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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.

Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent protein kinase A (PKA) and ATP-regulated chloride channel. Here, we demonstrate that nucleoside diphosphate kinase B (NDPK-B, NM23-H2) forms a functional complex with CFTR. In airway epithelia forskolin/IBMX significantly increases NDPK-B co-localisation with CFTR whereas PKA inhibitors attenuate complex formation. Furthermore, an NDPK-B derived peptide (but not its NDPK-A equivalent) disrupts the NDPK-B/CFTR complex in vitro (19-mers comprising amino acids 36-54 from NDPK-B or NDPK-A). Overlay (Far-Western) and Surface Plasmon Resonance (SPR) analysis both demonstrate that NDPK-B binds CFTR within its first nucleotide binding domain (NBD1, CFTR amino acids 351-727). Analysis of chloride currents reflective of CFTR or outwardly rectifying chloride channels (ORCC, DIDS-sensitive) showed that the 19-mer NDPK-B peptide (but not its NDPK-A equivalent) reduced both chloride conductances. Additionally, the NDPK-B (but not NDPK-A) peptide also attenuated acetylcholine-induced intestinal short circuit currents. In silico analysis of the NBD1/NDPK-B complex reveals an extended interaction surface between the two proteins. This binding zone is also target of the 19-mer NDPK-B peptide, thus confirming its capability to disrupt NDPK-B/CFTR complex. We propose that NDPK-B forms part of the complex that controls chloride currents in epithelia.

BACKGROUND: TRPV6 ion channels are key mediators of regulated transepithelial absorption of Ca2+ within the small intestine. Trpv6-/- mice were reported to have lower bone density than wild-type littermates and significant disturbances in calcium homeostasis that suggested a role for TRPV6 in osteoblasts during bone formation and mineralization. TRPV6 and molecules related to transepithelial Ca2+ transport have been reported to be expressed at high levels in human and mouse osteoblasts. RESULTS: Transmembrane ion currents in whole cell patch clamped SaOS-2 osteoblasts did not show sensitivity to ruthenium red, an inhibitor of TRPV5/6 ion channels, and 45Ca uptake was not significantly affected by ruthenium red in either SaOS-2 (P=0.77) or TE-85 (P=0.69) osteoblastic cells. In contrast, ion currents and 45Ca uptake were both significantly affected in a human bronchial epithelial cell line known to express TRPV6. TRPV6 was expressed at lower levels in osteoblastic cells than has been reported in some literature. In SaOS-2 TRPV6 mRNA was below the assay detection limit; in TE-85 TRPV6 mRNA was detected at 6.90±1.9×10(-5) relative to B2M. In contrast, TRPV6 was detected at 7.7±3.0×10(-2) and 2.38±0.28×10(-4) the level of B2M in human carcinoma-derived cell lines LNCaP and CaCO-2 respectively. In murine primary calvarial osteoblasts TRPV6 was detected at 3.80±0.24×10(-5) relative to GAPDH, in contrast with 4.3±1.5×10(-2) relative to GAPDH in murine duodenum. By immunohistochemistry, TRPV6 was expressed mainly in myleocytic cells of the murine bone marrow and was observed only at low levels in murine osteoblasts, osteocytes or growth plate cartilage. CONCLUSIONS: TRPV6 is expressed only at low levels in osteoblasts and plays little functional role in osteoblastic calcium uptake.