M I N I R E V I E WPhosphorylation and dephosphorylation of histidine residues in proteins Susanne Klumpp and Josef Krieglstein Abteilung Biochemie und Institut fu¨r Pharmakologie und To
Trang 1M I N I R E V I E W
Phosphorylation and dephosphorylation of histidine residues
in proteins
Susanne Klumpp and Josef Krieglstein
Abteilung Biochemie und Institut fu¨r Pharmakologie und Toxikologie, Fachbereich Pharmazie, Philipps-Universita¨t Marburg, Germany
Protein phosphorylation is a key mechanism for intracellular
signal transduction in both prokaryotic and eukaryotic cells
Vertebrate proteins are prevalently phosphorylated on side
chains that contain a hydroxyl group, such as serine,
thre-onine and tyrosine residues In the past decade, however, an
increasing number of examples of histidine phosphorylation
has been described Because acid treatment of
phospho-proteins during purification and detection of phosphoamino
acid analysis is routine, O-phosphomonoesters have been
studied more often, and the existence of acid-labile
phos-phates has been largely overlooked The latter class of
N-phosphoamidates may well be more widespread than is
generally believed, even though the O-phosphates remain the
major class in terms of quantity and extent of distribution in
proteins Phosphohistidine currently is estimated to be 10- to 100-fold more abundant than phosphotyrosine, but less abundant than phosphoserine [Matthews, H.R (1995) Pharmac Ther 67, 323–350.] This minireview briefly summarizes the extensive knowledge of the key mechanisms and functions of phosphohistidine in bacteria It also des-cribes the still limited, yet increasing, data from homologs of the bacterial two-component system Finally, novel mechanisms of phosphorylation and dephosphorylation of histidine residues not related to the two-component system are described
Keywords: signal transduction; histidine; kinase; phos-phatase; two-component system
C H E M I S T R Y , S T A B I L I T Y A N D
D E T E C T I O N O F P H O S P H O H I S T I D I N E
Whereas phosphorylation of serine, threonine or tyrosine
results in the formation of a phosphoester linkage,
phos-phorylation of histidine residues occurs on nitrogen atoms,
producing a phosphoramidate bond Phosphohistidines
have a large standard free energy of hydrolysis making
them the most unstable of any known phosphoamino acid
(reviewed in [1]) This explains why phosphohistidines are
utilized as enzyme intermediates, for example, in the
catalytic mechanism of succinyl-CoA-synthetase or
glucose-6-phosphatase On the other hand, in histone H4,
1-phosphohistidine at residue 75 is relatively stable, having a
half-life of 12 days at room temperature and pH 7.6 [2]
Those examples clearly demonstrate that the stability of
phosphoramidate bonds in proteins is influenced by
neigh-boring amino-acid residues and thus strongly varies
depending on the nature of the protein Accordingly,
histidine phosphatases may be needed or not
Phosphohistidine mostly goes undetected in
conven-tional studies of protein phosphorylation because of
instability of the phosphate-nitrogen bond in acid solutions
as used for routine phosphoamino acid analysis In
contrast, phosphoserine, phosphothreonine and
phospho-tyrosine resist acid treatment (Table 1) [3] The detection of O-phosphates in acid hydrolysates of proteins is possible because the hydrolysis of the phosphomonoester bonds is considerably slower than the hydrolysis of peptide bonds Accordingly, no significant loss of phosphoryl groups is observed after precipitation of O-phosphorylated proteins
by trichloroacetic acid as commonly used for phosphatase activity measurements Phosphoserine and phosphothreo-nine are labile under alkaline conditions unlike phospho-tyrosine, which provides a means to differentiate between these molecules Phosphoramidates are extremely acid labile but relatively base stable, except for phosphoargi-nine In contrast to the O-phosphates, all N-phosphates are unstable to neutral hydroxylamine, and pyridine also catalyzes their hydrolysis [3]
One- and two-dimensional thin layer chromatography procedures for separation of phosphohistidine from phos-phoserine, phosphothreonine and phosphotyrosine have been established previously [1,3] Free phosphohistidine is rapidly hydrolyzed, and is thus not commercially available
It can be synthesized easily using polyhistidine/phosphoryl-chloride or L-histidine/potassium phosphoramidate [4] Ion-exchange resins and reversed-phase column chroma-tography are useful to separate phosphohistidine from all other phosphoamino acids and also suitable to resolve the isomers of phosphohistidine [1,4]
The simplest approach to detect phosphohistidine in
32P-labeled proteins is to fractionate the proteins on a poly-acrylamide gel and blot the separated proteins to a poly (vinylidene difluoride) membrane This membrane is then placed directly in 1MKOH and incubated at 55°C for 2 h, dried and autoradiographed [3,5] Bands remaining repre-sent phosphotyrosine and phosphohistidine/phospholysine
Correspondence to S Klumpp, Abteilung Biochemie, Fachbereich
Pharmazie, Marbacher Weg 6, D-35032 Marburg, Germany.
