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Immunohistochemistry (IHC): Chromogenic Detection of 3-Phosphohistidine Proteins in Formaldehyde-Fixed, Frozen Mouse Liver Tissue Sections

  • Natalie Luhtala
  • Tony HunterEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2077)

Abstract

The development of antibodies that specifically detect histidine-phosphorylated proteins is a recent achievement and allows potential roles of histidine phosphorylated proteins in pathological and physiological conditions to be characterized. Immunohistochemical analyses enable the detection of proteins in tissues and can reveal alterations to the quantity and/or localization of these proteins through comparisons of normal and diseased specimens. However, the sensitivity of phosphohistidine modifications to phosphatases, acidic pH, and elevated temperatures poses unique challenges to the detection process and requires a protocol that bypasses traditional procedures utilizing paraffin-embedding and antigen-retrieval methods. Here, we detail a method for a brief fixation by 4% (v/v) paraformaldehyde on freshly collected tissues in the presence of PhosSTOP to block phosphatase activity, followed by a float on sucrose to protect the tissue prior to freezing. Specimens are then embedded in a cryopreservation medium in molds and frozen using an isoflurane, dry ice bath to best preserve the tissue morphology and phosphohistidine signal. We validate this technique in normal mouse liver using SC44-1, a monoclonal anti-3-pHis antibody used to uncover a role for a protein histidine phosphatase as a tumor suppressor in the liver. Furthermore, we demonstrate that the antibody signal can be eliminated by preincubating SC44-1 with a peptide treated with phosphoramidate to phosphorylate histidine residues. Thus, we present an IHC protocol suitable for specific detection of 3-phosphohistidine proteins in mouse liver tissue, and suggest that this can be used as a starting point for optimization of IHC using other phosphohistidine antibodies or in other tissue types, generating information that will enhance our understanding of phosphohistidine in models of disease.

Key words

Immunohistochemistry IHC Phosphohistidine pHis Histidine phosphorylation pHis antibodies Histidine phosphorylation antibodies 

References

  1. 1.
    Fuhs SR, Meisenhelder J, Aslanian A, Ma L, Zagorska A, Stankova M, Binnie A, Al-Obeidi F, Mauger J, Lemke G, Yates JR 3rd, Hunter T (2015) Monoclonal 1- and 3-phosphohistidine antibodies: new tools to study histidine phosphorylation. Cell 162(1):198–210.  https://doi.org/10.1016/j.cell.2015.05.046CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Kee JM, Oslund RC, Couvillon AD, Muir TW (2015) A second-generation phosphohistidine analog for production of phosphohistidine antibodies. Org Lett 17(2):187–189.  https://doi.org/10.1021/ol503320pCrossRefPubMedGoogle Scholar
  3. 3.
    Kee JM, Oslund RC, Perlman DH, Muir TW (2013) A pan-specific antibody for direct detection of protein histidine phosphorylation. Nat Chem Biol 9(7):416–421.  https://doi.org/10.1038/nchembio.1259CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Kee JM, Villani B, Carpenter LR, Muir TW (2010) Development of stable phosphohistidine analogues. J Am Chem Soc 132(41):14327–14329.  https://doi.org/10.1021/ja104393tCrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Hindupur SK, Colombi M, Fuhs SR, Matter MS, Guri Y, Adam K, Cornu M, Piscuoglio S, Ng CKY, Betz C, Liko D, Quagliata L, Moes S, Jenoe P, Terracciano LM, Heim MH, Hunter T, Hall MN (2018) The protein histidine phosphatase LHPP is a tumour suppressor. Nature 555(7698):678–682.  https://doi.org/10.1038/nature26140CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Cai X, Srivastava S, Surindran S, Li Z, Skolnik EY (2014) Regulation of the epithelial Ca(2)(+) channel TRPV5 by reversible histidine phosphorylation mediated by NDPK-B and PHPT1. Mol Biol Cell 25(8):1244–1250.  https://doi.org/10.1091/mbc.E13-04-0180CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Khan I, Steeg PS (2018) The relationship of NM23 (NME) metastasis suppressor histidine phosphorylation to its nucleoside diphosphate kinase, histidine protein kinase and motility suppression activities. Oncotarget 9(12):10185–10202.  https://doi.org/10.18632/oncotarget.23796CrossRefPubMedGoogle Scholar
  8. 8.
    Panda S, Srivastava S, Li Z, Vaeth M, Fuhs SR, Hunter T, Skolnik EY (2016) Identification of PGAM5 as a mammalian protein histidine phosphatase that plays a central role to negatively regulate CD4(+) T cells. Mol Cell 63(3):457–469.  https://doi.org/10.1016/j.molcel.2016.06.021CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Srivastava S, Li Z, Soomro I, Sun Y, Wang J, Bao L, Coetzee WA, Stanley CA, Li C, Skolnik EY (2018) Regulation of KATP channel trafficking in pancreatic beta-cells by protein histidine phosphorylation. Diabetes 67(5):849–860.  https://doi.org/10.2337/db17-1433CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Srivastava S, Panda S, Li Z, Fuhs SR, Hunter T, Thiele DJ, Hubbard SR, Skolnik EY (2016) Histidine phosphorylation relieves copper inhibition in the mammalian potassium channel KCa3.1. eLife 5.  https://doi.org/10.7554/eLife.16093
  11. 11.
    Srivastava S, Zhdanova O, Di L, Li Z, Albaqumi M, Wulff H, Skolnik EY (2008) Protein histidine phosphatase 1 negatively regulates CD4 T cells by inhibiting the K+ channel KCa3.1. Proc Natl Acad Sci U S A 105(38):14442–14446.  https://doi.org/10.1073/pnas.0803678105CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Matthews HR, MacKintosh C (1995) Protein histidine phosphatase activity in rat liver and spinach leaves. FEBS Lett 364(1):51–54CrossRefGoogle Scholar
  13. 13.
    Kowalewska K, Stefanowicz P, Ruman T, Fraczyk T, Rode W, Szewczuk Z (2010) Electron capture dissociation mass spectrometric analysis of lysine-phosphorylated peptides. Biosci Rep 30(6):433–443.  https://doi.org/10.1042/BSR20090167CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Ek P, Ek B, Zetterqvist O (2015) Phosphohistidine phosphatase 1 (PHPT1) also dephosphorylates phospholysine of chemically phosphorylated histone H1 and polylysine. Ups J Med Sci 120(1):20–27.  https://doi.org/10.3109/03009734.2014.996720CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Wei YF, Matthews HR (1991) Identification of phosphohistidine in proteins and purification of protein-histidine kinases. Methods Enzymol 200:388–414CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Molecular and Cell Biology LaboratorySalk Institute for Biological StudiesLa JollaUSA

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