Evidence of histidine and aspartic acid phosphorylation in human prostate cancer cells

Meisenhelder

Aslanian

et al. 2015

Cell

167

205

Summary Histidine phosphorylation (pHis) is well studied in bacteria; however, its role in mammalian signaling remains largely unexplored due to the lack of pHis-specific antibodies and the lability of the phosphoramidate (P-N) bond. Both imidazole nitrogens can be phosphorylated, forming 1-phosphohistidine (1-pHis) or 3-phosphohistidine (3-pHis). We have developed monoclonal antibodies (mAbs) that specifically recognize 1-pHis or 3-pHis; they do not cross-react with phosphotyrosine or the other pHis isomer. Assays based on the isomer-specific auto-phosphorylation of NME1 and phosphoglycerate mutase were used with immunoblotting and sequencing IgG variable domains to screen, select and characterize anti-1-pHis and anti-3-pHis mAbs. Their sequence independence was determined by blotting synthetic peptide arrays, and they have been tested for immunofluorescence staining and immunoaffinity purification, leading to putative identification of pHis-containing proteins. These reagents should be broadly useful for identification of pHis substrates and functional study of pHis using a variety of immunological, proteomic and biological assays.

Section: Resultsmentioning

confidence: 94%

Section: Figurementioning

confidence: 99%

Monoclonal 1- and 3-Phosphohistidine Antibodies: New Tools to Study Histidine Phosphorylation

Meisenhelder

Aslanian

et al. 2015

Cell

167

205

“…The identities of the pHis proteins we identified suggest that pHis may play a role in mammalian cell signaling [32][33][34] . Indeed, a direct correlation of pHis protein levels with carcinogenesis has recently been reported 30,[35][36][37] . Interestingly, we identified 20 N-phosphate sites on proteins that were reported to be enriched by IAP by Hindupur et al from mTOR-driven hepatocellular carcinoma mouse tumors as potential LHPP substrates, including BCLAF1, which in our studies was found to contain pLys (K391) with a reasonable probability of localization of 79.6%.…”

Section: Discussionmentioning

confidence: 95%

A non-acidic method using hydroxyapatite and phosphohistidine monoclonal antibodies allows enrichment of phosphopeptides containing non-conventional phosphorylations for mass spectrometry analysis

Adam

Meisenhelder

et al. 2019

Preprint

Four types of phosphate-protein linkage generate nine different phosphoresidues in living organisms. Histidine phosphorylation is a long-time established but largely unexplored post-translational modification, mainly because of the acid-lability of the phosphoramidate bonds. This lability means that standard phosphoproteomic methods used for conventional phosphate esters (phospho-Ser/Thr/Tyr) must be modified to analyze proteins containing the phosphoramidate-amino acids -phospho-His/Arg/Lys. We show that a non-acidic method allows enrichment of non-conventional phosphoresiduecontaining peptides from tryptic digests of human cell lines, using hydroxyapatite binding and/or immobilized 1-pHis and 3-pHis monoclonal antibodies for enrichment. 425 unique non-conventional phosphorylation sites (i.e. pHis, pLys and pArg) were detected with a high probability of localization by LC-MS/MS analysis and identified using a customized MaxQuant configuration, contributing to a new era of study in post-translational modification and cell signaling in humans. This is the first fully non-acidic method for phosphopeptide enrichment which uses immunoaffinity purification and remains compatible with mass spectrometry analysis for a wider coverage of potential protein phosphorylation events. Introduction:Phosphoproteomics has contributed to tremendous progress in the field of life science and revealed the extensive use of phosphorylation as one of the most important post-translational modifications in eukaryotic organisms. The identification and characterization of phosphorylation sites has been revolutionized by mass spectrometry (MS) with faster and more sensitive instruments. Progress in phosphosite-mapping with tandem mass spectrometry (MS/MS) has allowed improved localization and phosphorylation assignment to a single residue with high confidence. The development of bioinformatics tools for large-scale phosphoproteome data analysis was an equally important step and permitted the integration of different parameters searching for complex combinatorial possibilities 1,2 .Up until now, only one class of phosphate-bond containing phosphoamino acid, corresponding to the conventional serine, threonine and tyrosine phosphorylation (phosphate-ester or O-phosphate) has been systematically considered for phosphorylation site searching. But nine out of the 20 amino acids have the

“…An alternative strategy of elevating LC pH to 5 (without enrichment) was employed as a compromise between stabilization of pHis and ionization of peptides. 20 pHis phosphopeptides were identified; however, these have not yet been validated at the protein level [65,66]. Negative electron-transfer dissociation (NETD) developed by the Coon lab, which is run under alkaline conditions, is a promising new technique that has the potential to solve these issues [67–69].…”

Section: Introductionmentioning

confidence: 99%

pHisphorylation: the emergence of histidine phosphorylation as a reversible regulatory modification

Current Opinion in Cell Biology

Hunter

2017

139

106

Histidine phosphorylation is crucial for prokaryotic signal transduction and as an intermediate for several metabolic enzymes, yet its role in mammalian cells remains largely uncharted. This is primarily due to difficulties in studying histidine phosphorylation because of the relative instability of phosphohistidine (pHis) and lack of specific antibodies and methods to preserve and detect it. The recent synthesis of stable pHis analogs has enabled development of pHis-specific antibodies and their use has started to shed light onto this important, yet enigmatic posttranslational modification. We are beginning to understand that pHis has broader roles in protein and cellular function including; cell cycle regulation, phagocytosis, regulation of ion channel activity and metal ion coordination. Two mammalian histidine kinases (NME1 and NME2), two pHis phosphatases (PHPT1 and LHPP), and a handful of substrates were previously identified. These new tools have already led to the discovery of an additional phosphatase (PGAM5) and hundreds of putative substrates. New methodologies are also being developed to probe the pHis phosphoproteome and determine functional consequences, including negative ion mode mass spectroscopy and unnatural amino acid incorporation. These new tools and strategies have the potential overcome the unique challenges that have been holding back our understanding of pHis in cell biology.