Despite the structural conservation of PTEN with dual-specificity phosphatases, there have been no reports regarding the regulatory mechanisms that underlie this potential dual-phosphatase activity. Here, we report that K27-linked polyubiquitination of PTEN at lysines 66 and 80 switches its phosphoinositide/protein tyrosine phosphatase activity to protein serine/threonine phosphatase activity. Mechanistically, high glucose, TGF-β, CTGF, SHH, and IL-6 induce the expression of a long non-coding RNA, GAEA (Glucose Aroused for EMT Activation), which associates with an RNA-binding E3 ligase, MEX3C, and enhances its enzymatic activity, leading to the K27-linked polyubiquitination of PTEN. The MEX3C-catalyzed PTENK27-polyUb activates its protein serine/threonine phosphatase activity and inhibits its phosphatidylinositol/protein tyrosine phosphatase activity. With this altered enzymatic activity, PTENK27-polyUb dephosphorylates the phosphoserine/threonine residues of TWIST1, SNAI1, and YAP1, leading to accumulation of these master regulators of EMT. Animals with genetic inhibition of PTENK27-polyUb, by a single nucleotide mutation generated using CRISPR/Cas9 (PtenK80R/K80R), exhibit inhibition of EMT markers during mammary gland morphogenesis in pregnancy/lactation and during cutaneous wound healing processes. Our findings illustrate an unexpected paradigm in which the lncRNA-dependent switch in PTEN protein serine/threonine phosphatase activity is important for physiological homeostasis and disease development.
Access optionsAccess options
Subscribe to Journal
Get full journal access for 1 year
We are sorry, but there is no personal subscription option available for your country.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Song, M. S., Salmena, L. & Pandolfi, P. P. The functions and regulation of the PTEN tumour suppressor. Nat. Rev. Mol. Cell Biol. 13, 283–296 (2012).
Denu, J. M. & Dixon, J. E. A catalytic mechanism for the dual-specific phosphatases. Proc. Natl Acad. Sci. USA 92, 5910–5914 (1995).
Lee, J. O. et al. Crystal structure of the PTEN tumor suppressor: implications for its phosphoinositide phosphatase activity and membrane association. Cell 99, 323–334 (1999).
Maehama, T. & Dixon, J. E. The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate. J. Biol. Chem. 273, 13375–13378 (1998).
Tamura, M. et al. Inhibition of cell migration, spreading, and focal adhesions by tumor suppressor PTEN. Science 280, 1614–1617 (1998).
Blanco-Aparicio, C., Renner, O., Leal, J. F. & Carnero, A. PTEN, more than the AKT pathway. Carcinogenesis 28, 1379–1386 (2007).
Robinson, V. L. Rethinking the central dogma: noncoding RNAs are biologically relevant. Urol. Oncol. 27, 304–306 (2009).
Guttman, M. et al. Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals. Nature 458, 223–227 (2009). nature07672 [pii].
Hrdlickova, B., de Almeida, R. C., Borek, Z. & Withoff, S. Genetic variation in the non-coding genome: Involvement of micro-RNAs and long non-coding RNAs in disease. Biochim. Biophys. Acta 1842, 1910–1922 (2014).
St Laurent, G., Wahlestedt, C. & Kapranov, P. The Landscape of long noncoding RNA classification. Trends Genet. 31, 1910–1922 (2014).
Wilusz, J. E., Sunwoo, H. & Spector, D. L. Long noncoding RNAs: functional surprises from the RNA world. Genes Dev. 23, 1494–1504 (2009). 23/13/1494 [pii].
Johnsson, P., Lipovich, L., Grander, D. & Morris, K. V. Evolutionary conservation of long non-coding RNAs; sequence, structure, function. Biochim. Biophys. Acta 1840, 1063–1071, (2014).
Stone, R. C. et al. Epithelial-mesenchymal transition in tissue repair and fibrosis. Cell Tissue Res. 365, 495–506, (2016).
Kalluri, R. & Neilson, E. G. Epithelial-mesenchymal transition and its implications for fibrosis. J. Clin. Invest. 112, 1776–1784, (2003).
Kalluri, R. & Weinberg, R. A. The basics of epithelial-mesenchymal transition. J. Clin. Invest. 119, 1420–1428, (2009).
