Histidine phosphorylation, the so-called hidden phosphoproteome, is a poorly characterized post-translational modification of proteins1,2. Here we describe a role of histidine phosphorylation in tumorigenesis. Proteomic analysis of 12 tumours from an mTOR-driven hepatocellular carcinoma mouse model revealed that NME1 and NME2, the only known mammalian histidine kinases, were upregulated. Conversely, expression of the putative histidine phosphatase LHPP was downregulated specifically in the tumours. We demonstrate that LHPP is indeed a protein histidine phosphatase. Consistent with these observations, global histidine phosphorylation was significantly upregulated in the liver tumours. Sustained, hepatic expression of LHPP in the hepatocellular carcinoma mouse model reduced tumour burden and prevented the loss of liver function. Finally, in patients with hepatocellular carcinoma, low expression of LHPP correlated with increased tumour severity and reduced overall survival. Thus, LHPP is a protein histidine phosphatase and tumour suppressor, suggesting that deregulated histidine phosphorylation is oncogenic.
This is a preview of subscription content
Subscribe to Nature+
Get immediate online access to the entire Nature family of 50+ journals
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Kee, J.-M. & Muir, T. W. Chasing phosphohistidine, an elusive sibling in the phosphoamino acid family. ACS Chem. Biol. 7, 44–51 (2012)
Fuhs, S. R. et al. Monoclonal 1- and 3-phosphohistidine antibodies: new tools to study histidine phosphorylation. Cell 162, 198–210 (2015)
Llovet, J. M. et al. Hepatocellular carcinoma. Nat. Rev. Dis. Primers 2, 16018–16023 (2016)
Schulze, K. et al. Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets. Nat. Genet. 47, 505–511 (2015)
Kenerson, H. L. et al. Akt and mTORC1 have different roles during liver tumorigenesis in mice. Gastroenterology 144, 1055–1065 (2013)
Guri, Y. et al. mTORC2 promotes tumorigenesis via lipid synthesis. Cancer Cell 32, 807–823 (2017)
Yokoi, F., Hiraishi, H. & Izuhara, K. Molecular cloning of a cDNA for the human phospholysine phosphohistidine inorganic pyrophosphate phosphatase. J. Biochem. 133, 607–614 (2003)
Hiraishi, H., Yokoi, F. & Kumon, A. 3-phosphohistidine and 6-phospholysine are substrates of a 56-kDa inorganic pyrophosphatase from bovine liver. Arch. Biochem. Biophys. 349, 381–387 (1998)
Hiraishi, H., Ohmagari, T., Otsuka, Y., Yokoi, F. & Kumon, A. Purification and characterization of hepatic inorganic pyrophosphatase hydrolyzing imidodiphosphate. Arch. Biochem. Biophys. 341, 153–159 (1997)
Ek, P. et al. Identification and characterization of a mammalian 14-kDa phosphohistidine phosphatase. Eur. J. Biochem. 269, 5016–5023 (2002)
Klumpp, S. et al. Protein histidine phosphatase: a novel enzyme with potency for neuronal signaling. J. Cereb. Blood Flow Metab. 22, 1420–1424 (2002)
Panda, S. et al. Identification of PGAM5 as a mammalian protein histidine phosphatase that plays a central role to negatively regulate CD4+ T Cells. Mol. Cell 63, 457–469 (2016)
Cai, X., Srivastava, S., Surindran, S., Li, Z. & Skolnik, E. Y. Regulation of the epithelial Ca2+ channel TRPV5 by reversible histidine phosphorylation mediated by NDPK-B and PHPT1. Mol. Biol. Cell 25, 1244–1250 (2014)
Hartsough, M. T. et al. Nm23-H1 metastasis suppressor phosphorylation of kinase suppressor of Ras via a histidine protein kinase pathway. J. Biol. Chem. 277, 32389–32399 (2002)
Wagner, P. D. & Vu, N. D. Phosphorylation of ATP-citrate lyase by nucleoside diphosphate kinase. J. Biol. Chem. 270, 21758–21764 (1995)
Fuhs, S. R. & Hunter, T. pHisphorylation: the emergence of histidine phosphorylation as a reversible regulatory modification. Curr. Opin. Cell Biol. 45, 8–16 (2017)
