Quantitation of phosphohistidine in proteins in a mammalian cell line by 31P NMR

Abstract

There is growing evidence to suggest that phosphohistidines are present at significant levels in mammalian cells and play a part in regulating cellular activity, in particular signaling pathways related to cancer. Because of the chemical instability of phosphohistidine at neutral or acid pH, it remains unclear how much phosphohistidine is present in cells. Here we describe a protocol for extracting proteins from mammalian cells in a way that avoids loss of covalent phosphates from proteins, and use it to measure phosphohistidine concentrations in human bronchial epithelial cell (16HBE14o-) lysate using 31P NMR spectroscopic analysis. Phosphohistidine is determined on average to be approximately one third as abundant as phosphoserine and phosphothreonine combined (and thus roughly 15 times more abundant than phosphotyrosine). The amount of phosphohistidine, and phosphoserine/phosphothreonine per gram of protein from a cell lysate was determined to be 23 μmol/g and 68 μmol/g respectively. The amount of phosphohistidine, and phosphoserine/phosphothreonine per cell was determined to be 1.8 fmol/cell, and 5.8 fmol/cell respectively. Phosphorylation is largely at the N3 (tele) position. Typical tryptic digest conditions result in loss of most of the phosphohistidine present, which may explain why the amounts reported here are greater than is generally seen using mass spectroscopy assays. The results further strengthen the case for a functional role of phosphohistidine in eukaryotic cells.

Chemical Tools for Studying Phosphohistidine: Generation of Selective τ‐Phosphohistidine and π‐Phosphohistidine Antibodies**

“…The signals 8.15 ppm and −0.90 arise from an unknown impurity present in potassium phosphoramidate that was carried forward. The respective chemical shift values of τ‐pHis and π‐pHis do not change significantly between pH 10‐ 12 [11,30] …”

“…The respective chemical shift values of τ-pHis and π-pHis do not change significantly between pH 10-12. [11,30] combination of alanine glycine peptides containing triazolyl side chains 6 were used (Figure 5). [18] In the report which described the generation of τ-pHis (SC56-2) and π-pHis (SC1-1, SC50-3) monoclonal antibodies, the selectivity of these anti-bodies were assessed by dot blot/western blots of peptides containing pTyr side chain 2, triazolyl side chains 3 and 6, and in vitro phosphorylated NME1/NME2 and PGAM1 but not ELISA.…”

“…), 65 : 22 : 8); 1 H NMR (400 MHz, D 2 O) 3.40 (t, J = 5.5 Hz, 2H), 4.40 (t, J = 5.5 Hz, 2H), 7.62 (m, 1H), 7.72 (d, J = 2.5 Hz, 1 H); 13 The characterization data are comparable to the literature. [19] Methyl (2S)-3-(3-bromopyridin-4-yl)-2-[(tertbutoxycarbonyl)amino]propanoate (11). A round bottom flask containing rapidly stirred zinc dust (0.980 g, 15.0 mmol, 3.0 equiv.)…”

“…[2] There is now increasing evidence for the involvement of pHis in many eukaryotic cellular processes [3] and it is implicated in diseases states such as cancer. [4][5][6][7] Furthermore, there is mass spectrometry (MS) [8][9][10] and NMR spectroscopy [11] evidence that His phosphorylation is both abundant and more widely occurring than previously thought. It is therefore important to develop a range of methods for the detection of pHis and chemical tools which will allow for pHis isomer distinction, and the study of pHis proteins in vitro and in vivo.…”

Ionic polyphosphorylation of histidine repeat proteins by inorganic polyphosphate

“…Having demonstrated that 13H peptide is sufficient to undergo iH-PPn, we used this peptide for NMR studies. Phosphohistidine (pHis) is known to exhibit a characteristic P-N phosphoramidate bond peak in 31 P NMR at approximately -5 ppm, 31,32 which we would expect to observe if polyP attachment to histidine was covalent as it is presumed to be for K-PPn. A clear phosphoramidate peak at -5 ppm is visible for the pHis positive control, 33 but the 13H peptide treated with polyP displays no such peak (Figure 3D).…”