(Received for publication, April 27, 1995; and in revised form, July 20, 1995)
From the
The awd gene of Drosophila melanogaster encodes a nucleoside diphosphate kinase. Killer of prune
(Kpn) is a mutation in the awd gene which substitutes Ser
for Pro at position 97 and causes dominant lethality in individuals
that do not have a functional prune gene. This lethality is
not due to an inadequate amount of nucleoside diphosphate (NDP) kinase
activity. In order to understand why the prune/Killer of prune combination is lethal, even in the presence of an adequate NDP
kinase specific activity level, and to understand the biochemical basis
for the conditional lethality of the awd mutation, we generated second site mutations which revert
this lethal interaction. All of the 12 revertants we recovered are
second site mutations of the awd
gene.
Three revertants have deletions of the awd
protein coding region. Two revertants have substitutions of
the initiator methionine and do not accumulate KPN protein. Seven
revertants have amino acid substitutions of conserved residues that are
likely to affect the active site: five of these have no enzymatic
activity and two have a very low level of specific activity. These data
suggest that an altered NDP kinase activity is involved in the
mechanism underlying the conditional lethality of the awd
mutation.
Nucleoside diphosphate kinases (NDP kinase; EC
2.7.4.6) are ubiquitous enzymes that catalyze the formation of
nucleoside triphosphates and maintain equilibrium between nucleoside
triphosphate and nucleoside diphosphate pools. Cytosolic and
mitochondrial NDP kinases have been purified from a variety of tissue
sources(1) . cDNAs encoding several of these NDP kinases have
been cloned and sequenced. In some species two distinct cytosolic NDP
kinases have been cloned: human nm23-H1 and nm23-H2(2, 3) , rat
and
NDPK(4, 5) , mouse nm23-M1(6) and nm23-M2(7) , and spinach NDPK-I(8) and NDPK-II(9) . The two
cytosolic NDP kinases in each species are similar in amino acid
composition; the products of the two human genes, NDPK-A and NDPK-B,
are 88% identical in sequence, yet each has distinct apparent sizes on
SDS-PAGE gels and/or different isoelectric points. In Drosophila, cytosolic NDP kinase is encoded by the abnormal wing discs (awd) gene ((20) ;
GenBank(TM) accession number Y00226). The AWD protein is 78%
identical to each of the human NDP kinases, NDPK-A and NDPK-B. All NDP
kinases exist as multimers. The x-ray structure of the Myxococcus enzyme indicates that it exists as a tetramer(11) ,
whereas the x-ray structures of Dictyostelium(12) ,
and Drosophila enzymes(13) , indicate that these
enzymes form hexamers. Because Drosophila has only one NDP
kinase gene, the AWD/NDP kinase associates into homohexamers, whereas
the two human NDP kinases, NDPK-A and NDPK-B, form mixed
hexamers(14) .
NDP kinases act via a ping-pong mechanism.
The -phosphate of a nucleoside triphosphate is first transferred
to the active site histidine of the enzyme(15) , forming an
enzyme intermediate. The
-phosphate is then transferred from the
enzyme to a nucleoside diphosphate acceptor. It has been known for many
years that NDP kinases show little specificity toward the sugar or base
residue of nucleoside substrates. NDP kinases act upon purine or
pyrimidine ribonucleoside diphosphates or deoxyribonucleoside
diphosphates(1) . Recent crystallographic evidence has provided
an explanation for this broad range of substrate specificity. Unlike
the nucleotide binding sites found in GTP-binding proteins (16, 17) and other nucleotide-binding proteins, NDP
kinases have no glycine-rich phosphate binding loop or ``Rossmann
fold''(18) , and the enzyme has few direct contacts with
the base. The nucleotide substrate enters the active site, phosphate
first. The active site histidine is in a cleft, and conserved residues
lining the cleft primarily interact with the phosphate and ribose
groups of nucleotide substrates(11, 19) . This active
site configuration is conserved among the enzymes examined so far. Even
though the Myxococcus enzyme forms a tetramer and the Drosophila and Dictyostelium enzymes form hexamers,
the ADP binding site of the Myxococcus enzyme (11) is
remarkably similar to the ADP binding site of Dictyostelium NDP kinase (19) and Drosophila NDP
kinase(13) .