Kee, J.-M., Villani, B., Carpenter, L. R. & Muir, T. W. Development of stable phosphohistidine analogues. J. Am. Chem. Soc. 132, 14327–14329 (2010)
Riley, N. M. & Coon, J. J. Phosphoproteomics in the age of rapid and deep proteome profiling. Anal. Chem. 88, 74–94 (2016)
Attwood, P. V., Piggott, M. J., Zu, X. L. & Besant, P. G. Focus on phosphohistidine. Amino Acids 32, 145–156 (2007)
Besant, P. G. & Attwood, P. V. Detection and analysis of protein histidine phosphorylation. Mol. Cell. Biochem. 329, 93–106 (2009)
Makowska, Z. et al. Gene expression analysis of biopsy samples reveals critical limitations of transcriptome-based molecular classifications of hepatocellular carcinoma. J. Pathol. Clin. Res. 2, 80–92 (2016)
Vogelstein, B. et al. Cancer genome landscapes. Science 339, 1546–1558 (2013)
Vijayakrishnan, J. et al. A genome-wide association study identifies risk loci for childhood acute lymphoblastic leukemia at 10q26.13 and 12q23.1. Leukemia 31, 573–579 (2017)
Lesseur, C. et al. Genome-wide association analyses identify new susceptibility loci for oral cavity and pharyngeal cancer. Nat. Genet. 48, 1544–1550 (2016)
Neff, C. D. et al. Evidence for HTR1A and LHPP as interacting genetic risk factors in major depression. Mol. Psychiatry 14, 621–630 (2009)
CONVERGE consortium. Sparse whole-genome sequencing identifies two loci for major depressive disorder. Nature 523, 588–591 (2015)
Park, I.-H., Bachmann, R., Shirazi, H. & Chen, J. Regulation of ribosomal S6 kinase 2 by mammalian target of rapamycin. J. Biol. Chem. 277, 31423–31429 (2002)
Chong, P. S. Y. et al. LEO1 is regulated by PRL-3 and mediates its oncogenic properties in acute myelogenous leukemia. Cancer Res. 74, 3043–3053 (2014)
Lo, Re, O. et al. Induction of cancer cell stemness by depletion of macrohistone H2A1 in hepatocellular carcinoma. Hepatology 67, https://doi.org/10.1002/hep.29519 (2018)
Chen, M. et al. High-Mobility Group Box 1 promotes hepatocellular carcinoma progression through miR-21-mediated matrix metalloproteinase activity. Cancer Res. 75, 1645–1656 (2015)
Kwiatkowski, D. J. et al. A mouse model of TSC1 reveals sex-dependent lethality from liver hemangiomas, and up-regulation of p70S6 kinase activity in Tsc1 null cells. Hum. Mol. Genet. 11, 525–534 (2002)
Horie, Y. et al. Hepatocyte-specific Pten deficiency results in steatohepatitis and hepatocellular carcinomas. J. Clin. Invest. 113, 1774–1783 (2004)
Postic, C. & Magnuson, M. A. DNA excision in liver by an albumin-Cre transgene occurs progressively with age. Genesis 26, 149–150 (2000)
Dephoure, N. & Gygi, S. P. A solid phase extraction-based platform for rapid phosphoproteomic analysis. Methods 54, 379–386 (2011)
Tyanova, S. et al. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat. Methods 13, 731–740 (2016)
Tusher, V. G., Tibshirani, R. & Chu, G. Significance analysis of microarrays applied to the ionizing radiation response. Proc. Natl Acad. Sci. USA 98, 5116–5121 (2001)
Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008)
Gao, J. et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci. Signal. 6, pl1 (2013)
Cerami, E. et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov. 2, 401–404 (2012)
Lek, M. et al. Analysis of protein-coding genetic variation in 60,706 humans. Nature 536, 285–291 (2016)
M.N.H. acknowledges the Louis Jeantet Foundation, the Swiss National Science Foundation, SystemsX.ch, and the European Research Council (MERiC). T.H. acknowledges USPHS grants CA080100, CA082683 and CA194584 from the NCI. T.H. is an American Cancer Society Professor, and holds the Renato Dulbecco Chair in Cancer Research. S.P. acknowledges the Swiss National Science Foundation (Ambizione grant number PZ00P3_168165).
