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The Phosphatase PP2A Links Glutamine to the Tumor Suppressor p53
Molecular Cell
Previews The Phosphatase PP2A Links Glutamine to the Tumor Suppressor p53 Dana Gwinn1 and E. Alejandro Sweet-Cordero1,* 1Cancer Biology Program, Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2013.04.010
In this issue of Molecular Cell, Reid et al. (2013) show that glutamine withdrawal causes PP2A-mediated activation of p53 through its regulator EDD, linking levels of a critical metabolite to an important regulator of cell survival and proliferation. The amino acid glutamine plays several important roles in the metabolism of mammalian cells: it can donate nitrogen for the synthesis of purines and pyrimidines, and in the process is converted to glutamate by glutaminase, which can then act as a nitrogen donor for the synthesis of nonessential amino acids (Wise and Thompson, 2010). Glutamate can then be converted into a-ketoglutarate and thereby enter the tricarboxylic acid (TCA) cycle, or it can be converted to glutamylcysteine for glutathione (GSH) biosynthesis, which participates in maintenance of ROS levels. TCA cycle intermediates derived from glutamine can also contribute to NADPH production, yielding pyruvate and replenishing cellular reducing equivalents (DeBerardinis et al., 2007). The importance of glutamine metabolism is underscored by the transcriptional regulation of glutaminase isoforms by several cancer-associated genes, including oncogenic c-myc (Gao et al., 2009) and the tumor suppressor p53 (Hu et al., 2010; Suzuki et al., 2010). Given its multiple roles in metabolism, it is perhaps not surprising that glutamine itself can induce cellular proliferation and differentiation through regulation of certain transcription factors such as c-myc and c-jun (Brasse-Lagnel et al., 2009). However, pathways activated specifically in response to glutamine withdrawal are not well understood. In this issue of Molecular Cell, Reid et al. (2013) describe a mechanism through which glutamine deprivation leads to activation of the transcription factor p53 through regulation of the serine/threonine protein phosphatase 2A (PP2A), thus establishing a link between glutamine levels and a key tumor suppressor pathway (Figure 1).
The PP2A complex consists of three subunits: one of two isoforms of the ‘‘C’’ catalytic subunits, one of two isoforms of the ‘‘A’’ scaffolding subunits, and one of sixteen isoforms of the ‘‘B’’ regulatory subunits (Seshacharyulu et al., 2013). The diversity of assembly of these subunits allows for the generation of more than 60 distinct heterotrimeric holoenzymes. While substrate specificity of the PP2A holoenzyme is determined by the B subunit, regulation of phosphatase activity itself can be dictated by formation of the holoenzyme. Previous work demonstrated that the C subunit can be sequestered from the A and B subunits by binding to a4 (TAP42 in yeast), which renders PP2A catalytically inactive while protecting it from proteasomal degradation (Kong et al., 2009). In this setting, deletion of a4 leads to increased phosphorylation of multiple DNA response genes, such as p53 and histone H2AX. In the present work, Reid et al. (2013) reasoned that a4 might also play a role in regulating responses to nutrient-deprivation-induced stress in mammalian cells. To evaluate a potential role for a4 in the response to nutrient deprivation, the authors assessed the ability of overexpression of a4 to protect cells against various metabolic stress conditions including glutamine, glucose, leucine, and total amino acid deprivation. Overexpression of a4 protected against apoptosis only during glutamine withdrawal, suggesting that PP2A plays a specific role in the cellular response to glutamine starvation. To identify a specific PP2A B subunit that acts during glutamine deprivation, they assessed the expression levels of each of the 16 B subunit isoforms of PP2A during glutamine deprivation and found that only
the mRNA coding for the B55a subunit was induced. Furthermore, expression of B55a was required for the survival advantage induced by a4 overexpression during glutamine deprivation. The induction of B55a expression upon glutamine deprivation could be suppressed with antioxidants, suggesting that the upregulation of B55a mRNA upon glutamine withdrawal is mediated by increases in reactive oxygen species (ROS), a well-known consequence of glutamine withdrawal. To identify relevant substrates of the PP2A-B55a complex, the authors immunoprecipitated B55a and identified the HECT domain-containing E3 ubiquitin ligase EDD, which has previously been implicated in p53 regulation (Ling and Lin, 2011), as a B55a-interacting protein. As p53 has previously been implicated in metabolic control, they then examined whether p53 is regulated upon glutamine deprivation in a B55a-EDD-dependent manner. Upon glutamine withdrawal, phosphorylation of p53 at serine 18 increased, correlating with an increase in expression of p53 target genes. Overexpression of a4 enhanced the induction of p53 phosphorylation upon glutamine deprivation in a B55a-dependent manner. Additionally, when EDD expression was knocked down, loss of B55a no longer blocked suppression of p53 phosphorylation after glutamine deprivation. Thus, EDD appears to be a negative regulator of p53 that is in turn inhibited by expression of B55a, thus allowing for increased p53 activation under conditions of glutamine withdrawal. To determine whether the response to glutamine withdrawal by B55a is relevant during oncogenesis, human fibrosarcoma cells with or without knockdown of B55a
Molecular Cell 50, April 25, 2013 ª2013 Elsevier Inc. 157
Molecular Cell
Previews
Figure 1. When Glutamine Is Present, It Is Used to Fuel the TCA Cycle and Also Contributes to ROS Homeostasis, among Other Roles Under these conditions, EDD is phosphorylated and does not exert negative regulation of p53. However, when glutamine is withdrawn, TCA cycle flux decreases and ROS accumulates. Under these conditions, the PP2A subunit, B55a, is upregulated, leading to dephosphorylation of EDD, allowing for increased p53 activity, including expression of the cell-cycle inhibitor, p21, as well as glutaminase 2 (Gls2).
