Phosphohistidine phosphatase 1 (PHPT1) also dephosphorylates phospholysine of chemically phosphoryla


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Phosphohistidine phosphatase 1 (PHPT1) also
dephosphorylates phospholysine of chemically
phosphorylated histone H1 and polylysine

Pia Ek, Bo Ek & Örjan Zetterqvist

To cite this article: Pia Ek, Bo Ek & Örjan Zetterqvist (2015) Phosphohistidine phosphatase
1 (PHPT1) also dephosphorylates phospholysine of chemically phosphorylated
histone H1 and polylysine, Upsala Journal of Medical Sciences, 120:1, 20-27, DOI:
10.3109/03009734.2014.996720

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Upsala Journal of Medical Sciences. 2015; 120: 20–27

ORIGINAL ARTICLE

Phosphohistidine phosphatase 1 (PHPT1) also dephosphorylates
phospholysine of chemically phosphorylated histone H1 and polylysine

PIA EK1, BO EK2 & ÖRJAN ZETTERQVIST1

1Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden, and
2Department of Chemistry, Uppsala University, Uppsala, Sweden

Abstract
Background. Phosphohistidine phosphatase 1 (PHPT1), also named protein histidine phosphatase (PHP), is a eukaryotic
enzyme dephosphorylating proteins and peptides that are phosphorylated on a histidine residue. A preliminary finding that
histone H1, which lacks histidine, was phosphorylated by phosphoramidate and dephosphorylated by PHPT1 prompted the
present investigation.
Methods. Histone H1 and polylysine were phosphorylated at a low concentration (3.9 mM) of phosphoramidate. Their
dephosphorylation by recombinant human PHPT1 was investigated by using a DEAE-Sepharose spin column technique
earlier developed by us for studies on basic phosphoproteins and phosphopeptides. Determination of protein-bound,
acid-labile phosphate was performed by a malachite green method. Mass spectrometry (MS) was used to investigate the
occurrence of N-e-phospholysine residues in a phosphorylated histone H1.2 preparation, and to measure the activity of
PHPT1 against free N-w-phosphoarginine.
Results. Histone H1.2,which lackshistidine, was phosphorylated byphosphoramidate on severallysine residues, asshownbyMS.
PHPT1wasshowntodephosphorylatephosphohistoneH1ataratesimilartothatpreviouslydescribedforthedephosphorylation
of phosphohistidine-containing peptides. In addition, phosphopolylysine was an equally good substrate for PHPT1. However,
no dephosphorylation of free phosphoarginine by PHPT1 could be detected.
Conclusion. The finding that PHPT1 can dephosphorylate phospholysine in chemically phosphorylated histone H1 and
polylysine demonstrates a broader specificity for this enzyme than known so far.

Key words: Histone H1, phosphohistidine phosphatase, phospholysine, phospholysine phosphatase, PHP, PHPT1,
protein histidine phosphatase

Introduction

The discovery of protein phosphorylation on histi-
dine (1) was made 52 years ago (2). Most of the work
relatedtophosphohistidinehassofarbeenperformedon
the bacterial phosphoenolpyruvate-glucose phospho-
transferase system (3) and two-component systems
(4).Ineukaryoticcells,protein-boundphosphohistidine
constitutes a significant amount of the total phospho-
amino acid (5). Still, only a very low number of
phosphohistidine-containing proteins have been identi-
fied in contrast to the great number of proteins phos-
phorylated on serine, threonine and tyrosine (2,6,7).

Theproteinsphosphorylatedonhistidinearetoalarge
extent represented by catalytic intermediates of met-
abolic enzymes, such as the extensively studied nucle-
oside diphosphate kinase (NDPK) that transiently
forms 1-phosphohistidine (8,9) and ATP-citrate lyase
and succinyl-CoA synthetase (succinate thiokinase)
that similarly form 3-phosphohistidine in their active
sites(10-15). Theslow progressineukaryoticphospho-
histidine research can be explained by the lability of the
N–P bonds of phosphohistidines at the acid conditions
thatareroutinelyusedinmostphosphoproteinresearch
(5,16-18). However, recent developments of specific
antibodies in combination with mass-spectrometric

Correspondence: Pia Ek, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden. E-mail: Pia.Ek@imbim.uu.se

