(Received for publication, June 7, 1995; and in revised form, September 19, 1995)
From the
Nucleoside-diphosphate kinase (ATP:nucleoside- diphosphate
phosphotransferase, EC 2.7.4.6; NDP kinase) is an important enzyme for
the maintenance of the correct cellular levels of nucleoside
triphosphates (NTPs) and their deoxy derivatives (dNTPs) and is
involved in the regulation of cellular development. The enzyme is under
the dual control of algR2 and algH in Pseudomonas
aeruginosa. We report here the purification and characterization
of a protein that dephosphorylates the phosphorylated intermediate form
of the P. aeruginosa NDP kinase (Ndk). Dephosphorylation of
Ndk phosphate leads to loss of its enzymatic activity. The 10.1-kDa
polypeptide shows 77% homology at the N terminus with the Spo0E
phosphatase, identified as a negative regulator of sporulation in Bacillus subtilis and 66% with the human Bax protein,
identified as an effector of programmed cell death. The phosphatase
termed Npp showed varied specificity toward phosphorylated Ndks from
different sources including human erythrocyte Ndk phosphate. Its
activity toward other histidine phosphates such as CheA or the
-subunit of succinyl-CoA synthetase or phosphoesters such as p-nitrophenyl phosphate was quite limited. Npp was stable at
room temperature up to 2 h and required Mg
for
activity. The presence of a phosphatase capable of dephosphorylating
the phosphorylated form of P. aeruginosa Ndk represents an
interesting and efficient mode of post-translational modification of an
enzyme crucial to cellular development.
Nucleoside-diphosphate kinase (ATP:nucleoside-diphosphate
phosphotransferase, EC 2.7.4.6: Ndk) is an important enzyme that
catalyzes the reversible transfer of the 5`-terminal phosphate from
NTPs to NDPs (or dNTPs to dNDPs) by a ping-pong enzyme
mechanism(1) . The enzyme utilizes an autophosphorylated
reaction intermediate (2) and catalyzes the final step in NTP
and dNTP synthesis, converting -phosphate bond energy (in the form
of ATP) from oxidative phosphorylation into synthesis of DNA and RNA
precursors and appears to be essential for growth of most types of
cells under aerobic conditions (3) . Ndk has been implicated in
regulating or effecting developmental changes in eukaryotic cells.
Reduced transcript levels for the human Ndk gene called nm23 were found to be associated with higher metastatic potential in
tumor cells(4, 5) . Expression of nm23 from a
constitutive promoter in highly metastatic murine tumor cell was found
to suppress tumor metastasis(6) , defining its role as a
suppresser of cancer metastasis. Null mutations in the ndk gene of Drosophila, termed awd, cause
abnormalities in development of the larvae leading to tissue necrosis
and death at the prepupal stage(5, 7) . In the slime
mold Dictyostelium discoideum, the ndk gene is
developmentally regulated with a sharp decrease in ndk transcript levels coinciding with the onset of the
starvation-induced developmental cycle(8, 9) . A gene
encoding a DNA-binding protein, PuF, which is required for the
expression of c-myc in vitro, is highly homologous to the
human ndk gene nm23-H2(10) . This implies
that an alternate form of Ndk, nm23-H2, may be involved in the
regulation of c-myc. The intermediate in NTP synthesis by Ndk
is a phosphorylated form of the enzyme where an active site histidine
is involved(2) , although it is now known that the enzyme
undergoes phosphorylation at internal serine residues as well, albeit
at a lower level than histidine
phosphorylation(11, 12) .
Very little is known
about how Ndk formation or its activity is regulated in eukaryotic and
prokaryotic cells. We have recently described the purification and
characterization of Ndk from P. aeruginosa that forms a
complex with succinyl-CoA synthetase (Scs) (13) . Both Ndk and
the -subunit of Scs require phosphorylation for their enzymatic
activity(13) . We have also shown that formation of Ndk and Scs
is positively regulated by two separate genes, algR2 and algH in P. aeruginosa(14) . In the algR2
algH double mutant, which has extremely low Ndk levels, NTP
formation is mediated by an alternative kinase, which is sensitive to
Tween 20. Thus while the wild type cells grow readily in the presence
of Tween 20, the algR2 algH double mutant cannot grow in its
presence, suggesting that either Ndk or the alternate kinase is
essential for NTP synthesis and therefore for cellular
growth(14) . In this paper, we report the presence of a
phosphatase that is highly active on the phosphorylated form of P.
aeruginosa Ndk and may regulate intracellular Ndk activity through
a phosphorylation/dephosphorylation mechanism.