were used to generate xenografts in immunocompromised mice, demonstrating that knockdown of B55a suppressed tumor growth. Furthermore, comparison of samples from the core of the xenografts with samples from the periphery demonstrated that cells in the core had lower levels of glutamine and GSH. Supporting their proposed model, the levels of B55a expression and p53 phosphorylation were elevated in the core, correlating with the lower levels of glutamine. The work of Reid et al. (2013) provides compelling evidence for a novel pathway through which glutamine withdrawal leads to p53 activation, linking levels of a critical metabolite to an important regulator of cell survival and proliferation. As this pathway responds to changes in ROS levels, it is likely to be involved in other oxidative stress responses. The work points to regulation of phosphorylation of EDD as a potential important signal integrator that deserves further investigation. For
example, it is possible that regulation of EDD kinases is also important in response to metabolic alteration. While the B55a-EDD pathway is clearly involved in p53 activation upon glutamine withdrawal, there may also be additional mechanisms to regulate p53 upon glutamine withdrawal, as p53 targets are still upregulated in glutamine deprivation in the absence of B55a. Lastly, since many tumors lose p53 function, it remains unclear how p53-deficient tumors respond to altered levels of glutamine. It will be interesting to determine to what extent across human cancers loss of p53 leads to inability to respond to acute loss of glutamine. REFERENCES Brasse-Lagnel, C., Lavoinne, A., and Husson, A. (2009). FEBS J. 276, 1826–1844. DeBerardinis, R.J., Mancuso, A., Daikhin, E., Nissim, I., Yudkoff, M., Wehrli, S., and Thompson, C.B. (2007). Proc. Natl. Acad. Sci. USA 104, 19345–19350.
158 Molecular Cell 50, April 25, 2013 ª2013 Elsevier Inc.
Gao, P., Tchernyshyov, I., Chang, T.C., Lee, Y.S., Kita, K., Ochi, T., Zeller, K.I., De Marzo, A.M., Van Eyk, J.E., Mendell, J.T., and Dang, C.V. (2009). Nature 458, 762–765. Hu, W., Zhang, C., Wu, R., Sun, Y., Levine, A., and Feng, Z. (2010). Proc. Natl. Acad. Sci. USA 107, 7455–7460. Kong, M., Ditsworth, D., Lindsten, T., and Thompson, C.B. (2009). Mol. Cell 36, 51–60. Ling, S., and Lin, W.C. (2011). J. Biol. Chem. 286, 14972–14982. Reid, M.A., Wang, W.-I., Rosales, K.R., Welliver, M.X., Pan, M., and Kong, M. (2013). Mol. Cell 50, this issue, 200–211. Seshacharyulu, P., Pandey, P., Datta, K., and Batra, S.K. (2013). Cancer Lett. Published online February 20, 2013. http://dx.doi.org/10.1016/j. canlet.2013.02.036. Suzuki, S., Tanaka, T., Poyurovsky, M.V., Nagano, H., Mayama, T., Ohkubo, S., Lokshin, M., Hosokawa, H., Nakayama, T., Suzuki, Y., et al. (2010). Proc. Natl. Acad. Sci. USA 107, 7461–7466. Wise, D.R., and Thompson, C.B. (2010). Trends Biochem. Sci. 35, 427–433.
