Project Summary/Abstract
Phosphorylation of histidine (His) in proteins has a 60-year history, initially identified as a P-enzyme
intermediate, but subsequently as a regulatory mechanism in bacteria essential for signal transduction by
surface receptors that sense nutrients. Such signaling systems are lacking in mammals, but phosphohistidine
(pHis) is not only a key P-enzyme intermediate in mammalian enzymes (e.g. NME1/2, ACLY), but also occurs
as a reversible end-state protein modification, e.g. pHis18 in histone H4. His phosphorylation is labile to acid
and heat making it challenging to study, and to circumvent this our group developed a series of monoclonal
antibodies (mAbs) that recognize the 1-pHis or 3-pHis isoforms in a sequence-independent manner. These
mAbs were used to detect pHis in cells by immunoblotting and immunofluorescence (IF) staining, and for
affinity enrichment of pHis proteins for MS analysis, revealing ~700 potential pHis proteins and implying the
existence of large “hidden” phosphoproteome not detectable by conventional methods. In a collaborative study,
these mAbs were used to demonstrate site and isoform specific His phosphorylation of the KCa3.1 K+ channel
and deduce how pHis triggers channel opening. In a second collaborative study, the mAbs were used to
demonstrate increased levels of pHis proteins in mouse and human hepatocellular carcinoma (HCC), and
show that the increase was due to reduced expression of the LHPP pHis phosphatase in the tumors,
suggesting that LHPP acts as a tumor suppressor, and that elevated His phosphorylation plays a driver role in
this cancer. On this basis, studies are planned to investigate whether increased His phosphorylation plays a
broader role in human cancer. Initially, our structures of mAb-derived Fab fragments bound to pHis peptides
will be exploited to develop better tools for studying His phosphorylation in cancer - mAbs with higher affinity
and scFvs for intracellular expression to localize and perturb pHis proteins, sequence specific pHis antibodies
for studying individual proteins, and improved MS-based pHis site identification. In parallel, in-depth studies
with existing 1/3-pHis mAbs, as well as new pHis reagents as they come online, will be conducted on three
selected tumor types - HCC, pediatric neuroblastoma and pancreatic cancer, where there is evidence that
aberrant His phosphorylation may play a role. Immunoblotting, and IF and IHC staining will be performed on
tumor tissues/cell lines and normal controls, combined with use of optimized pHis peptide enrichment and site
identification protocols to define changes in His phosphorylation unique to tumor tissues. Where warranted, the
function of individual pHis sites in cancer will be studied by site-directed mutagenesis in tumor cell lines. In
each case, further experiments will be guided by the identity and function of pHis proteins found in a particular
cancer. Overall, it is anticipated that comparative studies on HCC, neuroblastoma and PDAC will shed light on
whether His phosphorylation plays a general role in cancers, whether there are common mechanisms, and
whether targeting His phosphorylation could be a viable new therapeutic approach.