The authors declare no competing financial interests.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, Liver weight (LW) to body weight (BW) ratio (percentage) of 20-week-old L-dKO mice (n = 9) and age-matched control mice (n = 9). ****P < 0.0001, two-sided unpaired t-test. b, mRNA expression analysis in 20-week-old L-dKO tumours (n = 3) compared to livers from age-matched control mice (n = 3). CD90 (also known as Thy1; *P = 0.0104), CD133 (also known as Prom1; *P = 0.0227), Cd44 (*P = 0.0473) and Aldh1 (also known as Aldh1a1; *P = 0.0107), two-sided unpaired t-test. Data are mean ± s.d. c, Diagram showing the scheme used for mass spectrometry-based proteomic analysis. Hepatic proteomes from control mice (n = 6) were combined and used as a pooled control against tumours (n = 12) from four different L-dKO mice. d, Graph showing number of proteins upregulated (red), downregulated (blue) and not regulated (grey) in 12 tumours from c. ANOVA-based two-sample t-test with a FDR of 2%, S0 = 1 was used to determine regulation (n = 12). e, List of kinases and phosphatases (including putative phosphatases) regulated in a minimum of 10 out of 12 tumours. f, Lhpp mRNA expression analysis in 20-week-old L-dKO tumours compared to livers from age-matched control mice (n = 4). P = 0.0022, two-sided unpaired t-test. Data are mean ± s.d.
a, LHPP protein is highly conserved in eukaryotes, from worm to human. b, Human LHPP protein shows weak homology to known human histidine phosphatases PHPT1 and PGAM5.
a, Quantification of immunoblot (from Fig. 2a) indicates reduced LHPP (***P = 0.0004) and increased NME1 (*P = 0.0254) and NME2 (*P = 0.0105) expression in tumours compared to age-matched control littermates (band intensities in each lane are normalized to intensity of corresponding calnexin protein levels), control mice (n = 4), L-dKO tumours (n = 8). P values are from a two-sided unpaired t-test. Data are mean ± s.d. b, Immunoblot analysis of liver tumour (n = 6) and non-tumour (n = 3) tissue from three independent 20-week-old L-dKO mice shows tumour-specific loss of LHPP expression. Ponceau S image of the whole blot is also included. For full scan, see Supplementary Fig. 3. c, Immunohistochemistry analyses showing increased NME1 expression in L-dKO liver compared to liver from control mice. Although L-dKO tumours displayed reduced expression of LHPP, hepatic LHPP expression in the non-tumour region was comparable to control mice. Parallel tissue sections that were processed similarly (without primary antibody) served as antibody control. Similar results were obtained in livers from 4 independent control mice and L-dKO mice. d, Immunoblot analysis of liver from control mice and L-dKO liver tumours treated with INK128 (1 mg kg−1 body weight; two intraperitonial injections in 24 h; n = 4 mice per condition, lysates from two mice are pooled into one lane). LHPP expression remains unchanged, but NME1 and NME2 expression is reduced considerably in L-dKO mice after INK128 treatment. For full scans, see Supplementary Fig. 4.