Previews The Phosphatase PP2A Links Glutamine to the Tumor Suppressor p53 Dana Gwinn1 and E. Alejandro Sweet-Cordero1,* 1Cancer Biology Program, Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2013.04.010
In this issue of Molecular Cell, Reid et al. (2013) show that glutamine withdrawal causes PP2A-mediated activation of p53 through its regulator EDD, linking levels of a critical metabolite to an important regulator of cell survival and proliferation. The amino acid glutamine plays several important roles in the metabolism of mammalian cells: it can donate nitrogen for the synthesis of purines and pyrimidines, and in the process is converted to glutamate by glutaminase, which can then act as a nitrogen donor for the synthesis of nonessential amino acids (Wise and Thompson, 2010). Glutamate can then be converted into a-ketoglutarate and thereby enter the tricarboxylic acid (TCA) cycle, or it can be converted to glutamylcysteine for glutathione (GSH) biosynthesis, which participates in maintenance of ROS levels. TCA cycle intermediates derived from glutamine can also contribute to NADPH production, yielding pyruvate and replenishing cellular reducing equivalents (DeBerardinis et al., 2007). The importance of glutamine metabolism is underscored by the transcriptional regulation of glutaminase isoforms by several cancer-associated genes, including oncogenic c-myc (Gao et al., 2009) and the tumor suppressor p53 (Hu et al., 2010; Suzuki et al., 2010). Given its multiple roles in metabolism, it is perhaps not surprising that glutamine itself can induce cellular proliferation and differentiation through regulation of certain transcription factors such as c-myc and c-jun (Brasse-Lagnel et al., 2009). However, pathways activated specifically in response to glutamine withdrawal are not well understood. In this issue of Molecular Cell, Reid et al. (2013) describe a mechanism through which glutamine deprivation leads to activation of the transcription factor p53 through regulation of the serine/threonine protein phosphatase 2A (PP2A), thus establishing a link between glutamine levels and a key tumor suppressor pathway (Figure 1).
The PP2A complex consists of three subunits: one of two isoforms of the ‘‘C’’ catalytic subunits, one of two isoforms of the ‘‘A’’ scaffolding subunits, and one of sixteen isoforms of the ‘‘B’’ regulatory subunits (Seshacharyulu et al., 2013). The diversity of assembly of these subunits allows for the generation of more than 60 distinct heterotrimeric holoenzymes. While substrate specificity of the PP2A holoenzyme is determined by the B subunit, regulation of phosphatase activity itself can be dictated by formation of the holoenzyme. Previous work demonstrated that the C subunit can be sequestered from the A and B subunits by binding to a4 (TAP42 in yeast), which renders PP2A catalytically inactive while protecting it from proteasomal degradation (Kong et al., 2009). In this setting, deletion of a4 leads to increased phosphorylation of multiple DNA response genes, such as p53 and histone H2AX. In the present work, Reid et al. (2013) reasoned that a4 might also play a role in regulating responses to nutrient-deprivation-induced stress in mammalian cells. To evaluate a potential role for a4 in the response to nutrient deprivation, the authors assessed the ability of overexpression of a4 to protect cells against various metabolic stress conditions including glutamine, glucose, leucine, and total amino acid deprivation. Overexpression of a4 protected against apoptosis only during glutamine withdrawal, suggesting that PP2A plays a specific role in the cellular response to glutamine starvation. To identify a specific PP2A B subunit that acts during glutamine deprivation, they assessed the expression levels of each of the 16 B subunit isoforms of PP2A during glutamine deprivation and found that only
the mRNA coding for the B55a subunit was induced. Furthermore, expression of B55a was required for the survival advantage induced by a4 overexpression during glutamine deprivation. The induction of B55a expression upon glutamine deprivation could be suppressed with antioxidants, suggesting that the upregulation of B55a mRNA upon glutamine withdrawal is mediated by increases in reactive oxygen species (ROS), a well-known consequence of glutamine withdrawal. To identify relevant substrates of the PP2A-B55a complex, the authors immunoprecipitated B55a and identified the HECT domain-containing E3 ubiquitin ligase EDD, which has previously been implicated in p53 regulation (Ling and Lin, 2011), as a B55a-interacting protein. As p53 has previously been implicated in metabolic control, they then examined whether p53 is regulated upon glutamine deprivation in a B55a-EDD-dependent manner. Upon glutamine withdrawal, phosphorylation of p53 at serine 18 increased, correlating with an increase in expression of p53 target genes. Overexpression of a4 enhanced the induction of p53 phosphorylation upon glutamine deprivation in a B55a-dependent manner. Additionally, when EDD expression was knocked down, loss of B55a no longer blocked suppression of p53 phosphorylation after glutamine deprivation. Thus, EDD appears to be a negative regulator of p53 that is in turn inhibited by expression of B55a, thus allowing for increased p53 activation under conditions of glutamine withdrawal. To determine whether the response to glutamine withdrawal by B55a is relevant during oncogenesis, human fibrosarcoma cells with or without knockdown of B55a
Molecular Cell 50, April 25, 2013 ª2013 Elsevier Inc. 157
Molecular Cell
Previews
Figure 1. When Glutamine Is Present, It Is Used to Fuel the TCA Cycle and Also Contributes to ROS Homeostasis, among Other Roles Under these conditions, EDD is phosphorylated and does not exert negative regulation of p53. However, when glutamine is withdrawn, TCA cycle flux decreases and ROS accumulates. Under these conditions, the PP2A subunit, B55a, is upregulated, leading to dephosphorylation of EDD, allowing for increased p53 activity, including expression of the cell-cycle inhibitor, p21, as well as glutaminase 2 (Gls2).