(Received 18 October 2014; accepted 4 December 2014)

ISSN 0300-9734 print/ISSN 2000-1967 online � 2015 Informa Healthcare
DOI: 10.3109/03009734.2014.996720

http://informahealthcare.com/journal/ups
mailto:Pia.Ek@imbim.uu.se


methods adapted to the detection of phosphohistidine
(19) give hope for a change in the near future.
In 2002, a new tool for studies of eukaryotic histidine

phosphorylation was obtained in the form of a 14 kDa
phosphohistidine phosphatase (PHPT1) that was
independently discovered and characterized by
Ek et al. (20) and Klumpp et al. (21). The latter group
named the enzyme protein histidine phosphatase
(PHP). No activity of this enzyme could be detected
toward a set of O-phosphorylated peptides containing
phosphoserine, phosphothreonine, or phosphotyro-
sine (20). The activity was independent of divalent
cations, and okadaic acid did not inhibit the enzyme
(20,21). By mutational studies of the recombinant
human enzyme, His-53 was shown to be essential
for the phosphatase activity toward phosphohistidine
(22). The 3D structure of the enzyme was determined
both by X-ray crystallography (23) and NMR (24).
ATP-citrate lyase and the b-subunit of G-protein were
soon identified as potential physiological substrates
(25,26). Convincing evidence of a physiological role
for PHPT1 has been obtained from studies of the
potassium channel KCa3.1 that is phosphorylated by
nucleoside diphosphate kinase B (NDPK-B) on histi-
dine residue 358 (27,28). In these experiments, ion
transportation was activated by the phosphorylation
and deactivated by a PHPT1-dependent dephosphor-
ylation. Similar results have been obtained for the
calcium channel TRPV5, although a direct proof of
the formation of phosphohistidine in this case is still
lacking (29). The interesting possibility of NDPK
acting as a protein kinase with PHPT1 as the balancing
phosphoprotein phosphatase has been discussed in a
review by Wieland et al. (30). Furthermore, evidence
for a possible role of PHPT1 in cytoskeletal reorgani-
zation (31,32) and in hepatocellular carcinoma cell
proliferation (33) has been presented. In several of the
experiments above, the possibilities to modulate the
expression of the PHPT1 gene have been crucial.
Another approach to study the substrate specificity

of PHPT1 was described by Attwood et al. in their
study of chemically phosphorylated histone H4 pep-
tides containing His-18 and His-75 (34). These
phosphopeptides were good substrates of PHPT1. In
parallel, our group designed a new method for the
determination of phosphohistidine phosphatase activ-
ity based on highly basic substrates, such as chemically
phosphorylated histone H4 and Ac-VRLKHRKLR-
pNA, a peptide representing the amino acid sequence
around His-358 of KCa3.1 (35). During preliminaries
to the latter work several histone preparations were
tested beside histone H4. Commercial preparations of
histone H1 from calf thymus, in spite of their lack
of histidine, were phosphorylated to the highest degree
of all histones tested and were dephosphorylated by

PHPT1 at high rates. These unexpected results have
been investigated in the present study.

Materials and methods

Materials

Human recombinant phosphohistidine phosphatase
(PHPT1) was expressed and purified as described
by Ma et al. (22). Calf thymus histone H1 (IIIS) and
polylysine were from Sigma-Aldrich (Stockhom,
Sweden). Calf thymus histone H1 was also from
Calbiochem(Stockhom,Sweden),Abcam(Cambridge,
UK), Santa Cruz (Heidelberg, Germany), and
SignalChem (Stockhom, Sweden). Trypsin was
from Promega (Stockhom, Sweden). N-w-phospho-
L-arginine was from Sigma-Aldrich. Non-radioactive
phosphoramidate was synthesized by applying the
method described for [32P]phosphoramidate by
Buckler and Stock (36). Malachite green reagent
was Biomolgreen from AH Diagnostics (Stockholm,
Sweden). DEAE-Sephacel was from GE Health Care
(Uppsala, Sweden). Micro Bio-Spin columns were
obtained from BioRad (Stockholm, Sweden).