Fax: + 49 6421 282 6645, Tel.: + 49 6421 282 6646,
E-mail: klumpp@mailer.uni-marburg.de
(Received 6 August 2001, revised 21 September 2001, accepted 26
September 2001)
Trang 2Additional treatment of the membrane with 6M HCl at
55°C for 2 h leaves only phosphotyrosine Diethyl
pyro-carbonate is the most frequently used histidine modifying
reagent preventing subsequent phosphorylation of histidine
residues
Screening for proteins containing N-linked phosphate
with antibodies to phosphohistidine is not yet possible
Attempts to prepare antibodies to derivatives or conjugates
of phosphohistidine have not been successful However,
many of the commercially available anti-phosphotyrosine Ig
also recognize phosphohistidine
P H O S P H O H I S T I D I N E I N B A C T E R I A :
T H E T W O - C O M P O N E N T S Y S T E M S
In prokaryotic signaling, the predominant phosphorylation scheme is referred to as Ôtwo-componentÕ system [6,7]
It frequently is involved in linking an extracellular stimulus such as changing osmolarity, oxygen, nitrogen or phospho-rus levels to gene regulating events, but also affects differentiation and other functions, such as chemotaxis Two-component systems are abundant in most eubacte-ria, in which they typically constitute at least 1% of encoded proteins The Escherichia coli genome encodes 62 two-component proteins Two-two-component systems are present
in both Gram positive and Gram negative bacteria
In addition to housekeeping functions, they also control expression of toxins and other proteins important for pathogenesis
The two-component signal transduction pathway consists
of a sensor that is connected to a regulator through histidine phosphorylation and a subsequent phosphotransfer event
to aspartate The response time is quite fast The two-component system comprises several characteristic domains usually structured on two conserved proteins: a histidine kinase/sensor and a response regulator that are phosphor-ylated at histidine and aspartate residues, respectively (Fig 1) Stimuli, detected by a sensor domain of the histidine kinase, regulate histidine kinase activities The histidine kinase catalyzes an ATP-dependent trans-auto-phosphorylation reaction in which one subunit of the dimer phosphorylates a specific histidine residue within the other
Table 1 Chemical stability of phosphorylated amino acids +, stable
phosphoamino acid; –, labile phosphoamino acid.
Stability in Nature of phosphoamino acid Acid Alkali
O-Phosphates
N-Phosphates
Acyl-Phosphate
Fig 1 The two-component phosphotransfer scheme This typically consists of a dimeric transmembrane sensor histidine kinase (component I) and a cytoplasmic response regulator (component II) However, there are variations, e.g the cytosolic histidine kinases CheA, and multicomponent phosphorelay systems, which consist of even three proteins: a hybrid histidine kinase with an additional response regulator domain at the C-terminus; a separate histidine-containing phosphotransfer protein that serves as a histidine phosphate intermediate; and the response regulator The two-component histidine kinase domain is a module of 250 amino acids that has four conserved blocks of amino-acid sequences located within the ATP-binding domain (N, G 1 , F and G2) Similarly, the response regulator domain can be identified from the number and spacing of conserved aspartate, lysine, and hydrophobic residues in a module of 120 amino acids Abbreviation: TM, transmembrane.