The Killer of prune (Kpn)
mutation of Drosophila is a point mutation in the awd gene (20) resulting in a substitution of proline at
position 97 for serine ((21) ; this paper). KPN/NDP kinase is
enzymatically active in extracts from awd individuals and the awd
mutation causes no phenotype by itself. The prune gene of Drosophila has been cloned but the biochemical
function of its product is unknown(22, 23) .
Individuals that cannot make a functional prune gene product
are viable, but they have prune-colored eyes rather than red-colored
eyes due to decreased amounts of red drosopterin pigments(24) .
In the absence of a functional prune gene product, the awd
mutation is lethal. Furthermore,
only one copy of the mutant awd
gene is
required for lethality of prune flies(25) . The cause
of this lethality is unknown. Lascu et al.(21) found
that purified KPN/NDP kinase is less thermostable at 56 °C than
purified wild-type AWD/NDP kinase and suggested that in awd
mutant larvae an accumulation of
KPN monomers occurs. The Pro
residue that is altered by
the awd
mutation occurs in the Kpn
loop, a structure that is conserved in all NDP
kinases(11, 12, 13) . In Dictyostelium(12) and in Drosophila(13) the hexamer
can be divided into a ``top'' trimer and a
``bottom'' trimer. The three Kpn loops from the top trimer
are in close proximity on the top of the hexamer as are the three Kpn
loops on the bottom of the hexamer. Part of the Kpn loop is thus found
at trimer subunit interfaces and may play a role in hexamer
stabilization. This structural information provides an explanation for
the reduced thermostability of KPN/NDP kinase purified from awd
mutant flies and its reduced
ability to renature after urea treatment(21) .
The Kpn loops
of Myxococcus, Drosophila and Dictyostelium have some
residues in the active site of the molecule. For example, Arg of Drosophila and the corresponding Arg
of Dictyostelium and Arg
of Myxococcus are
within the Kpn loop and form hydrogen bonds with the
phosphate of
ADP(11, 12, 13) . Indeed Tepper et al.(26) have shown that mutating Arg
to Ala in Dictyostelium NDP kinase reduces the specific activity to 0.5%
of normal. In this paper we describe a mutation at this site in Drosophila that also reduces NDP kinase specific activity.
Thus the Kpn loop may be important for both the integrity of the
hexamer and the active site. In order to understand the biochemical
basis of prune/Killer of prune lethality, we recovered second
site mutations which revert the prune/Killer of prune lethal
interaction and analyzed the molecular basis of these mutations as well
as the effects of these mutations on AWD/NDP kinase activity.
Figure 1: Purification of recombinant AWD protein. The AWD protein was purified as described under ``Experimental Procedures'' with samples of each purification step reserved for analysis on polyacrylamide gels. Proteins were fractionated on 15% polyacrylamide-SDS gels and detected by Coomassie Blue staining.
Figure 2:
Specificity of HA1 antibody. Extracts of
larvae were fractionated by SDS-PAGE (A) or IEF-agarose (B). Proteins were transferred to filters and immunoblotted
with HA1 antibody. AWD/NDP kinase is detected in wild-type larvae
(+) but not in mutant larvae(-). Mutant larvae were
homozygous for awd which is missing
the entire awd coding region.
Figure 3: Acidic modification of AWD/NDP kinase by incubation with dCTP. Purified AWD/NDP kinase was incubated with dCTP at indicated molar concentrations, fractionated by isoelectric focusing in an agarose gel, and immunoblotted with HA1 antibody. Arrows indicate pI of each species calculated from the position of markers.
Figure 4:
Reversal of dCTP acidic modification of
AWD/NDP kinase by incubation with ADP. Purified AWD/NDP kinase was
incubated with 10M dCTP and then incubated
with ADP at indicated molar concentrations. The protein was
fractionated by isoelectric focusing in an agarose gel and
immunoblotted with HA1 antibody.