Figure 1:
Purification of Npp, molecular mass
determination, and N-terminal sequence homology. The phosphatase was
purified as described under ``Experimental Procedures.'' The
-fold purification is described in Table 1. Active protein was
used either as such in assays or dialyzed against 3 1-liter
changes of buffer A containing 50% glycerol and stored frozen at
-70 °C until use. A, lane 1, molecular mass
markers; lane 2, 5 µg; lane 3, 10 µg of Npp. B, results of N-terminal sequence analysis (9 amino acids) of
the Npp and its homology with the Spo0E phosphatase as well as the
human Bax protein.
Figure 2:
Titration of the activity of the purified
Npp. A, dephosphorylation activity of Npp on Ndk-P from P.
aeruginosa. Lane 1, phosphorylated Ndk incubated without Npp for 5
min; lanes 2-9 represent incubation of the
phosphorylated Ndk with 2, 4, 6, 8, 10, 12, 14, and 16 ng of Npp,
respectively, for 5 min. B, plot of the dephosphorylation of P. aeruginosa Ndk-P with increasing concentrations of Npp,
demonstrating release of inorganic phosphate
(P).
Figure 3:
Effect of Npp on the 16- and 12-kDa forms
of Ndk. The two forms of Ndk were prepared as described under
``Experimental Procedures.'' The 16- and 12-kDa forms of Ndk
were autophosphorylated with [-
P]ATP,
unreacted ATP was removed by biospin column chromatography and the two
forms were subjected to different concentrations of phosphatase
treatment for 5 min. After the incubation period, samples were mixed
with 5.5 µl each of 4
SDS stop buffer, electrophoresed on a
15% SDS-polyacrylamide gel, and visualized by autoradiography on a
Kodak X-OMAT-AR film at room temperature after exposure for 1 h. Lane 1, 25 pmol of 16-kDa
P-labeled Ndk; lane
2, +9 pmol of Npp; lane 3, +20 pmol of Npp; lanes 4, 25 pmol of
P-labeled 12-kDa Ndk; lane 5, +9 pmol Npp; lane 6, +20 pmol
Npp.
Figure 4:
Effect
of Npp on the phosphorylated forms of Ndks from different sources.
Nucleoside-diphosphate kinase enzymes from P. aeruginosa, E. coli, Human erythrocytes, and Saccharomyces cerevisiae were labeled with [-
P]ATP according to
the protocol standardized for the Ndk from P. aeruginosa (as
described under ``Experimental Procedures''). The
[
-
P] ATP was removed by biospin column
chromatography, and the phosphorylated Ndks were treated with varying
concentrations of Npp for 5 min. Samples were electrophoresed on a 15%
SDS-polyacrylamide gel and visualized after autoradiography. A, lane 1, 25 pmol of P. aeruginosa Ndk,
autophosphorylated; lanes 2-5, incubated with 2, 4, 10,
and 14 ng of Npp, respectively. B, lane 1, 25 pmol of
human erythrocytes Ndk, autophosphorylated; lanes 2-5,
incubated with 2, 4, 10, and 14 ng of Npp, respectively. C, lane 1, 25 pmol of E. coli Ndk, autophosphorylated; lanes 2-5, incubated with 2, 4, 10, and 14 ng of
phosphatase, respectively. D, lane 1, 25 pmol of S. cerevisiae Ndk, autophosphorylated; lanes
2-5, incubated with 2, 4, 10, and 14 ng of Npp,
respectively. E, quantitative representation of the activity
of Npp on Ndk-phosphates from various
sources.
The results are shown in Fig. 5, A-C. Fourteen ng of Npp is able to completely
dephosphorylate 25 pmol of the P. aeruginosa Ndk phosphate (Fig. 2A) in 5 min. In the case of phosphorylated CheA (Fig. 5A), complete dephosphorylation required a
7-8-fold higher amount of Npp. For the dephosphorylation of the
-subunit of succinyl-CoA synthetase, even a 10-fold higher
concentration of Npp was not able to completely dephosphorylate the
protein within the 5-min period (Fig. 5B). Npp had no
effect on p-nitrophenyl phosphate, while the reaction with the E. coli alkaline phosphatase Type III (Sigma) was linear with
respect to time and substrate under the experimental conditions (Fig. 5C).
