Although the importance of protein histidine phosphorylation in mammals has been a subject of increasing interest, few chemical probes are available for monitoring and manipulating PHP activity. Here, we present an optimized and validated protocol for assaying the activity of PHPT1 using the fluorogenic substrate DiFMUP. The kinetic parameters of our optimized assay are significantly improved as compared with other PHPT1 assays in the literature, with a kcat of 0.39 ± 0.02 s–1, a Km of 220 ± 30 μM, and a kcat/Km of 1800 ± 200 M–1 s–1. In addition, the assay is significantly more sensitive as a result of using a fluorescent probe, requiring only 109 nM enzyme as compared with 2.4 μM as required by previously published assays. In the process of assay optimization, we discovered that PHPT1 is sensitive to a reducing environment and inhibited by transition-metal ions, with one apparent Cu(II) binding site with IC50 value of 500 ± 20 μM and two apparent Zn(II) binding sites with IC50 values of 25 ± 1 and 490 ± 20 μM.
Protein histidine phosphorylation was first discovered in 1962,(1) nearly 20 years before protein tyrosine phosphorylation.(2)
Despite this significant time advantage and the higher incidence of
pHis (accounting for perhaps 6% or more of the total phosphoamino acids
in the proteome)(3) as compared to pTyr (estimated at less than 1% of total phosphosites),(4)
the roles of histidine phosphorylation in mammalian cells are virtually
unknown compared to the roles of tyrosine phosphorylation, which are in
turn much less well understood than serine and threonine
phosphorylation. Nonetheless, it has become clear that histidine
phosphorylation is important in mammalian cells. For example, histidine
phosphatase activity is known to be important in several biological
processes, including regulation of T-cell receptor signaling,(5) G-protein coupled receptor signaling,(6) and potassium channel activation.(7) In addition, elevated levels of PHPT1 have been found in hepatocellular carcinoma(8) and lung cancer(9)
tissue when compared to noncancerous tissue, and high expression of
PHPT1 in clear-cell renal cell carcinoma has been negatively correlated
with patient survival.(10)
Although interest in the biological roles of histidine phosphorylation in mammals continues to grow,(11,12)
the lack of chemical tools available to study pHis and the enzymes that
regulate it is a significant roadblock to the field. Highly sensitive,
continuous fluorogenic assays for tyrosine phosphatase activity have
been invaluable, providing substrates for enzyme assays and high
throughput inhibitor screening(13−16) and tools for monitoring PTP activity in cells,(17−19) which have yielded insights into the roles of PTPs in biology.(17,20)
In contrast, no commercially available inhibitors and few enzyme assays
exist for studying PHP activity. The histidine phosphatase PHPT1 has
been shown to hydrolyze para-nitrophenylphosphate(21) (pNPP, a colorimetric substrate commonly used to monitor general phosphatase activity,(13) see Figure 1) and small pHis containing peptides using an HPLC-based assay.(22)
However, these substrates suffer from modest turnover, low sensitivity,
and discontinuous assay readout methodologies. Adding to the complexity
of the problem, the assay conditions that have been used seem to have
been borrowed from the literature on other protein phosphatases and have
not been optimized for monitoring PHP activity. For example, some
assays include DTT, which is required for protein tyrosine phosphatase
activity to reduce the catalytic cysteine residue,(23) while other assays include MgCl2, which is required for serine and threonine phosphatase activity.(24)
However, the PHPs are believed to be neither cysteine dependent
hydrolyases nor metallohydrolases, but rather to utilize a histidine
residue either as a general base to activate a water molecule to serve
as the hydrolytic nucleophile(21) or to participate in phosphoryl transfer directly.(25,26)
All of these issues ultimately limit the widespread use of the few
existing PHP substrates and result in a high barrier of entry to
studying the biochemistry and biology of the PHPs.
Given the current state of the field, an ideal PHP activity probe would
be readily available from commercial sources and provide a highly
sensitive readout. Fluorogenic substrates provide direct, continuous
readouts and would greatly facilitate the study of these enzymes in
vitro, the development of inhibitors, and advance our understanding of
the biological roles of histidine phosphatases. On the basis of a
preliminary report that PHPT1 can hydrolyze the highly sensitive
fluorogenic phosphatase substrate 6,8-difluoromethylumbelliferyl
phosphate (DiFMUP),(27,28)
we investigated the utility of DiFMUP as a substrate for PHPT1 activity
and here provide an optimized and validated PHPT1 activity assay
protocol.
2) https://pubs.acs.org/doi/full/10.1021/acs.analchem.9b00734
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Recent technological advances have made it possible to investigate the hitherto rather elusive protein histidine phosphorylation. However, confident site-specific localization of protein histidine phosphorylation remains challenging. Here, we address this problem, presenting a mass-spectrometry-based approach that outperforms classical HCD fragmentation without compromising sensitivity. We use the phosphohistidine immonium ion as a diagnostic tool as well as ETD-based fragmentation techniques to achieve unambiguous identification and localization of histidine-phosphorylation sites. The work presented here will allow more confident investigation of the phosphohistidine proteome to reveal the roles of histidine phosphorylation in cellular signaling events.
Gaining Confidence in the Elusive Histidine Phosphoproteome
- Clement M. Potel
- Miao-Hsia Lin et al.
Conclusion
ARTICLE SECTIONS
To
summarize, we presented evidence that an optimized novel
phosphohistidine-immonium-ion-triggering method can be used to extend
the coverage of the still largely elusive phosphohistidine proteome. By
combining two layers of evidence (i.e., the presence of the immonium
diagnostic ion and the use of ETD-based fragmentation techniques), this
method enhances confident identification of protein histidine
phosphosites. Current knowledge of the biological role of histidine
phosphorylation, in prokaryotes but also in eukaryotes, represents only
the tip of the iceberg. In our view, our method should help the
community gain in-depth insight in the biological function and abundance
of histidine phosphorylation in organisms across the whole tree of
life.
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