Figure 5: Comparison of immunoreactivity and enzyme activity of the different isoforms. Extracts of wild-type larvae were fractionated by isoelectric focusing in an agarose gel, transferred to a filter, and either immunoblotted with HA1 antibody (left) or overlaid with a reaction mixture to detect NDP kinase activity colorimetrically (right).
Figure 6:
The distributions of different isoforms is
not altered in prune, awd, or prune/Killer of prune larvae. Larval extracts of the indicated
genotype were assayed for protein concentration. Equal amounts of
protein were fractionated by isoelectric focusing in an agarose gel,
transferred to a filter, and immunoblotted with HA1 antibody. Lane
1, yellow prune
/Y viable male
larvae; lane 2, yellow
prune
/Y;
awd
/awd
viable male larvae; lane 3, yellow
prune/yellow
prune
;
awd
/awd
viable female larvae; lane 4, yellow
prune
/Y;
awd
/awd
lethal
male larvae; lane 5, Canton-S wild-type male larvae; lane
6, homozygous null mutant yellow/Y;
awd
/awd
lethal male larvae; lane 7, purified recombinant
AWD/NDP kinase; lane 8, Canton-S wild-type male
larvae.
The
remaining nine revertants have a normal-size PstI fragment.
The initiator methionine in two of these revertants, awd and awd
, is changed
to lysine and valine, respectively, and these two revertants fail to
accumulate AWD/KPN protein. The remaining seven revertants contain
single point mutations which result in single amino acid changes ( Table 2and Fig. 7) in addition to the Kpn mutation. In awd
, the arginine at position
89 is changed to cysteine. In awd
, arginine at
position 106 is changed to cysteine. In awd
the
glutamic acid at position 130 is changed to lysine. In both awd
and awd
, the
aspartic acid residue at position 15 is changed to asparagine. In awd
, alanine at position 127 is changed to
threonine; and in awd
, aspartic acid at
position 122 is changed to asparagine (Table 2, Fig. 7).
Figure 7:
Amino acid replacements in awd revertants. Nucleotide changes and
names of mutations are indicated above the wild-type sequence; amino
acid replacements are indicated below the sequence. The revertants awd
and awd
have the identical nucleotide
change but are of independent origin.
Extracts from control larvae and each of these nine point mutant
revertants were fractionated by IEF and immunoblotted with HA1. All of
the revertants and controls were assayed as hemizygotes; they had one
revertant copy of the awd gene and one copy of awd
. We used awd
as a
positive control, since all of the point mutant revertants were induced
in an awd
mutant background. Three major
isoforms were detected in awd
hemizygotes with
approximate isoelectric points of pH 7.4, 7.1, and 6.8 (Fig. 8).
We used the awd coding region deletion allele awd
as a negative control. A very faint band is
detected in awd
homozygotes with an approximate
isoelectric point of pH 7.4 (Fig. 8). We interpret this faint
band to be residual maternally supplied NDP kinase(31) . The
same faint band is also detected in another negative control, awd
, and in all of the revertants. The mutant, awd
, has a normal awd coding region
but a mutation in the regulatory region which severely reduces awd transcription. (
)Neither awd
nor awd
revertant larvae accumulate AWD
protein (Fig. 8), which is expected from the loss of an
initiator methionine mutation. The level of NDP kinase specific
activity in these extracts is not greater than in awd
larvae (Table 2). The AWD protein in awd
revertant larvae (Arg
Cys, Table 2) and
in awd
revertant larvae (Arg
Cys, Table 2) gives rise to a single band on IEF
agarose gels which is indicative of nonphosphorylated subunits (Fig. 8). The level of NDP kinase activity in extracts from
either of these revertants is as low as the specific activity of the
null awd
mutant. The isoelectric point of awd
and awd
AWD protein
(6.1) is more acidic than unphosphorylated wild-type AWD due to the
replacement of Arg with Cys. The AWD protein in the awd
revertant (Glu
Lys, Table 2) produces multiple bands on IEF agarose gels, which is
indicative of phosphorylated subunits. However, this mutant has a NDP
kinase specific activity as low as that of the awd
null mutant. A more basic isoelectric point is expected for the
AWD protein in awd
revertant larvae because of
the replacement of an acidic residue with a basic residue, and indeed
in some preparations we detect a pI 8.0 isoform which is more basic
than the most basic isoform in control awd
larvae. The AWD protein in awd
and awd
revertant larvae (Asp
Asn, Table 2) gives rise to only a single band on IEF agarose
gels which is indicative of nonphosphorylated subunits (Fig. 8).