Trang 3subunit resulting in a phosphoimidazole The response
regulator then catalyzes transfer of the phosphoryl group
from the phosphohistidine to one of its own aspartate
residues Phosphorylation of the conserved regulatory
domain of the response regulator activates an effector
domain that elicits the specific output response, for example,
change in flagella motion or change in transcription
Structures of the bacterial histidine protein kinase
cata-lytic domains are unlike those of any previously
character-ized serine-, threonine- or tyrosine kinase [8,9] However, the
histidine kinase structures are related to ATPase domains of
the type II topoisomerase gyrase B and the chaperone
Hsp90 A highly conserved glutamic acid residue is
predic-ted to be involved in the catalytic mechanism of the ATPase
enzymes This glutamic acid is not present in the histidine
kinase active site, which might explain why members of this
superfamily function as kinases and others as ATPases
T W O - C O M P O N E N T S Y S T E M S I N Y E A S T ,
A M O E B A , F U N G I A N D P L A N T
Although two-component pathways are common in
bacteria, evidence for their existence in eukaryotes is scarce
Starting no more than a decade ago, the field of reversible
histidine phosphorylation in eukaryotes finally is beginning
to emerge By aligning members of the bacterial
two-component histidine kinase family, oligonucleotide primers
were designed to amplify the kinase domain This approach
proved successful to clone homologs to the bacterial
histidine kinases and response regulators in yeast, amoeba,
fungi and plants
The best documented linkage between prokaryotic and
eukaryotic two-component signal transduction mechanisms
is for the osmoregulation system in yeast The SLN1 and
SSK1 proteins of Saccharomyces cerevisiae have sequence
similarities to both the histidine kinase and the response
regulator proteins from bacteria, respectively [10,11]
Gen-etic analysis of site-directed mutants indicates that SLN1/
SSK1 act in the same manner as do its bacterial
counter-parts, i.e through formation of a phosphohistidyl enzyme
followed by phosphotransfer to an aspartyl residue on its
response regulator Interestingly, this histidine protein
kinase from yeast is part of a signaling cascade in which
traditional eukaryotic mitogen-activated protein kinases
participate as downstream elements (HOG1 MAPK
cascade)
The gene dokA from the slime mold Dictyostelium
discoideum codes for a homolog of the bacterial hybrid
histidine kinase family which is defined by the presence of
conserved amino-acid sequence motifs corresponding to an
N-terminal receptor domain, a central histidine kinase and a
C-terminal response regulator [12] DokA mutants are
deficient in the osmoregulatory pathway, resulting in
premature cell death of this amoeba under high osmotic
stress The predicted protein sequence of the gene nik-1 from
the fungus Neurospora crassa also shares homology with
both the kinase and response regulator modules of
two-com-ponent signaling proteins [13] Deletion studies revealed that
Nik-1 is involved in proper hyphal development In plants,
the simple gas ethylene (C2H4) serves as a hormone with
profound effects on growth and development The
C-terminal half of the ethylene response protein ETR1 in
the plant Arabidopsis thaliana is similar in sequence to both
components of the prokaryotic family of signal transducers known as the two-component system [14,15] Similar to SLN1, ETR1 also is involved in a MAPK cascade One has to bear in mind, however, that the statement of dealing with analogs of the prokaryotic two-component systems so far is based on homology cloning resulting in amino-acid similarity and based on functional studies employing deletion/mutations Histidine phosphorylation
of the isolated or recombinant proteins, as extensively performed for the bacterial histidine kinase CheA, has not been studied So far, only one protein histidine kinase has been purified from eukaryotes on the basis of activity measurements The enzyme was isolated from S cerevisiae [16]
H I S T I D I N E P H O S P H O R Y L A T I O N
A N D D E P H O S P H O R Y L A T I O N
I N M A M M A L I A N S Y S T E M S
Until now, two-component proteins have not been identi-fied in animals and are not encoded by worm, fly or human genomes It therefore has been suggested that the building blocks of the two-component systems in lower organisms may provide a target for the development of antibiotics directed against both fungal and bacterial pathogens in vertebrates
Two eukaryotic genes have been discovered whose predicted products exhibit limited structural homology to the bacterial histidine protein kinases, however, functional homology could not be demonstrated The gene which encodes branched-chain a-ketoacid dehydrogenase kinase (BCKDH kinase) was cloned in 1992 [17] The sequence of this enzyme shows no resemblance to any eukaryotic protein kinase The closest homologs to BCKDH kinase are found among the histidine protein kinases of bacteria Direct evidence for histidine phosphorylation of BCKDH kinase still is lacking In contrast, BCKDH kinase auto-phosphorylates on serine and auto-phosphorylates its physiolo-gical substrate, branched-chain a-ketoacid dehydrogenase,
on a pair of serine residues as well Pyruvate dehydrogenase kinase from rats is another example of a protein with weak resemblance to histidine protein kinases from bacteria As described for BCKDH kinase, pyruvate dehydrogenase kinase phosphorylates proteins on hydroxyl amino acids Although there is no evidence for the existence of two-component systems in vertebrates yet, there are numerous reports on histidine phosphorylation in vertebrate proteins The description of phosphohistidine in mammals started in the 1970s when Smith’s group [18] studied a rat nuclear protein kinase that phosphorylates histone H4 on histidine
