Over the past several decades research into signal transduction and regulation of protein function by protein phosphorylation has transformed the fields of molecular and cellular biology and led to breakthroughs in cancer therapeutics. Although 9/20 amino acids can be phosphorylated including histidine (His), arginine (Arg) and lysine (Lys), the majority of attention has been focused on phosphorylation of serine (Ser), threonine (Thr), and tyrosine (Tyr); these hydroxyamino acids form acid-stable, phosphoester (P-O) bonds. In contrast, His, Lys, and Arg form heat- and acid-labile phosphoramidate (P-N) bonds. Phosphospecific antibodies (Abs) and phosphatase inhibitors have enabled the routine study of phosphoester protein phosphorylation, and the use of MS-proteomics has identified >200,000 non-redundant sites of phosphorylation. In contrast, the lack of specific Abs and phosphatase inhibitors to study phosphoramidate protein phosphorylation and the relative instability of the P-N bond under typical conditions used for proteomics have made it impossible to determine the prevalence of this class of protein phosphorylation. Current biochemical and proteomic technologies have been optimized for preservation and detection of phosphoester amino acids (pSer, pThr and pTyr), and there is a need for analogous technology to study phosphorylated basic amino acids, and in particular phosphohistidine (pHis), which have remained largely invisible and underappreciated. The development of specific Abs and methods for detection of pHis will increase awareness of this posttranslational modification in the scientific community, and provide a renewable resource that will allow discovery and functional analysis of novel sites of protein phosphorylation. This technology has the potential to uncover new signal transduction pathways and identify therapeutic targets for human diseases. To enable the study of histidine phosphorylation as a regulatory process in mammalian cells, the following tools and technologies will be developed and employed: 1. Sequence-independent anti-1-pHis and 3-pHis monoclonal antibodies will be generated using degenerate peptide libraries containing stable analogues of 1-pHis and 3-pHis as antigens. 2. Proteomic techniques for the study of His phosphorylation using these MAbs will be optimized and used to survey His phosphorylation by mass spectrometry and other methods, including immunoblotting and immuno- fluorescence, in normal and cancer cell lines, to understand its role in normal cell physiology and in cancer. 3. pHis-binding domains will be identified, and their role in histidine phosphorylation signaling will be studied. 4. pHis protein phosphatase inhibitors will be developed to elevate histidine phosphorylation levels in cells. 5. Unnatural amino acid technology will be used to incorporate stable pHis mimetics into proteins at specific sites in vivo to study the consequences of histidine phosphorylation. The overarching goal is to develop tools needed to study histidine phosphorylation in mammalian cells and begin to determine its importance as a regulatory process in normal cells and in diseases, such as cancer.

Public Health Relevance

The studies proposed in this application seek to elucidate at the molecular level how the reversible attachment of phosphate to the amino acid histidine in proteins regulates their activity. By analogy with addition of phosphate to serine, threonine and tyrosine, the phosphorylation of histidine is likely to play an important role in normal cell functon and in human disease, and in particular cancer.

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National Cancer Institute (NCI)
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Molecular and Integrative Signal Transduction Study Section (MIST)
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Knowlton, John R
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Salk Institute for Biological Studies
La Jolla
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Hindupur, Sravanth K; Colombi, Marco; Fuhs, Stephen R et al. (2018) The protein histidine phosphatase LHPP is a tumour suppressor. Nature 555:678-682
Lu, Zhimin; Hunter, Tony (2018) Metabolic Kinases Moonlighting as Protein Kinases. Trends Biochem Sci 43:301-310
Adam, Kevin; Hunter, Tony (2018) Histidine kinases and the missing phosphoproteome from prokaryotes to eukaryotes. Lab Invest 98:233-247
Luhtala, Natalie; Aslanian, Aaron; Yates 3rd, John R et al. (2017) Secreted Glioblastoma Nanovesicles Contain Intracellular Signaling Proteins and Active Ras Incorporated in a Farnesylation-dependent Manner. J Biol Chem 292:611-628
Fuhs, Stephen Rush; Hunter, Tony (2017) pHisphorylation: the emergence of histidine phosphorylation as a reversible regulatory modification. Curr Opin Cell Biol 45:8-16
Panda, Saswati; Srivastava, Shekhar; Li, Zhai et al. (2016) Identification of PGAM5 as a Mammalian Protein Histidine Phosphatase that Plays a Central Role to Negatively Regulate CD4(+) T Cells. Mol Cell 63:457-69
Srivastava, Shekhar; Panda, Saswati; Li, Zhai et al. (2016) Histidine phosphorylation relieves copper inhibition in the mammalian potassium channel KCa3.1. Elife 5:
Fuhs, Stephen Rush; Meisenhelder, Jill; Aslanian, Aaron et al. (2015) Monoclonal 1- and 3-Phosphohistidine Antibodies: New Tools to Study Histidine Phosphorylation. Cell 162:198-210