were used to generate xenografts in immunocompromised mice, demonstrating that knockdown of B55a suppressed tumor growth. Furthermore, comparison of samples from the core of the xenografts with samples from the periphery demonstrated that cells in the core had lower levels of glutamine and GSH. Supporting their proposed model, the levels of B55a expression and p53 phosphorylation were elevated in the core, correlating with the lower levels of glutamine. The work of Reid et al. (2013) provides compelling evidence for a novel pathway through which glutamine withdrawal leads to p53 activation, linking levels of a critical metabolite to an important regulator of cell survival and proliferation. As this pathway responds to changes in ROS levels, it is likely to be involved in other oxidative stress responses. The work points to regulation of phosphorylation of EDD as a potential important signal integrator that deserves further investigation. For
example, it is possible that regulation of EDD kinases is also important in response to metabolic alteration. While the B55a-EDD pathway is clearly involved in p53 activation upon glutamine withdrawal, there may also be additional mechanisms to regulate p53 upon glutamine withdrawal, as p53 targets are still upregulated in glutamine deprivation in the absence of B55a. Lastly, since many tumors lose p53 function, it remains unclear how p53-deficient tumors respond to altered levels of glutamine. It will be interesting to determine to what extent across human cancers loss of p53 leads to inability to respond to acute loss of glutamine. REFERENCES Brasse-Lagnel, C., Lavoinne, A., and Husson, A. (2009). FEBS J. 276, 1826–1844. DeBerardinis, R.J., Mancuso, A., Daikhin, E., Nissim, I., Yudkoff, M., Wehrli, S., and Thompson, C.B. (2007). Proc. Natl. Acad. Sci. USA 104, 19345–19350.
158 Molecular Cell 50, April 25, 2013 ª2013 Elsevier Inc.
Gao, P., Tchernyshyov, I., Chang, T.C., Lee, Y.S., Kita, K., Ochi, T., Zeller, K.I., De Marzo, A.M., Van Eyk, J.E., Mendell, J.T., and Dang, C.V. (2009). Nature 458, 762–765. Hu, W., Zhang, C., Wu, R., Sun, Y., Levine, A., and Feng, Z. (2010). Proc. Natl. Acad. Sci. USA 107, 7455–7460. Kong, M., Ditsworth, D., Lindsten, T., and Thompson, C.B. (2009). Mol. Cell 36, 51–60. Ling, S., and Lin, W.C. (2011). J. Biol. Chem. 286, 14972–14982. Reid, M.A., Wang, W.-I., Rosales, K.R., Welliver, M.X., Pan, M., and Kong, M. (2013). Mol. Cell 50, this issue, 200–211. Seshacharyulu, P., Pandey, P., Datta, K., and Batra, S.K. (2013). Cancer Lett. Published online February 20, 2013. http://dx.doi.org/10.1016/j. canlet.2013.02.036. Suzuki, S., Tanaka, T., Poyurovsky, M.V., Nagano, H., Mayama, T., Ohkubo, S., Lokshin, M., Hosokawa, H., Nakayama, T., Suzuki, Y., et al. (2010). Proc. Natl. Acad. Sci. USA 107, 7461–7466. Wise, D.R., and Thompson, C.B. (2010). Trends Biochem. Sci. 35, 427–433.