Methods

Protein phosphorylation and dephosphorylation. Histone
H1 and polylysine were chemically phosphorylated by
3.9 mM phosphoramidate as described for histone
H4 (35). Thus, 50 mg of histone H1, 100 mg of 30 kDa
polylysine, or 100 mg of 90 kDa polylysine, each
dissolved in 25 mL 10 mM HCl, were separately
mixed with 25 mL 25 mM Tris/HCl (pH 8.5) and
1 mL 0.2 M phosphoramidate to give pH 7.0 and
incubated for at least 24 h. Tween 20 was then added
to give the final concentration 0.04% (w/v), and the
incubation was interrupted, either by freezing at 80�C
or by centrifugation for 2 min at 900 g on a 200-mL
DEAE-Sephacel spin column equilibrated in 25 mM
Tris/HCl (pH 8.5) as described (35). The eluted
volume was immediately centrifuged on a second
DEAE-Sephacel column, and the eluted volume
from the latter column was used for dephosphoryla-
tion experiments and analysis of the acid-labile phos-
phate bound to the basic protein, as described (35).
Biomolgreen was used as the malachite green reagent.
Dephosphorylation was performed at pH 7.5 and

30�C by incubating a 50-mL aliquot of the eluted
volume from the second spin column with 5 pmol
of PHPT1 in 1 ml 25 mM Hepes (pH 7.5) for
indicated times. The incubation was interrupted by
an immediate centrifugation at 900 g on a DEAE-
Sephacel spin column to bind PHPT1 and any
released orthophosphate. The eluted volume from

PHPT1 also dephosphorylates phospholysine 21



this column was analyzed for remaining acid-labile,
protein-bound phosphate as described above. Histone
H1 and polylysine concentrations were estimated by
UV spectrometry at 280 nm and 215 nm, respectively.
Investigation of PHPT1 activity against N-w-phos-

phoarginine. N-w-phosphoarginine (250 mM) was
incubated for 40 min with recombinant PHPT1 in
the same buffers and volumes as described above for
phosphohistone H1 and phosphopolylysine. N-w-
phosphoarginine and arginine were analyzed by
MS, as described below.
Mass-spectrometric analysis. Chromatographically

purified histone H1 from SignalChem was chemically
phosphorylated as described above for the H1 prepa-
ration from Sigma-Aldrich, and then diluted with nine
volumes of 0.1 M ammonium hydrogen carbonate and
digested with 1 mg trypsin/100 mg histone for 3 h. The
digestion was interrupted by freezing the sample at
–20�C. A 1-mL thawed sample was run on a Thermo
Orbitrap Velos (Termo Scientific, Stockholm,
Sweden) in 0.1% (w/v) formic acid, using a gradient
from 4% to 30% acetonitrile in 120 min, followed by a
steep gradient to 48% in 10 min, and finally by a
washing step at 80% acetonitrile. MS1 resolution
was held at 60,000 (full width at half maximum), and
fragments were measured between 300 and 1800 Da.
Fragmentation was generated with collision-induced
dissociation (CID). The LC-column was a Thermo
Easy column C18 (100 mm � 75 mm and 3 mm beads)
from Termo Scientific. Buffer A was 0.1% formic acid
in H2O, and buffer B was 0.1% formic acid in aceto-
nitrile. The instrument was using lock-mass for
improved mass accuracy. The resulting raw-format
files were analyzed by XTandem over the web. Beside
the default settings, phosphorylated serine, threonine,
and lysine were chosen as possible modifications.
For MS analysis of phosphoarginine and arginine, a

Thermo Velos equipment was used in infusion mode.
A syringe pump delivered 3 mL/min of 50% methanol
(no acid) into which the sample was diluted with four
partsofsolvent.Theinstrument’sstandardcapillaryinlet
was used, and when the analysis seemed stable several
manualrecordingsof1minweremade.Thespectrawere
averaged, and the 255.08 Da peak was used for compar-
ison of the plus with the minus dephosphorylation
samples.Tostrengthenfurthertheidentificationofphos-
phoarginine, a brief fragmentation was also recorded
showing the characteristic loss of HPO3 (80 Da).