Fischer, K. R. et al. Epithelial-to-mesenchymal transition is not required for lung metastasis but contributes to chemoresistance. Nature 527, 472–476, (2015).
Okubo, T. et al. Down-regulation of promoter 1.3 activity of the human aromatase gene in breast tissue by zinc-finger protein, snail (SnaH). Cancer Res. 61, 1338–1346 (2001).
Masur, K. et al. Diabetogenic glucose and insulin concentrations modulate transcriptome and protein levels involved in tumour cell migration, adhesion and proliferation. Br. J. Cancer 104, 345–352, (2011).
Saville, M. K. et al. Regulation of p53 by the ubiquitin-conjugating enzymes UbcH5B/C in vivo. J. Biol. Chem. 279, 42169–42181, (2004).
Katoh, S., Tsunoda, Y., Murata, K., Minami, E. & Katoh, E. Active site residues and amino acid specificity of the ubiquitin carrier protein-binding RING-H2 finger domain. J. Biol. Chem. 280, 41015–41024, (2005).
Polge, C. et al. UBE2B is implicated in myofibrillar protein loss in catabolic C2C12 myotubes. J. Cachexia Sarcopenia Muscle 7, 377–387, (2016).
Zhang, L., Xu, M., Scotti, E., Chen, Z. J. & Tontonoz, P. Both K63 and K48 ubiquitin linkages signal lysosomal degradation of the LDL receptor. J. Lipid Res. 54, 1410–1420, (2013).
Wu, T. et al. UBE2S drives elongation of K11-linked ubiquitin chains by the anaphase-promoting complex. Proc. Natl Acad. Sci. USA 107, 1355–1360, (2010).
Dandona, P., Chaudhuri, A., Ghanim, H. & Mohanty, P. Proinflammatory effects of glucose and anti-inflammatory effect of insulin: relevance to cardiovascular disease. Am. J. Cardiol. 99, 15B–26B, (2007).
Lamouille, S., Xu, J. & Derynck, R. Molecular mechanisms of epithelial-mesenchymal transition. Nat. Rev. Mol. Cell Biol. 15, 178–196, (2014).
Sonnylal, S. et al. Connective tissue growth factor causes EMT-like cell fate changes in vivo and in vitro. J. Cell. Sci. 126, 2164–2175, (2013).
Yoo, Y. A. et al. Sonic hedgehog pathway promotes metastasis and lymphangiogenesis via activation of Akt, EMT, and MMP-9 pathway in gastric cancer. Cancer Res. 71, 7061–7070, (2011).
Sullivan, N. J. et al. Interleukin-6 induces an epithelial-mesenchymal transition phenotype in human breast cancer cells. Oncogene 28, 2940–2947 (2009).
Chan, D. A. et al. Targeting GLUT1 and the Warburg effect in renal cell carcinoma by chemical synthetic lethality. Sci. Transl. Med. 3, 94ra70, (2011).
Biswas, S. et al. Endothelin-1 regulation is entangled in a complex web of epigenetic mechanisms in diabetes. Physiol. Res. 67, S115–S125 (2018).
Zhang, Z. Y. et al. Substrate specificity of the protein tyrosine phosphatases. Proc. Natl Acad. Sci. USA 90, 4446–4450 (1993).
Donella Deana, A. et al. An investigation of the substrate specificity of protein phosphatase 2C using synthetic peptide substrates; comparison with protein phosphatase 2A. Biochim. Biophys. Acta 1051, 199–202 (1990).
Pinna, L. A. & Donella-Deana, A. Phosphorylated synthetic peptides as tools for studying protein phosphatases. Biochim. Biophys. Acta 1222, 415–431 (1994).
Brown-Shimer, S. et al. Molecular cloning and chromosome mapping of the human gene encoding protein phosphotyrosyl phosphatase 1B. Proc. Natl Acad. Sci. USA 87, 5148–5152 (1990).
Stone, S. R. et al. The nucleotide sequence of the cDNA encoding the human lung protein phosphatase 2A alpha catalytic subunit. Nucleic Acids Res. 16, 11365 (1988).
Stevenson, L. F. et al. The deubiquitinating enzyme USP2a regulates the p53 pathway by targeting Mdm2. EMBO J. 26, 976–986, (2007).
