprivation, hypoxia and low pH. p66Shc translocates to mitochondria and increases mitochondrial membrane permeability by increased oxidative stress, and thus plays an important role in the mitochondria-dependent oxidative balance [15]. We showed nutrient-deprivation-induced p66Shc expression (Fig. 1A–C), which is connected to reactive oxygen species production. By contrast, silencing p66Shc decreases cell proliferation (Fig. 1E) [10]. In this case, as a mediator of autophagy and apoptosis, downregulation of p66Shc in cancer cells may delay autophagy and benefit cell survival under stress conditions.

Second, we show that phosphorylation of Erk1/2 (Thr202/Tyr204), but not phosphorylated Akt1 (Ser473), is sustained in p66Shc knockdown cells by nutrient deprivation (Fig. 3A–D). In normal epithelial cells, Erk activity is transient and returns to basal levels within hours upon growth factor stimulation. However, there is controversial evidence to suggest that levels of Erk1/2 phosphorylation in tumor cells are very variable. In human cancer cells, prolonged Erk1/2 activity can promote cell death by apoptosis or autophagy [16]. However, a recent study shows that the activity of Erk2, but not Erk1, is specifically related to cell proliferation and epithelial-mesenchymal transition (EMT) [17]. Therefore, loss of p66Shc further inhibited the proliferation of nutrient-deprived A549 cells through the activation of long-term autophagic signaling pathways, which has greater influence than the activation of proliferative signals. These results also show p66Shc as a key regulator and balance point for autophagic cell death and survival under long-term nutrient deprivation. The most prominent regulator of autophagy is the mTOR pathway, which acts downstream of Akt activation. High nutrient levels and growth factor stimulation lead to activation of mTOR, which in turn inhibits autophagy by phosphorylation, thus inactivating a complex containing the two kinases uncoordinated family member-51–like kinase 1 or 2 (ULK1 or ULK2), as well as autophagy-related 1 [18]. Akt plays a significant role in cAMP response element-binding protein phosphorylation and activation, leading to improved cell survival [19]. The protein kinase Akt positively regulates the activity of the mTORC1 complex by phosphorylating and inhibiting TSC2 and the proline-rich Akt substrate of 40 kDa. Thus, Akt inhibition decreases mTORC1 activity and promotes autophagy [20]. Inhibition of the Akt pathway or mTOR by physiological or pharmacological means activates autophagy by derepressing ULK1 or ULK2 and their downstream targets in the Beclin 1 complex [21]. However, activation of Akt also leads to multiple inhibitory effects on the apoptotic machinery such as phosphorylation of Erk1/2 as well as inhibition of Forkhead transcription factors [22]. Therefore, cross-regulation of ERK activation by AKT pathways is inhibitory or stimulating, depending on cell types and ligands [23]. Recent studies also showed that bifaceted oncogenic Ras functions on autophagy for tumorigenic progression. Oncogenic Ras induces autophagy and autophagic cell death by upregulation of the BH3-only protein NOXA [24]. Interestingly, another study showed that oncogenic Ras-driven tumors become addicted to autophagy to maintain healthy mitochondria [25]. However, Ras signaling can also exert proneoplastic effects through downregulation of Beclin 1, thus, suppressing autophagy pathways [26]. Importantly, A549 cells are fully transformed with an activating mutation in k-Ras (Fig. S1), possibly explaining k-Ras-driven autophagy by nutrient deprivation. We previously reported that p66Shc restrictes k-Ras activition in primary human lung epithelial cells [10], and downregulation of p66Shc expression may not show its inhibition effect of oncogenic k-Ras signaling in A549 cells.

Third, silencing p66Shc increases apoptosis resistance by nutrient deprivation. During nutrient deprivation, an autophagy-inducing stimulus, reactive oxygen species-induced DNA damage activates PARP, leading to ATP depletion [27] or DNA repair [28]. Nutrient deprivation also disrupts the Bim–Beclin 1 interaction by dissociating dynein light chain 1 (DYNLL1/LC8) and Bim, which is phosphorylated and activated by c-Jun N-terminal kinase (JNK) and leads to apoptosis [29, 30]. Based on caspase analysis, we found that the cleaved caspase 7 and cleaved PARP were inhibited by p66Shc depletion and low-nutrient treatment (Fig. 4A,B). By contrast, cleavage of caspase 6 and caspase 9 (Asp315) is reduced independent of silencing p66Shc after 1 day of low fetal bovine serum, but increases again at 3 days (Fig. 4C). It has also been reported that caspases can cleave Beclin 1, thereby destroying its proautophagic activity and mediating mitochondrion-mediated apoptosis by Bim [31]. However, long-term co-downregulation of caspase 3/7 enhances the induction of autophagy [32]. In addition, class III phosphatidylinositol 3-kinase is a substrate of caspase 7, demonstrating complicated cross-talk between apoptosis and autophagy [33], which are likely exploitable for cancer therapy [34]. Interestingly, downregulation of caspase 7 by p66Shc knockdown was sustained for 3 days with nutrient deprivation (Fig. 4A,B), suggesting that an alternative cell death pathway other than apoptosis may occur.

