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PTEN is a protein tyrosine phosphatase for IRS1

Nature Structural & Molecular Biology volume 21, pages 522–527 (2014)Cite this article

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Abstract

The biological function of the PTEN tumor suppressor is mainly attributed to its lipid phosphatase activity. This study demonstrates that mammalian PTEN is a protein tyrosine phosphatase that selectively dephosphorylates insulin receptor substrate-1 (IRS1), a mediator of insulin and IGF signals. IGF signaling was defective in cells lacking NEDD4, a PTEN ubiquitin ligase, whereas AKT activation triggered by EGF or serum was unimpaired. Defective IGF signaling caused by NEDD4 deletion, including phosphorylation of IRS1 and AKT, was rescued by PTEN ablation. We demonstrate the nature of PTEN as an IRS1 phosphatase by direct biochemical analysis and cellular reconstitution, showing that NEDD4 supports insulin-mediated glucose metabolism and is required for the proliferation of IGF1 receptor–dependent but not EGF receptor–dependent tumor cells. Thus, PTEN is a protein phosphatase for IRS1, and its antagonism by NEDD4 promotes signaling by IGF and insulin.

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Figure 1: NEDD4 specifically regulates signaling by IGF and insulin but not by EGF or serum.
Figure 2: NEDD4 functions through PTEN in the IGF1 signaling pathway.
Figure 3: PTEN is a protein phosphatase for IRS1 in vitro.
Figure 4: PTEN is a protein phosphatase for IRS1 in cells.
Figure 5: NEDD4 is required for growth of IGF1R-dependent tumor cells.
Figure 6: NEDD4 regulates insulin-induced glucose metabolism.
Figure 7: The IGF but not the EGF signaling pathway is regulated by the NEDD4-PTEN circuitry.

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Acknowledgements

We thank B. Yang (University of Iowa) for providing NEDD4−/− MEFs and paired NEDD4+/+ MEFs, J. Lee for excellent technical support and N. Pavletich for discussing the structural basis of the defects of various PTEN mutants. We also thank members of Jiang laboratory and D. Marks for discussing and reading the manuscript. This work is supported by an American Cancer Society scholar award (to X.J.) and by funding from Mr. William H. and Mrs. Alice Goodwin and the Commonwealth Foundation for Cancer Research of the Experimental Therapeutics Center of Memorial Sloan-Kettering Cancer Center (to X.J.) and the Geoffrey Beene Cancer Research foundation (to X.J., N.R. and S.C.).

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Authors and Affiliations

  1. Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA

    Yuji Shi, Junru Wang & Xuejun Jiang

  2. Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA

    Sarat Chandarlapaty

  3. Cancer Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA

    Justin Cross & Craig Thompson

  4. Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA

    Neal Rosen

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Contributions

Y.S., N.R. and X.J. designed the study; Y.S., J.W. and J.C. performed the experiments; Y.S., S.C., N.R. and X.J. wrote the paper; and all authors were involved in data analysis and interpretation.

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Correspondence to Xuejun Jiang.

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Integrated supplementary information

Supplementary Figure 1 NEDD4 specifically regulates IGF but not EGF or serum signaling.

(a) Western blotting analysis of (AKT phosphorylation) upon different stimuli. NEDD4+/+ and NEDD4-/- MEFs were serum starved for 3 hrs, followed by 5 min stimulation with 10% serum, IGF1 (50 ng/ml), or 50 ng/ml, 100 ng/ml IGF2. β-actin is used as a loading/sample preparation control. (b) Western blotting analysis similar as in (a), with different amounts of EGF; EGFR phosphorylation was also assessed. γ-tubulin is used as loading control. (c) Western blots analyses of time course of the effect of NEDD4 elimination on insulin signaling. MEFs harboring Dox-inducible NEDD4 shRNA construct were treated with or without 1 μg/ml Dox for 3 days, serum-starved for 3 hrs, and then stimulated for the indicated amount of time with insulin (100 ng/ml).