Results

Phosphorylation of histone H1

In the pre-study of commercial histones mentioned in
the Introduction, the chemical phosphorylation by

phosphoramidate was found to be highest for histone
H1 from calf thymus. Under the conditions used, all
histone H1 preparations could be chemically phos-
phorylated to a level of 1–1.5 mol phosphate/mol
protein. By amino acid analysis, performed by the
Amino Acid Analysis Center at Uppsala University,
Sweden, the histidine content of the histone
H1 preparation from Sigma-Aldrich (IIIS) was found
to be less than 0.2 mol/mol protein and could thus
not account for the observed level of acid-labile
phosphorylation. This histone is obtained from the
supernatant after precipitation of nuclear fraction with
5% trichloroacetic acid (37). The amino acid analysis
data were compatible with reported primary struc-
tures of histones H1.2–5. Therefore, this histone
H1 preparation was judged to be suitable for the
present phosphorylation/dephosphorylation experi-
ments. Although the content of arginine was found
to be 4.4 mol/mol protein, the possibility of arginine
phosphorylation by phosphoramidate can probably be
excluded (38). Since, however, a phosphorylation of
peptide-bound lysine residues by phosphoramidate
has been demonstrated (38), a phosphorylation of
some of the numerous lysine residues (e.g. 59 lysine
residues/mol protein in histone H1.2) was considered
possible. Compared to this high number of lysine
residues, the phosphorylation of histone H1 is quite
low. This can be explained by the initial concentra-
tions used for phosphoramidate (3.9 mM) and
histone H1 (0.05 mM), which are much lower than
used for histidine-containing peptides (20,34) and
lysine-containing peptides (38). The low initial
concentration of phosphoramidate was, however, a
deliberate choice in order not to exceed the binding
capacity of the DEAE-Sephacel spin columns used as
described under Methods.

Dephosphorylation by PHPT1 of chemically
phosphorylated histone H1

All preparations of phosphorylated histone H1
could be dephosphorylated by PHPT1. One set of
experiments is described in Figure 1. The initial rate
of the PHPT1-catalyzed dephosphorylation was
1.0 ± 0.1 mol/s per mol of enzyme (mean and SD
of seven separate experiments). This rate is in the
same order of magnitude as previously described for
the dephosphorylation of chemically phosphorylated
histone H4 (35) and a chemically phosphorylated
histidine-containing peptide (20). In the absence of
PHPT1, phosphorylated histone H1 was stable under
the conditions used, i.e. the mean remaining phos-
phohistone H1 for seven separate experiments at 0, 5,
and 10 min was 101% ± 2%. Other commercial
histone H1 preparations, phosphorylated to about

22 P. Ek et al.



the same degree, were dephosphorylated by PHPT1
at similar initial rates (data not shown).

Phosphorylated polylysine and its dephosphorylation by
PHPT1

To confirm that protein-bound lysine can be
N-e-phosphorylated by phosphoramidate and then
dephosphorylated by PHPT1, polylysine (30 kDa)
was investigated. When phosphorylated under
conditions identical to those used for histone H1,
a phosphorylation of 1.5–2 mol/mol polylysine
was usually obtained. Phosphopolylysine was depho-
sphorylated by PHPT1 at a rate that was 70% ± 8%
(n = 3) of that of phosphorylated histone
H1 (Figure 1). In the absence of PHPT1, phospho-
polylysine was stable under the conditions used,
i.e. the mean remaining phosphopolylysine for three
separate experiments at 0, 5, and 10 min was
100% ± 1%.
To exclude the possibility that N-terminal N-a-

phospholysine in the 30 kDa polylysine was the only
substrate of PHPT1, polylysine with a greater mean
molecular size (90 kDa) was investigated together
with the 30 kDa polylysine. Under the conditions
of this experiment, performed three times, the phos-
phorylation was 6.0 ± 2.6 and 1.6 ± 0.5 mol/mol
protein for the 90 kDa and 30 kDa polylysine,
respectively. Upon extended incubation of both types

of phosphorylated polylysines with PHPT1, all phos-
phate was released (data not shown). This shows that
beside any N-terminal phospholysine, several lysine
residues were N-e-phosphorylated and could be
dephosphorylated by PHPT1.