In summary, our data show that the adaptor protein p66Shc acts as a positive regulator of nutrient-deprivation-induced autophagy process, which also explains how loss of p66Shc in human lung cancers underlies the onset of apoptotic resistance. Owing to the relevant role of p66Shc in nutrient deprivation, we reasoned that the assessment of p66Shc expression in lung cancer specimens can be a valuable prognostic biomarker for the risk of lung cancer progression and the enforced function of p66Shc might provide a potential intervention strategy for lung cancer treatment.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Reagents and immunoblot

Bafilomycin A1 and 3-MA were purchased from Sigma-Aldrich (St. Louis, MO, USA). Blots were performed for LC3B (Sigma-Aldrich), phospho-Akt1 (Ser473) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-Erk1/2 (Thr202/Tyr204), phospho-p38 MAPK (Thr180/Tyr182), p62, cleaved caspase antibody sampler kit (Cell Signaling, Beverly, MA, USA), ShcA (BD Biosciences, San Jose, CA, USA) or horseradish peroxidase-conjugated secondary antibodies (Bio-Rad, Hercules, CA, USA), followed by ECL detection reagents (GE Healthcare, Piscataway, NJ, USA). Blots were stripped and reprobed for total Erk, Akt1 (Cell Signaling), or β-actin (Chemicon, Temecula, CA, USA). RFP–LC3B was a gift from Lance Terada (UT Southwestern Medical Center at Dallas, TX, USA).

Cell culture and viral infection

Phoenix-293 and A549 cells were obtained from American Type Culture Collection (Manassas, VA, USA). Autophagy was induced in cells by culturing them in low serum (0.1%)-containing medium with amino acid deprivation. A549 cells were grown to 60–80% confluence prior to lentiviral transduction. For lentiviral transduction, phoenix-293 cells were cotransfected with the transfer constructs and the third-generation packaging plasmids pMD2.VSVG, pMDLg/pRRE and pRSV–REV, and fresh supernatant was used for infection. After 8 h infection, A549 cells were washed and allowed to recover for 24 h prior to further procedure. Target sequences for human p66Shc (shRNA(1) nucleotides 42–60 and shRNA(2) nucleotides 252–270, respectively) were used to create oligonucleotides creating shRNA loops at a multiplicity of infection of 100 for 16 h as described previously [10, 35].

Ras GTPase activity

Ras activation was performed using a pulldown technique as described previously [10]. Briefly, the Ras-binding domain (RBD) of Raf fused to GST (Raf–GST) was expressed in BL21–RP Escherichia coli (Stratagene, La Jolla, CA, USA) and affinity-purified on GSH–Sepharose (GE Healthcare). A549 cell lysate was centrifuged at 10 000 g; two-thirds of the supernatants were used for pulldown with the respective fusion and immunoblotted for k-Ras, and one-third of the lysate was precipitated in ice-cold acetone (1 : 1) for assessment of total k-Ras.

Microscopy

Cells were plated on 35-mm coverslip-bottom dishes coated with fibronectin. RFP chromophores were excited with 488 nm (argon) or 543 nm (HeNe) laser lines using fluorescence microscope (Nikon Inc., Melville, New York, USA). Images were obtained with a CCD camera (Coolsnap ES, Roper Scientific, Martinsried, Germany) using metamorph software (Molecular Devices, Sunnyvale, CA, USA). p66Shc knockdown A549 cells were transfected with RFP–LC3B and RFP–LC3B fluorescent intensity was measured as previously described [35]. At least 50 cells from more than 10 fields were counted for statistical analysis.

Cell death and proliferation assays

Appropriate culture medium containing 10% fetal bovine serum or 0.1% fetal bovine serum with amino acids deprivation for nutrient deprivation was added and cells were allowed to proliferate for 24 h. All cells were harvested (nonadherent and adherent) and washed with NaCl/Pi. Cell death was assessed as DNA fragmentation using a cell death enzyme-linked immunosorbent assay (Roche Diagnostics GmbH, Mannheim, Germany). Absorbance was normalized for cell number. Proliferation was assessed using a BrdU incorporation enzyme-linked immunosorbent assay (Roche), again normalized for cell number.

Statistical analysis

Data are expressed as mean ± SEM from at least three independent experiments. Statistical analysis was performed with ANOVA for multiple variables and with t-tests for comparison of two groups with normal distribution.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

This project has been supported in part by National Natural Science Foundation of China (No. 81071730, 91019012 and 31071128), the Tianjin Municipal Science and Technology Commission (No. 11JCZDJC18700, 11JCZDJC19000, and 13JCQNJC11300), specialized Fund for the Doctoral Program of Higher Education (No. 20121202110001), and the Ministry of Science and Technology of China (No. 2009CB918903).

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  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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febs12416-sup-0001-FigS1.zipZip archive216K

Fig. S1. Nutrient starvation does not change k-Ras activity in A549 cells. p66Shc knockdown A549 cells as well as the control cells were nutrient starved for 0, 8 or 24 h. The cell lysates were used for Raf-GST pull down and active k-Ras was immunoblotted as described in the Experimental procedures.

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