In the 1990s, Motojima & Goto [19] and Hedge & Das [20] were studying the proteins p36 and p38, respectively The proteins may be identical: both proteins are phosphorylated
in response to the presence of Ras protein and guanine nucleotides, and the phosphorylation occurs on a histidine residue Noiman & Shaul [21] developed a protocol for rapid detection of histidine phosphoproteins in cellular crude extracts Running the phosphorylation reactions in the presence of 5 mMEDTA (instead of Mg2+) exclusively results in histidine phosphorylation
In 1995, a novel pathway for activation-dependent signal transduction in vertebrates was introduced [22] A ligand-induced cascade generates phosphohistidine in platelets
Trang 4In addition to well known phosphorylation on serine,
threonine and tyrosine residues, thrombin and collagen lead
to the phosphorylation of histidine in the cytoplasmic tail of
P-selectin The appearance and disappearance of
phos-phohistidine on P-selectin are very rapid
The next finding of an intracellular signaling system
involving histidine phosphorylation in mammals appeared
in 2000 [23] A 37-kDa protein from airway epithelia is
phosphorylated on histidine and was identified as annexin I
It is a member of a family of Ca2+-dependent
phospholipid-binding proteins whose phosphorylation is regulated by the
chloride-ion concentration The failure of annexin I to
autophosphorylate indicates that it is a substrate for a
distinct yet undiscovered histidine kinase
Histidine is an important catalytic residue in many
enzymes In some cases, the histidine is covalently modified
during the reaction In a further subset, the modified form is
phosphohistidine Such phosphohistidine intermediates are
not discussed in this article
D E P H O S P H O R Y L A T I O N
O F P H O S P H O H I S T I D I N E
In prokaryotes
Many bacterial two-component histidine kinases are
bifunctional, having both kinase activity (acting on
histi-dine) and phosphatase activity (acting on
phosphoaspar-tate) Response regulators are the targets for the histidine
kinase phosphatases In the case of EnvZ, the osmosensor in
E coliand its cognate response regulator OmpR [24], it has
been suggested that OmpR-P is regulated by the OmpR-P
phosphatase activity, whereas the OmpR kinase activity is
maintained at a constant level Similarly, in the CheA
chemotaxis system, dephosphorylation of the
phospho-CheY response regulator is modulated by the CheZ
phosphatase
The SixA protein from E coli is a prokaryotic
phos-phohistidine phosphatase discovered recently [25] SixA
plays a role in down-regulation of the ArcB-to-ArcA
phosphorelay under certain anaerobic respiratory
condi-tions This histidine phosphatase activity is directed towards
the phosphotransmitter domain of the bacterial hybrid
histidine kinase SixA consists of 161 amino acids and has
an arginine-histidine-glycine signature at the N-terminus,
which presumably functions as a nucleophilic
phosphoac-ceptor
In mammals
Because a mammalian histidine kinase is not available yet,
all published studies of protein histidine phosphatases in
vertebrates so far have been carried out with histone H4
phosphorylated by the histidine kinase purified from yeast
Among the classical eukaryotic protein phosphatases,
protein tyrosine phosphatases and the serine/threonine
phosphatase type 2B (calcineurin) do not dephosphorylate
H4 phosphorylated on histidine The serine/threonine
protein phosphatases type 1, type 2A and type 2C, in
contrast, are highly active against phosphohistidine in
histone H4 (reviewed in [1]) Intriguingly, none of the
serine/threonine phosphatases acting on histidine
phosphor-ylated histone, hydrolyze phosphohistidine using the
bacterial histidine kinase CheA autophosphorylated on histidine 48 as a substrate [26]
In 2000, SixA was the first bacterial histidine phosphatase identified implicated in the histidine to aspartate phospho-relay (see above [25]) In the meantime, the first mammalian protein histidine phosphatase has also been discovered [26] Both enzymes are of low apparent molecular mass (17 and
14 kDa, respectively), hence similar in size to eukaryotic low molecular mass protein tyrosine phosphatases But the proteins are clearly distinct Vertebrate protein histidine phosphatase (PHP1) was isolated from rabbit liver Its amino-acid sequence shows no resemblance to any phatase described so far Furthermore, inhibitors of phos-phatases acting on phosphoserine, phosphothreonine and phosphotyrosine residues had no effect Protein histidine phosphatase is present in a variety of species from human to nematodes, but absent in bacteria It is highly expressed throughout different tissues ATP-citrate lyase known to undergo autophosphorylation on histidine [27] as well as external phosphorylation on histidine via nucleoside diphosphate kinase [28], is the first vertebrate substrate identified for protein histidine phosphatase [26]
S U M M A R Y
Knowledge of the phosphorylation and dephosphorylation
of histidine residues in bacterial proteins is vast In contrast, very little is known about the reversible phos-phorylation of histidine in vertebrates We have recently learned that the dogma of histidine phosphorylation/ dephosphorylation in bacteria on the one hand vs serine/ threonine and tyrosine phosphorylation/dephosphorylation
in vertebrates on the other, is no longer realistic [29]
It may be time to look for the more transiently phosphor-ylated amino acids, histidine and aspartic acid, as protein modifications resulting in mammalian signal transduction The few reports on that topic published within the last decade look promising
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