PHPT1 and phosphoarginine

No dephosphorylation of N-w-phosphoarginine by
PHPT1 could be detected, although the incubation
time was 4-fold that under which phosphohistidine
H1 and phosphopolylysine were significantly
dephosphorylated. The intensity reported for the
phosphoarginine peak was 1.33 � 106 for the control
without dephosphorylation and 1.52 � 106 after
attempting dephosphorylation, while the reported
intensity for arginine was negligible.

MS-sequencing of chemically phosphorylated histone H1:
identification of phosphorylated lysine residues

In order to control that the phosphorylation of histone
H1 really occurred on lysine residues, the calf thymus
histone H1 obtained from SignalChem was
phosphorylated to 1.5 mol phosphate/mol protein
by phosphoramidate as described above and subjected
to MS-sequencing after trypsination. This chromato-
graphically purified histone H1 preparation was cho-
sen in order to minimize the risk of interference with
the LC-MS/MS. From the MS-data, this histone
H1 was interpreted by the XTandem program to be
histone H1c, which corresponds to histone H1.2
according to an alternative nomenclature (39). The
high reliability of this interpretation is apparent from
the log(e) for this assignment, which was –683 and
defined as ‘the base-10 log of the expectation that any
particular protein assignment was made at random
(E-value)’. Sequence data are given in Figure 2. Seven
out of the 59 lysine residues were identified as targets
for the chemical phosphorylation and are highlighted.

0

20

40

60

80

100

120

0 2 4 6 8 10 12

P
ro

te
in

-b
o

u
n

d
 a

c
id

-l
a
b

il
e

p
h

o
s
p

h
a
te

 (
mM

)

Time (min)

Figure 1. Decrease in phosphate in phosphoramidate phosphor-
ylated histone H1 (&) and 30 kDa polylysine (^) during incu-
bation with PHPT1. The concentration was 1 mg/mL of
phosphohistone and 2 mg/mL of phosphopolylysine. An amount
of 5 pmol PHPT1 was added per 51 mL incubation volume. The
reaction was performed at pH 7.5 and 30�C during indicated times
and was interrupted by centrifugation of 50 mL of the reaction
mixture through a spin column containing 200 mL of DEAE-
Sepharose equilibrated in 25 mM Tris/HCl pH 8.5. The pro-
tein-bound, acid-labile phosphate in the final eluate was analyzed
as described under Methods. Each time point was analyzed in
duplicate.

1 MSETAPAAPA AAPPAEKTPV KKKAAKKPAG ARRKASGPPV
41 SELITKAVAA SKERSGVSLA ALKKALAAAG YDVEKNNSRI
81 KLGLKSLVSK GTLVQTKGTG ASGSFKLNKK AATGEAKPKA
121 KKAGAAKPKK AAGAAKKTKK ATGAATPKKT AKKTPKKAKK

161 PAAAAVTKKV AKSPKKAKAA KPKKAAKSAA KAVKPKAAKP

201 KVAKPKKAAP KKK

Figure 2. Amino acid sequence and phosphorylated sites of bovine
histone H1.2 as determined by mass spectrometry. Lysine residues
are marked red, and those identified as targets for the phosphor-
ylation by phosphoramidate (i.e. seven residues) are highlighted.
Out of the 305 peptides that were used to identify the protein,
14 peptides were reported to contain phospholysine.

PHPT1 also dephosphorylates phospholysine 23



Of the 305 tryptic peptides that were used to identify
the protein, 14 were reported to contain phospholy-
sine, and, of these, two contained phospholysine as
the C-terminal residue (both representing Lys-46).
This seems to be incompatible with the substrate
specificity of trypsin, and the interpretation of
Lys-46 as a target for phosphorylation may therefore
be questioned. However, a phosphorylation of at least
six different lysine residues seems to have been ver-
ified. Since only a fraction of each target lysine would
be phosphorylated at the low phosphoramidate con-
centration used, this number is compatible with the
total phosphorylation of 1.5 mol/mol protein.