Camps, M., Nichols, A. & Arkinstall, S. Dual specificity phosphatases: a gene family for control of MAP kinase function. FASEB J. 14, 6–16 (2000).
Leslie, N. R. & Downes, C. P. PTEN function: how normal cells control it and tumour cells lose it. Biochem. J. 382, 1–11 (2004).
Xu, Y. et al. Structure of the protein phosphatase 2A holoenzyme. Cell 127, 1239–1251 (2006).
Myers, M. P. et al. The lipid phosphatase activity of PTEN is critical for its tumor supressor function. Proc. Natl Acad. Sci. USA 95, 13513–13518 (1998).
Shi, Y. et al. PTEN is a protein tyrosine phosphatase for IRS1. Nat. Struct. Mol. Biol. 21, 522–527, (2014).
Davidson, L. et al. Suppression of cellular proliferation and invasion by the concerted lipid and protein phosphatase activities of PTEN. Oncogene 29, 687–697, (2010).
Lander, R. et al. Interactions between Twist and other core epithelial-mesenchymal transition factors are controlled by GSK3-mediated phosphorylation. Nat. Commun. 4, 1542, (2013).
Zhou, B. P. et al. Dual regulation of snail by GSK-3beta-mediated phosphorylation in control of epithelial-mesenchymal transition. Nat. Cell Biol. 6, 931–940, (2004).
Levy, D., Adamovich, Y., Reuven, N. & Shaul, Y. Yap1 phosphorylation by c-Abl is a critical step in selective activation of proapoptotic genes in response to DNA damage. Mol. Cell 29, 350–361, (2008).
Basu, S., Totty, N. F., Irwin, M. S. & Sudol, M. & Downward, J. Akt phosphorylates the Yes-associated protein, YAP, to induce interaction with 14-3-3 and attenuation of p73-mediated apoptosis. Mol. Cell 11, 11–23 (2003).
Yang, J. & Weinberg, R. A. Epithelial-mesenchymal transition: at the crossroads of development and tumor metastasis. Dev. Cell 14, 818–829 (2008).
Wang, W. et al. AMPK modulates Hippo pathway activity to regulate energy homeostasis. Nat. Cell Biol. 17, 490–499, (2015).
Yang, H. et al. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154, 1370–1379 (2013).
Kalluri, R. EMT: when epithelial cells decide to become mesenchymal-like cells. J. Clin. Invest. 119, 1417–1419, (2009).
Ganguli-Indra, G. Protocol for cutaneous wound healing assay in a murine model. Methods Mol. Biol. 1210, 151–159, (2014).
Nguyen, K. T. et al. Essential role of Pten in body size determination and pancreatic beta-cell homeostasis in vivo. Mol. Cell Biol. 26, 4511–4518, (2006).
Wang, Z. et al. Pten regulates development and lactation in the mammary glands of dairy cows. PLoS ONE 9, e102118, (2014).
Zhao, M. PTEN: a promising pharmacological target to enhance epithelial wound healing. Br. J. Pharmacol. 152, 1141–1144, (2007).
Miyoshi, K. et al. Epithelial Pten controls acute lung injury and fibrosis by regulating alveolar epithelial cell integrity. Am. J. Respir. Crit. Care Med. 187, 262–275, (2013).
Salmena, L., Carracedo, A. & Pandolfi, P. P. Tenets of PTEN tumor suppression. Cell 133, 403–414 (2008).
Holt, L. J. Regulatory modules: Coupling protein stability to phopshoregulation during cell division. FEBS Lett. 586, 2773–2777 (2012).
Yang, J. et al. Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell 117, 927–939 (2004).
Dang, C. V. MYC on the path to cancer. Cell 149, 22–35 (2012).
Wang, Y., Shi, J., Chai, K., Ying, X. & Zhou, B. P. The Role of Snail in EMT and Tumorigenesis. Curr. Cancer Drug Targets 13, 963–972 (2013).
Urata, Y. N., Takeshita, F., Tanaka, H., Ochiya, T. & Takimoto, M. Targeted knockdown of the kinetochore protein D40/Knl-1 inhibits human cancer in a p53 status-independent manner. Sci. Rep. 5, 13676 (2015).
