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A pan-specific antibody for direct detection of protein histidine phosphorylation

Abstract

Despite its importance in central metabolism and bacterial cell signaling, protein histidine phosphorylation has remained elusive with respect to its extent and functional roles in biological systems because of the lack of adequate research tools. We report the development of the first pan-phosphohistidine (pHis) antibody using a stable pHis mimetic as the hapten. This antibody was successfully used in ELISA, western blotting, dot blot assays and immunoprecipitation and in detection and identification of histidine-phosphorylated proteins from native cell lysates when coupled with MS analysis. We also observed that the amount of protein pHis in Escherichia coli lysates depends on carbon source and nitrogen availability in the growth medium. In particular, we found that the amount of pHis on phosphoenolpyruvate synthase (PpsA) is sensitive to nitrogen availability in vivo and that α-ketoglutarate inhibits phosphotransfer from phosphorylated PpsA to pyruvate. We expect this antibody to open opportunities for investigating other pHis proteins and their functions.

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Figure 1: Affinity purification of antibodies raised against pTze.
Figure 2: Affinity-purified antibodies raised against pTze are cross-reactive to pHis and are sequence independent.
Figure 3: Cross-reactivity of pHis antibody to pTyr.
Figure 4: Analysis of histidine phosphorylation on PtsI.
Figure 5: The amount of pHis in PpsA is sensitive to nitrogen availability and is regulated by α-KG.

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Acknowledgements

We would like to acknowledge S. Darst and M. Bick (The Rockefeller University) for providing KinB and J. Rabinowitz and M. Reaves (Princeton University) for providing NCM 3722 cells. We thank J. Rabinowitz, M. Reaves and B. Wang for stimulating discussions and S. Kyin and H. Shwe for their assistance in MS. This work was funded by the US National Institutes of Health (5R01GM095880). J.-M.K. and R.C.O. were supported by a postdoctoral fellowship from the Damon Runyon Cancer Research Foundation (DRG-2005-09) and a National Institutes of Health Research Service Award (1F32CA167901), respectively.

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J.-M.K., R.C.O. and T.W.M. designed the experiments. J.-M.K., R.C.O. and D.H.P. performed the experiments. J.-M.K. and R.C.O. prepared new reagents. Experimental results were analyzed and the manuscript was written by J.-M.K., R.C.O., D.H.P. and T.W.M.

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Correspondence to Tom W Muir.

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Competing interests

J.-M.K. and T.W.M. are co-inventors of a patent application on stable phosphohistidine analogs.

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Supplementary Results, Supplementary Notes 1–9 (PDF 43558 kb)

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Kee, JM., Oslund, R., Perlman, D. et al. A pan-specific antibody for direct detection of protein histidine phosphorylation. Nat Chem Biol 9, 416–421 (2013). https://doi.org/10.1038/nchembio.1259

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