MS spectra

Figure 3 shows the fragmentation pattern of a
phospholysine-containing peptide representing resi-
dues 82–90 of histone H1.2. The peptide was selected
as doubly charged, and if one follows the y-ion series an
almostcompleteladderisfound(onlytheC-terminalK
is missing). The bound phosphate seems to be quite
stable, and the expected phospho-fragments with
higher mass are all present both as doubly and singly
charged, implying that the phospholysine is capable of
carrying a plus charge even if phosphorylated. The
lysine-bound phosphate is removed between peaks
741.3 and 533.3 in the y-series, showing that the phos-
phorylated residue is Lys-85. On top of the figure the
small insert depicts the y- and b-ions series in a mini-
format, where the size of each staple is proportional to
the abundance of the corresponding fragment in the

main spectrum. The main spectrum also shows peaks
corresponding to 18 Da-losses of both y- and b-ions.

Discussion

The present study shows that phosphohistidine phos-
phatase (PHPT1) can dephosphorylate a chemically
phosphorylated histone H1 that does not contain
histidine residues. This is explained by the finding
that some of the lysine residues of histone H1 were
phosphorylated during the incubation with phosphor-
amidate and became good substrates of PHPT1.
Itisknownthatpeptide-bound lysineresiduescanbe

N-e-phosphorylatedbyphosphoramidate(38).Asseen
from the sequencing of the phosphorylated histone
H1.2 by MS (Figure 2), several lysine residues were
identified as targets for the phosphoramidate.
Kowalewska et al. found that fragmentation of phos-
pholysine peptides by CID gave a too low number of
fragments to establish the phosphorylation site and
suggested that this was caused by extensive neutral
losses of HPO3 (80 Da) and water (18 Da) (38). How-
ever,interpretationofthespectruminFigure3suggests
that not all phospholysine-containing peptides show
this extensive loss of HPO3 and water since it showed a
sufficientnumberofphosphorylatedfragmentstoallow
the identification of the phosphorylated site.
It is worth noting that most of the lysine residues of

histone H1, that were identified as targets for
phosphoramidate, are also subject to natural post-
translational modifications (39,40). Therefore, it is
reasonable to suppose that some of these sites may be

L L L K

533.3

7
4
1
.3

7
9
1
.3

9
1
1
.4

7
7
3
.4

8
9
3
.5

8
7
8
.4

7
2
3
.46
7
4
.4

6
9
2
.4

5
9
5
.2

5
7
9
.3

5
6
1
.3

4
9
4
.7

4
6
6
.3

4
4
6
.2

3
3
3
.1

2
8
4
.2

2
3
4
.1

2
1
6
.1

4
2
7
.7

4
9
2
.2 5
0
3
.7

4
5
6
.2

SVSKG

100

80

60

40

20

0
0 200 400 600 800 1000 –0.4 0.4

Error (Da)m/z

R
I

Figure 3. MS/MS spectrum showing the fragmentation pattern of one of the peptides obtained after trypsin treatment of histone H1 from
SignalChem. The y-ion series is shown in red, and the b-ions series is in blue. Also shown is y- and b-ions fitting with the loss of 18 Da in violet and
turquoise, respectively. Other observed but unspecified mached ions are grey. The small inset at the top shows only the y- and b-ions, with the
length of the staples representing the intensity of the ions.

24 P. Ek et al.



accessible also to enzyme-catalyzed phosphorylations,
e.g. by histone H1 kinases, such as those described by
Smith and co-workers (41-44) and Sikorska et al. (45).
The same sites, when phosphorylated, may then be
accessed also by PHPT1. One prerequisite for this to
happen would be that lysine-phosphorylated histone
H1, usually located in the nuclei, and PHPT1, usually
located in the cytosol, can meet at least temporarily in
the same cell compartment. Interestingly, it has
been shown by immunohistochemical methods that
part of the PHPT1 can be located also in the nuclei
(www.proteinatlas.org/search/PHPT1) (46).
PHPT1 could also dephosphorylate phosphopoly-

lysine at a rate similar to that of the phosphorylated
histone H1. Whether this means that a phospholysine
residue itself, rather than the amino acid sequence
around the phospholysine, defines the substrate
specificity of PHPT1 may be a subject of future
investigations. Of interest in this context is the finding
by Attwood et al. that the kinetics of the dephosphor-
ylation of short phosphohistidine-containing peptides
depends on the amino acid sequence around the
phosphohistidine (34).
A free a-amino group of an N-terminal amino acid