a, Diagram of NME1 and NME2 autophosphorylation on His118. The high-energy phosphate is later transferred from the histidine kinase dimer pair to the histidine on the substrate. b, CB1 cell lysate was electrophoresed and transferred onto a nitrocellulose membrane. Two sets of the membrane were treated with recombinant full-length LHPP (for 4 h) in TMD buffer (see Methods) and the other set of the membrane was treated with plain TMD buffer (pH 8.5). Immunoblotting analysis of both the membranes with 3-pHis antibody (clone SC44-1) indicates that LHPP is a protein pHis phosphatase. For full scans, see Supplementary Fig. 6. c, Immunoblotting shows no apparent change pSer/Thr or pTyr levels in CB1 cells infected with adenovirus containing LHPP compared to cells infected with RFP-only control. Similar result was observed in two independent experiments. For full scans, see Supplementary Fig. 9. d, e, Immunoblotting (d) and quantification (e) show a reduction in 3-pHis levels in SNU449 cells infected with adenovirus containing LHPP compared to control-infected cells. Similar results were observed in four independent biological experiments. For full scans, see Supplementary Fig. 8. A 50–60% confluent dish was first infected, 24 h after infection, the cells were trypsinized and 2 × 105 cells were seeded into a 6-well adherent tissue culture dish and 24 h later lysed in pHis compatible buffer (see Methods). The monoclonal antibodies used to detect 3-pHis and 1-pHis were SC44-1 and SC1-1, respectively (n = 4). **P = 0.0019, two-sided ratio paired t-test. Data are mean ± s.d. f, Colony-forming assay in CB1 cells (n = 3 biologically independent experiments) and SNU449 (n = 5 biologically independent experiments) cells shows reduced proliferation upon LHPP over expression. Twelve hours after infection, CB1 (2 × 104 per well) and SNU449 (4 × 104 per well) cells were seeded into a 6-well plate tissue culture plate and stained with crystal violet (2% crystal violet in 20% methanol) on day 7. **P = 0.0026, ****P < 0.0001, two-sided unpaired t-test. Data are mean ± s.d. See Methods for further details. g, Photomicrographs of LHPP-overexpressing CB1 cells (left) displayed reduced ability to form hepatospheres in ultralow attachment plates; n = 3 biologically independent experiments. **P = 0.0024, two-sided unpaired t-test. Data are mean ± s.d. (see Methods).
a, Left and middle, colony-forming assay in SNU449 cells shows increased proliferation upon LHPP knockdown using pooled short interfering RNA (siRNA). Then 36 h after infection, SNU449 cells were re-transfected with LHPP siRNA and 4 × 104 cells per well were seeded into a 6-well tissue culture plate and allowed to proliferate for another 3 days. At the end of the third day, cells were stained with crystal violet (2% crystal violet in 20% methanol), and similar results were obtained from five biologically independent experiments. **P = 0.0075, two-sided unpaired t-test. Data are mean ± s.d. Right, immunoblot analysis (n = 1) revealed a reduction in LHPP protein levels after siRNA transfection. Longer exposure of the LHPP blot relative to the blot in Extended Data Fig. 4d. In addition to immunobloting, a reduction in LHPP transcript levels after siRNA transfection was also confirmed in two independent experiments (data not shown). For full scans, see Supplementary Fig. 10. b, Immunoblot analysis (bottom) and quantification (top) of non-tumour and tumour liver lysates confirmed LHPP overexpression in 20-week-old AAV-LHPP-infected mice (as in Fig. 3e). For full scans, see Supplementary Fig. 11. c, Serum extracted from mice in (Fig. 3e) and analysed for ALT, AST and LDH levels shows a reduction in liver damage upon AAV-LHPP infection, unit per litre (U l−1) (control mice infected with AAV-control and AAV-LHPP virus (n = 4), L-dKO mice infected with AAV-control (n = 5) and AAV-LHPP virus (n = 8)). Data are mean ± s.d. P values are by one-way ANOVA with Tukey’s multiple-comparison test. ALT (adjusted ***P = 0.004, ****P < 0.0001), AST (adjusted **P = 0.013 for control mice versus L-dKO mice transfected with AAV-control; adjusted **P = 0.0075 for L-dKO mice transfected with AAV-control versus L-dKO mice transfected with AAV-LHPP), LDH (adjusted *P = 0.0196, **P = 0.0037).