Clevenger, C. V. Role of prolactin/prolactin receptor signaling in human breast cancer. Breast Dis. 18, 75–86 (2003).
Ayala, I. et al. Faciogenital dysplasia protein Fgd1 regulates invadopodia biogenesis and extracellular matrix degradation and is up-regulated in prostate and breast cancer. Cancer Res. 69, 747–752, (2009).
Keyomarsi, K., Tucker, S. L. & Bedrosian, I. Cyclin E is a more powerful predictor of breast cancer outcome than proliferation. Nat. Med. 9, 152, (2003).
Matise, M. P. & Joyner, A. L. Gli genes in development and cancer. Oncogene 18, 7852–7859, (1999).
Tamura, M., Gu, J., Takino, T. & Yamada, K. M. Tumor suppressor PTEN inhibition of cell invasion, migration, and growth: differential involvement of focal adhesion kinase and p130Cas. Cancer Res. 59, 442–449 (1999).
Ghosh, S., Varela, L., Sood, A., Park, B. H. & Lotan, T. L. mTOR signaling feedback modulates mammary epithelial differentiation and restrains invasion downstream of PTEN loss. Cancer Res. 73, 5218–5231, (2013).
Hutchinson, J. N., Jin, J., Cardiff, R. D., Woodgett, J. R. & Muller, W. J. Activation of Akt-1 (PKB-alpha) can accelerate ErbB-2-mediated mammary tumorigenesis but suppresses tumor invasion. Cancer Res. 64, 3171–3178 (2004).
Xue, G. et al. Akt/PKB-mediated phosphorylation of Twist1 promotes tumor metastasis via mediating cross-talk between PI3K/Akt and TGF-beta signaling axes. Cancer Discov. 2, 248–259, (2012).
Li, C. W. et al. AKT1 inhibits epithelial-to-mesenchymal transition in breast cancer through phosphorylation-dependent Twist1 degradation. Cancer Res. 76, 1451–1462, (2016).
Wilkes, E. H., Terfve, C., Gribben, J. G., Saez-Rodriguez, J. & Cutillas, P. R. Empirical inference of circuitry and plasticity in a kinase signaling network. Proc. Natl Acad. Sci. USA 112, 7719–7724, (2015).
Tyanova, S., Temu, T. & Cox, J. The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat. Protoc. 11, 2301–2319 (2016).
Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805, (2011).
Wen, B., Mei, Z., Zeng, C. & Liu, S. metaX: a flexible and comprehensive software for processing metabolomics data. BMC Bioinform. 18, 183, (2017).
Xing, Z. et al. lncRNA directs cooperative epigenetic regulation downstream of chemokine signals. Cell 159, 1110–1125 (2014).
Fey, A. et al. Establishment of a real-time PCR-based approach for accurate quantification of bacterial RNA targets in water, using Salmonella as a model organism. Appl. Environ. Microbiol. 70, 3618–3623 (2004).
We thank Mr. D. Aten for assistance with figure presentation and Peter K. Park and Sergey D. Egranov for manuscript preparation. This research work is partially supported by National Cancer Institute (NCI) CPTAC award [U24 CA210954], Cancer Prevention and Research Institutes of Texas [CPRIT RR160027], McNair Medical Institute at The Robert and Janice McNair Foundation to B.Z. This work was supported in part by Cancer Prevention Research Institute of Texas (CPRIT) grant number RP130397 and NIH grant number 1S10OD012304-01 to D.H.H. This work was supported by National Institutes of Health Pathway to Independence Award (R00CA166527), National Cancer Institute R01 award (1 R01 CA218036-01), Cancer Prevention Research Institute of Texas First-time Faculty Recruitment Award (R1218) grants, Department of Defense Breakthrough award (BC151465), Andrew Sabin Family Foundation Fellows award, and AACR-Bayer Innovation and Discovery Grants to L.Q.Y. and National Institutes of Health Pathway to Independence Award (R00DK094981), National Cancer Institute R01 award (1R01CA218025-01, 1R01CA231011-01), Department of Defense Breakthrough award BC180196, and Cancer Prevention Research Institute of Texas Individual Investigator Research Award (150094 and 180259) to C.R.L.
About this article
Nature Immunology (2019)