The AWD protein in these revertants is expected to be more basic due to
the loss of an acidic Asp residue; however, these revertant AWD
proteins have a more acidic isoelectric point than wild type (Fig. 8). Extracts from awd
and awd
have an NDP kinase specific activity as
low as that of the null awd
mutant. The AWD
protein in awd
revertant larvae (Ala
Thr, Table 2) has multiply phosphorylated hexamers
and has 20% of the enzyme activity of awd
.
Indeed we detect a major band with an isoelectric point of 7.4, which
is the same as the most basic band in awd
extracts. The AWD protein in awd
revertant larvae (Asp
Asn, Table 2) is
expected to be more basic due to the loss of an acidic Asp residue;
however, this revertant produces primarily a single more acidic
isoform. Extracts from awd
revertant larvae
have 20% of the awd
-specific enzyme activity.
For some of the revertants that should synthesize normal amounts of
protein, such as awd
, awd
, awd
, awd
, and awd
, reaction
with the antibody is more intense or less intense than with the
positive control (Fig. 8). This is either because the double
mutant protein has different stability than the KPN protein or because
the HA1 antibody preparation binds to the double mutant protein
differently than it does to the KPN protein.
Figure 8:
Revertants alter the accumulation or
isoelectric point of AWD/NDP kinase. Each lane represents a hemizygous
mutant larval extract. For each mutation indicated, larvae
transheterozygous for that mutation and awd were selected by their larval yellow phenotype. Mutant
larval extracts were assayed for protein concentration and equal
amounts of total protein were fractionated by agarose IEF and
immunoblotted with HA1 antibody. Samples of the positive control awd
/awd
and the negative control awd
/awd
were fractionated on both of two
gels.
The isoelectric point (8.3) of
the most basic isoform of purified, recombinant AWD/NDP kinase (Fig. 3) is more basic than the isoelectric point (7.7) of the
most basic isoform in extracts of fly larvae (Fig. 2B, Fig. 5, and Fig. 6). One possible explanation for this
difference in isoelectric point between AWD/NDP kinase produced in
bacteria and AWD/NDP kinase produced in flies is that the enzyme
produced in flies is stably phosphorylated on a residue other than the
active site histidine leading to a more acidic isoelectric point than
predicted by the amino acid composition. However, another possible
explanation for this difference is that the -amino group of the
amino-terminal residue of AWD/NDP kinase produced in flies is modified
which could also lead to a more acidic isoelectric point than that
predicted by the amino acid composition. This latter possibility is
made credible by the finding that the
-amino group of the
amino-terminal residue of rat NDP kinase-
is indeed
blocked(4) .
Seven
additional revertants accumulate mutant subunits with amino acid
substitutions. The mutant subunits in five of these seven awd, awd
, awd
, awd
, and awd
have no NDP kinase activity and as
hemizygotes in a prune
background, all five
of these mutations also cause an awd null phenotype
indistinguishable from awd
homozygotes. The
mutant subunits in the other two of the seven revertants that
accumulate KPN protein, awd
and awd
, have approximately 20% of the enzyme
activity of awd
. Both awd
and awd
heterozygotes are viable in a prune mutant background. So at a minimum 80% of KPN/NDP kinase
activity must be eliminated to escape lethality in the absence of prune gene function. In the presence of prune gene
function awd
hemizygotes are viable and have 28%
of wild-type NDP kinase activity; awd
hemizygotes produce some viable adults and have 6% of wild-type
NDP kinase activity. Thus the minimal amount of NDP kinase activity
required for viability is between 6 and 28% of the wild-type activity.
We arrived at a similar conclusion in a different experiment using a
promoter-awd cDNA transgene(31) .