Figure 5:
A,
effect of Npp on phosphorylated CheA. The phosphatase was used over a
wide range of concentrations for analyzing its effect on phosphorylated
CheA. 25 pmol of phosphorylated CheA was incubated with 10-100 ng
of Npp for 5 min, as specified under Fig. 4. Lane 1, 25
pmol of labeled CheY; lane 2, +10 ng of Npp; lane
3, +14 ng of Npp; lane 4, +50 ng of Npp; lane 5, +100 ng of Npp. B, effect of Npp on the
-subunit of succinyl-CoA synthetase. 25 pmol of the
autophosphorylated
-subunit of succinyl-CoA synthetase was
incubated with Npp in a slightly higher concentration range than tested
for CheA. Lane 1, 25 pmol of labeled Scs; lane 2,
+10 ng of Npp; lane 3, +14 ng of Npp; lane
4, +50 ng of Npp; lane 5, +100 ng of Npp; lane 6, +150 ng of Npp. C, effect of Npp on p-nitrophenyl phosphate. 500 pmol of PNPP was incubated with
increasing concentrations of either alkaline phosphatase or Npp in a
final reaction volume of 20 µl. Picomoles of p-nitrophenol
formed in each case were quantitated after diluting the reaction to 1
ml and reading absorbance at 420 nm.
Figure 6:
A, stability of Npp at room temperature.
10 ng of Npp was aliquoted out into the standard assay buffer for Npp
and preincubated at room temperatures for periods ranging from 1 to 8
h. 12.5 pmol of phosphorylated P. aeruginosa Ndk was added at
the end of each incubation time point, and the reaction continued for
another 30 s. Reaction products were analyzed as described previously. Lane 1, 12.5 pmol of Ndk-P with no Npp; lane 2,
+0 s preincubated Npp; lane 3, + 2 h preincubated
Npp; lane 4, +4 h preincubated Npp; lane 5,
+6 h preincubated Npp; lane 6, + 8 h preincubated
Npp. B, activity of Npp at various pH values. The Npp
reactions were reconstituted as described previously in 25 mM HEPES-KOH buffers of pH values 6.5, 7.0, 7.6, 8.2, 9.0, and 10.0.
MgCl was added to a final concentration of 10 mM.
Npp activity is shown at assay buffer pH values of 6.5 (lane
1), 7.0 (lane 2), 7.6 (lane 3), 8.2 (lane
4), 9.0 (lane 5), and 10.0 (lane 6). Lane 7 shows control, 25 pmol Ndk-P without any Npp. C, effect
of EDTA on the dephosphorylating activity of Npp. EDTA was added to
final concentrations ranging from 0 to 100 µM and
preincubated with Ndk-P prior to the addition of Npp. Reaction products
were analyzed as described previously. Lane 1, 25 pmol of
Ndk-P; lane 2, + 14 ng of Npp; lane 3, + 25
µM EDTA + 14 ng Npp; lane 4, + 50
µM EDTA + 14 ng of Npp; lane 5, + 100
µM EDTA + 14 ng of Npp. D, reactivation of
Npp by Na
, Mn
, or Mg
after inactivation by 100 µM EDTA. To the assay
system containing Ndk-P, 100 µM EDTA and Npp that was
apparently inactive, Na
, Mg
, or
Mn
were added to final concentrations of 5-75
µM in the assay. Lanes 1, 7, and 13, 25 pmol of control Ndk-P + 100 µM EDTA
+ 14 ng Npp; lanes 2-6, 5, 10, 25, 50, and 75
µM Na
; lanes 8-12, 5, 10,
25, 50, and 75 µM Mn
; lanes 14-18, 5, 10, 25, 50, and 75 µM
Mg
, respectively.