may be phosphorylated by phosphoramidate (47),
although to a lesser extent than the e-amino group
of lysine residues (38). We therefore cannot entirely
exclude the possibility of an N-terminal phosphory-
lation of histone H1 or polylysine. However, N-e-
phosphorylation actually occurred of histone H1, as
judged from the MS-spectra. A similar phosphoryla-
tion occurred of polylysine, as judged from the finding
that the phosphorylation increased almost four times
when a 90 kDa polylysine was compared to a 30 kDa
polylysine as phosphate acceptor.
The finding that PHPT1 shows activity toward

phospholysine in addition to phosphohistidine adds
to the fact that PHPT1 can also dephosphorylate free
phosphoramidate (20,34). This activity towards
phosphoramidate actually made Attwood and
Wieland suggest, in a recent review, that PHPT1
(PHP) ‘may not be quite so specific for phos-
phohistidine’ (48).
The enzyme is, however, not active against all N–P

bonds, since it did not cleave N-w-phosphoarginine
under conditions used in the present study. This
result raises the question of whether PHPT1 is in
fact similar to, or even identical with, the partially
characterized 13 kDa phosphoamidase described in
1999 by Hiraishi et al. (49). According to these
authors, the latter enzyme dephosphorylated NDPK
that was autophosphorylated on a histidine residue.
This is, however, in contrast to a result by
Klumpp et al. who, in their studies on the dephos-
phorylation of ATP citrate lyase, did not obtain any

dephosphorylation of autophosphorylated NDPK
by PHPT1 (25). Therefore, the current evidence
suggests that the two enzymes are not identical.
Other phosphatases, with activity against phospho-

lysine, have also been reported. Comprehensive
reviews on this matter have been published (50,51).
In addition to the 13 kDa phosphatase, Hiraishi et al.
have studied a bovine liver 56 kDa phosphatase that is
active toward free phosphohistidine and phospholy-
sine (52). The corresponding human enzyme has
been cloned and further characterized, and the chro-
mosomal location of its gene was determined to be
10q26.13 (53). According to a BLAST/Align search
at www.ncbi.nlm.nih.gov its amino acid sequence
shows no similarity to that of PHPT1. In addition,
the PHPT1 gene has a different chromosomal
location, i.e. 9q34.3 (20).
Wong et al. (54,55) investigated the presence of

phospholysine phosphatase activity in soluble rat tis-
sue extracts and in a partially purified form by using
phosphorylated polylysine as the probe. Interestingly,
the activity against this substrate in extracts from rat
liver corresponds to the activity of PHPT1 in pig liver
cytosol, as measured with a phosphohistidine peptide
as the substrate (20). To our knowledge, further
purification and characterization of the enzyme
described by Wong et al. have not been reported,
and its identity with PHPT1 is thus unknown.
In conclusion, the results of the present study

suggest that PHPT1 would be active against several
phospholysine-containing proteins in vivo. With the
exception of histone H1, which is already shown to be
enzymatically phosphorylated in vivo and in vitro
(41-44), such proteins remain to be identified. We
assume that the well-characterized phosphohistidine
phosphatase PHPT1 will become a valuable tool in
the investigation of the role also of phospholysine in
eukaryotic regulatory protein phosphorylation. The
value of this tool may add to future characterizations
of putative lysine kinases and to new phosphoproteo-
mic approaches of the type recently outlined for
phosphorylated lysine peptides (56).

Acknowledgements

Gunilla Pettersson and Elvy Netzel are gratefully
acknowledged for performing some of the experi-
ments in this work.

Funding

The work was supported by the Medical Faculty of
Uppsala University.

PHPT1 also dephosphorylates phospholysine 25

http://www.proteinatlas.org/search/PHPT1
http://www.ncbi.nlm.nih.gov


Declaration of interest: The authors report no
conflicts of interest. The authors alone are responsible
for the content and writing of the paper.

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	Abstract
	Introduction
	Materials and methods
	Materials
	Methods

	Results
	Phosphorylation of histone H1
	Dephosphorylation by PHPT1 of chemically phosphorylated histone H1
	Phosphorylated polylysine and its dephosphorylation by PHPT1
	PHPT1 and phosphoarginine
	MS-sequencing of chemically phosphorylated histone H1 identification of phosphorylated lysine residues
	MS spectra

	Discussion
	Funding
	Declaration of interest
	References