Supplementary Figure 2 Characterization of recombinant PTEN protein in lipid and protein phosphatase assays.

(a) WT PTEN but not the CS or GE mutant dephosphorylates PIP3. The assay was performed as described in Methods. 40 μM PIP3 and 0.1 μM PTEN (WT), 10 μM PTEN (CS) and 10 μM PTEN (GE) proteins were used in the assay. WT: wild-type; CS: C124S mutant; GE: G129E mutant. (b) Coomassie blue gel showing purified recombinant PTEN proteins. YL: Y138L mutant. (c) WT-PTEN or GE but not YL or CS mutant can dephosphorylate IRS1. Immunoprecipitated IRS1 was incubated with recombinant PTEN (WT, YL GE or CS mutant) protein (0.6 μM) for 1 hr at 30 °C. (d) Comparing PTEN (WT or mutants) activity in lipid phosphatase assay. The assay was performed similar as in (a), with 40 μM PIP3 and PTEN (WT/YL/CS/GE) proteins of indicated concentrations. (e) Comparison of PTP1B and PTEN activity in the in vitro protein phosphatase assays. The assay was performed similar as in (c), with substrates (IGF1R and IRS1) and enzymes (PTP1B and PTEN) as indicated, and quantitated by densitometry.

Supplementary Figure 3 WT and GE mutant but not CS mutant of PTEN can dephosphorylate IRS1 in cells.

(a)Validation of PTEN shRNA MEFs reconstituted with equal amount of GFP-S-tagged PTEN (WT/CS/GE). (b-e) The experiments were performed the same as that in Figure 4 except that both p-IRS1 (Y608) and p-IRS1 (Y989) were monitored here. (f) PI3K inhibitors block IGF1-induced phosphorylation of AKT but not that of IGF1R or IRS1. WT MEFs were pretreated for 1 hr with either 2 μM BYL-719 or 1 μM GDC0941, followed by 50 ng/ml IGF1 for 10 min.

Supplementary Figure 4 PTEN and AKT regulate IGF1R expression via feedback mechanism.

(a) PTEN-/- MEFs express lower level of IGF1R which can be increased by AKT inhibition. WT or PTEN-/- MEFs were treated with 1 μM AKTi for 12 or 24 hrs. γ-tubulin is used as loading control. (b) MEFs harboring long-term PTEN shRNA have a decreased IGF1R expression, which can be reversed by AKT inhibition. β-actin is used as loading control. (c) Expression of wild-type but not CS or GE mutant PTEN increased IGF1R expression in PTEN-/- MEFs. PTEN-/- MEFs were reconstituted with GFP or GFP-S-PTEN (WT/CS/GE), and treated with or without 1 μM AKTi for 24 hrs. (d) Stable PTEN shRNA restored IGF1-induced AKT activation in NEDD4-/- MEFs but had minimal effect on IRS1 phosphorylation. NEDD4-/- MEFs with control knockdown or stable PTEN knockdown were serum-starved, and then stimulated with 50 ng/ml IGF1 for 5 min, with or without 1 μM PI3K inhibitor

Supplementary Figure 5 NEDD4 is required for AKT activity in MCF7 but not MDA-MB-468 cells.

(a) The effect of IGF1R inhibitor, EGFR inhibitor on AKT activity in MCF7 and MDA-MB-468 cells. MCF7 and MDA-MB-468 cells were treated with either 5 μM IGF1Ri or 5 μM EGFRi for 3 hrs. γ-tubulin is used as loading control. (b) The effect of NEDD4 RNAi on AKT activity in MCF7 and MDA-MB-468 cells. MCF7 and MDA-MB-468 cells harboring Dox-inducible NEDD4 shRNA construct were treated with or without 1 μg/ml Dox for 3 days

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Shi, Y., Wang, J., Chandarlapaty, S. et al. PTEN is a protein tyrosine phosphatase for IRS1. Nat Struct Mol Biol 21, 522–527 (2014). https://doi.org/10.1038/nsmb.2828

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