Nucleoside-diphosphate kinase has been implicated in a variety of physiological and developmental effects in both prokaryotes as well as eukaryotes. However, the regulation of this enzyme in various systems is still far from understood. We have previously shown that the effect of mutations on two disparate genes, algR2 and algH, is also manifested in the form of a drastically reduced level of nucleoside-diphosphate kinase (Ndk) in P. aeruginosa(14) . We have recently reported that in the case of E. coli, the insertional inactivation of a gene rnk results in a drastic reduction of Ndk activity (18) and further that E. coli Ndk is primarily involved in GTP formation at low concentrations of NDPs(19) . The implication of Ndk in the preferential synthesis of GTP over other nucleotides is not entirely surprising. GTP plays a crucial role in regulating numerous cellular events such as signal transduction, elongation steps in protein biosynthesis, tubulin polymerization, and malignant transformation(20) . This nucleotide has also been proposed as having a general role in regulating anabolic processes involved in growth and cell proliferation(21) .
The fact that the levels of an enzyme of such importance to the cell need to be finely regulated need not be overemphasized. Moreover, for the type of reaction that this enzyme catalyzes, a remarkable degree of control and a highly stringent regulation can be achieved by regulating the ratio of phosphorylated enzyme to its non-phosphorylated counterpart. Recently, autophosphorylation on residues different from the active site histidine (notably serine) was reported for both the human (22) and the Myxococcus xanthus(2) enzymes. However, the manner in which phosphorylation/dephosphorylation activities modulate Ndk activity and consequently the ATP/ADP or the NTP/NDP ratios within the cell is unknown at present. The levels of any or all of these NTPs may vary at any given time of cell growth depending on a variety of factors including the oxygen tension and/or nutrient deprivation. The levels of enzymes that are constantly in demand by the cell can be more efficiently managed when placed under an efficient but fully reversible post-translational signal.
An intriguing but interesting aspect of the characterization of the P. aeruginosa Npp is its high level of identity at the N terminus with the human Bax protein, an effector of mammalian programmed cell death(16) . It has been suggested that both effector and repressor genes exist within each mammalian cell death pathway. One such mammalian gene has been identified, bcl-2, that functions as a repressor of programmed cell death(24) . Bcl-2 blocks cell death following a variety of stimuli. Bcl-2 conferred a death-sparing effect to certain hematopoietic cell lines following growth factor withdrawal(25, 26, 27) . Bcl-2 has also been shown to protect primary neuronal cell cultures from nerve growth factor withdrawal cell death(28) . Thus Bcl-2 may be needed to save the progenitor and long lived cells in a variety of cell lineages. Despite the progress in defining the physiological roles of Bcl-2, the biochemical basis of its actions remains largely unknown. Recent reports, however, have shown that another protein Bax can complex with Bcl-2 and that under conditions where Bax predominates, cell death is accelerated(16) . Nothing is known about the mode of action of Bcl-2 or Bax. It is tempting to speculate that the interaction of Bax and Bcl-2 is mediated by a phosphorylation/dephosphorylation cycle not very much unlike that seen in the case of the dephosphorylation of Ndk by the phosphatase.
The strong homology of N terminus amino acid sequence of Npp with the Spo0E phosphatase is also quite significant. The initiation of sporulation in Bacillus subtilis is under the control of the Spo0A transcription factor; this protein is a member of the response regulator class of the two component systems and is inactive unless phosphorylated(23) . Spo0A-P acts both as a repressor of certain vegetative genes and as an activator of certain genes required for the initiation of sporulation. The Spo0E protein, long known as a negative inhibitor of sporulation, was recently characterized as a phosphatase specific for Spo0A(23) . Overproduction of the Spo0E protein was known to severely inhibit sporulation, whereas deletion of this locus caused premature sporulation and accumulation of mutations in the phosphorelay. Given the central role that Ndk plays in the maintenance of NTP levels of the cell and the accumulating evidence that the enzyme is also crucial to development as well as differentiation, it is not entirely surprising that Npp shows significant N terminus sequence homology with a phosphatase that plays a pivotal role in the sporulation process in B. subtilis.
Isolation and characterization of the Npp gene is now ongoing in our laboratory, and it would be interesting to see if a similar gene might be characterized in the human cDNA library and also if there exists any type of functional identity between Npp and Spo0E. Given the highly conserved nature of Ndks from various sources (13) and the paucity of information on its regulation, further studies on the role of the Npp in energy metabolism are worth pursuing as well.