a, Representative immunohistochemistry images from the LHPP-stained tissue microarray. Scale bar, 100 μm. b, Representative images and staining intensities of LHPP staining in the tissue microarray. c, Tissue microarray (TMA) indicates that LHPP is significantly downregulated in human HCC compared to adjacent non-tumour tissue (n = 20 HCC patients, ****P < 0.0001, two-sided paired t-test). d, Box plots showing a significant reduction in LHPP expression in HCC tissue compared to adjacent non-tumour tissue (n = 59). ****P < 0.0001, two-sided Mann–Whitney U test. Non-tumour (minimum, −1.422, median, 0.03329, maximum, 0.6179, lower 95% confidence interval (CI) of mean −0.0931, upper 95% CI of mean 0.0931) and HCC (minimum, −1.797, median, −0.3729, maximum, 0.7683, lower 95% CI of mean −0.5328, upper 95% CI of mean −0.2506). Expression of PHPT1 and PGAM5 was not reduced in HCC. e, Box plots showing a strong reduction in LHPP expression in Edmondson grade III/IV tumours (aggressive tumours, n = 17) compared to Edmondson grade I/II tumours (less aggressive tumours, n = 42). **P = 0.0036, two-sided Mann–Whitney U test. Edmondson grade I/II (minimum, −1.592, median, −0.2594, maximum 0.7683, lower 95% CI of mean −0.3987, upper 95% CI of mean −0.0986) and III/IV (minimum, −1.797, median, −0.5281, maximum, −0.09671, lower 95% CI of mean −1.018, upper 95% CI of mean −0.4717). f, Lollipop plot showing the distribution of mutations in LHPP across pan-cancer datasets in the TCGA and ICGC portal. Solid white circles indicate missense mutations, red circles indicate inactivating mutations (including nonsense mutations, splice site mutations and frameshift deletions; detailed list of mutations is provided in Supplementary Table 5).
This file contains supplementary figures 1-13. (PDF 12380 kb)
This file contains Supplementary Table 1: Identification of proteins enriched with histidine phosphorylation in tumours. 1173 proteins were detected and enriched (min 2-fold) in 2 out of 2 tumours (100%), or 3 out of 3 tumours (100%), or 3/4 out of 4 tumours (75%, 100%). (XLSX 44 kb)
This file contains Supplementary Table 2: Identification of proteins enriched with histidine phosphorylation in control tissue. 823 proteins were detected and enriched (min 2-fold) in 2 out of 2 control tissues (100%), or 3 out of 3 control tissues (100%), or 3/4 out of 4 control tissues (75%, 100%). (XLSX 33 kb)
This file contains Supplementary Table 3: Differentially enriched proteins with histidine phosphorylation in tumours 236 proteins were detected and enriched (min 2-fold) in 2 out of 2 experiments (100%), or 3 out of 3 experiments (100%), or 3/4 out of 4 experiments (75%, 100%). (XLSX 17 kb)
This file contains Supplementary Table 4: Identification of proteins whose histidine phosphorylation decreased upon LHPP re-expression. Nine proteins (with a log fold change of 1.7) and 17 proteins (with a log fold change of 1.4) were more abundant in immunoprecipitates from CB1 cells lacking LHPP in at least 50% of the detections (out of 5 experiments). (XLSX 10 kb)
This file contains Supplementary Table 5: List of mutations detected in LHPP. It shows a list of LHPP mutations detected from TCGA and ICGC portal. Deleterious mutations are highlighted in red. (XLSX 66 kb)
About this article
Cite this article
Hindupur, S., Colombi, M., Fuhs, S. et al. The protein histidine phosphatase LHPP is a tumour suppressor. Nature 555, 678–682 (2018). https://doi.org/10.1038/nature26140
m6A methylation mediates LHPP acetylation as a tumour aerobic glycolysis suppressor to improve the prognosis of gastric cancer
Cell Death & Disease (2022)
Nature Methods (2022)
Cell Death & Disease (2022)
BMC Genomics (2021)
Resilience, plasticity and robustness in gene expression during aging in the brain of outbred deer mice
BMC Genomics (2021)