The seven
revertants we recovered that accumulate enzymatically inactive or less
active AWD subunits affect six different conserved residues. Two of
those residues, Arg and Arg
hydrogen-bond to
the
phosphate of bound
nucleotides(11, 19, 38) . Glu
positions the active site
His
(11, 19, 38) . Tepper et
al.(26) have shown by site-directed mutation of the Dictyostelium cytosolic NDP kinase that alteration of any of
these three residues dramatically reduces enzyme activity, in complete
agreement with our results. The Arg
Cys and
Arg
Cys mutant proteins (from awd
and awd
,
respectively) have no activity and do not accumulate phosphorylated
subunits, which suggests that the amino acid substitutions in these
mutant proteins severely affect the initial binding of NTP substrate.
The isoelectric points of these mutant forms migrate according to the
predicted value, which is another indication that the mutants are not
phosphorylated. The Glu
Lys mutant protein in the awd
revertant does accumulate phosphorylated
forms of the protein so binding of NTP substrates must occur. Since the
turnover rate for the NDP kinase phosphorylation reaction is much
slower than the turnover rate for the dephosphorylation
reaction(1) , and since phosphorylated forms of the Glu
Lys mutant protein do accumulate, we expected to observe a
reduced enzymatic activity for this mutant. Yet instead of a reduced
level of enzymatic activity, we detect no significant activity for this
mutant. Our interpretation of these observations is that the active
site histidine is capable of becoming phosphorylated in such a mutant
yet at a much reduced rate, because the active site histidine is no
longer positioned properly by Glu
. A reduction of the
dephosphorylation rate must also occur to completely eliminate activity
of the mutant enzyme. We propose three mechanisms by which the
dephosphorylation reaction rate could be reduced. 1) Suboptimal
positioning of the phosphorylated histidine by the mutant residue might
affect the rate of phosphate transfer to bound NDP. 2) Migration of the
phosphate residue from the 1 to the 3 position of histidine, which is
the more stable form, might occur in the mutant protein and this
isomeric form might have a reduced rate of phosphate transfer.
3-Phosphohistidine residues have been identified previously (1) , but the affect of 3-phosphohistidine on the catalytic
activity of the enzyme has not been thoroughly examined. 3) The fact
that we observe negligible NDP kinase specific activity with this
mutant rather than simply a reduction in NDP kinase specific activity
is probably also a reflection of the charge interference by the Lys
substitution at position 130. The mutant Lys residue might stabilize
the phosphorylated intermediate, preventing its transfer to NDP. The
other mutations that we have isolated, Asp
Asn,
Ala
Thr, and Asp
Asn, also
reduce enzyme activity, and we propose that they also disrupt substrate
binding and/or catalysis. From the crystal structure data it is known
that Lys
hydrogen bonds to the sugar residue of
nucleotides(11, 19, 38) . We suggest that
replacement of Asp
by Asn in awd
and awd
disrupts this interaction and
causes the loss of enzyme activity (Table 2). The fact that we do
not observe multiple phosphorylated forms with the two Asp
Asn revertants suggests that this is indeed the case and
that the Lys
hydrogen bond is very important for
nucleotide binding. The Asp
Asn mutant proteins
accumulate a more acidic form rather than the more basic form expected
from the loss of an acidic Asp residue. One possible explanation for
this is that the acidic form accumulating is actually the completely
phosphorylated hexamer. If this explanation is correct, then the
Asp
Asn substitution preferentially affects NDP
binding and subsequent dephosphorylation of the enzyme intermediate.
The replacement of Ala
by Thr in awd
results in an 80% reduction of enzyme activity. We suggest that
since Ala
and Glu
residues are on the same
face of the
4
-helix, the Ala
change might
disrupt the interaction of Glu
and His
. The
fact that we see multiply phosphorylated forms with the Ala
Thr protein and with the Glu
Lys
mutation is in agreement with this suggestion. Furthermore, since the
Ala
Thr substitution is much less drastic a change
than Glu
Lys, one would expect a less severe
consequence from an Ala
Thr substitution. This
expectation is met by a less reduced specific activity, a less severe
phenotype, and not as much accumulation of the phosphorylated
Ala
Thr enzyme intermediates as in the Glu
Lys revertant. In addition, no change in the isoelectric
banding pattern is predicted from this amino acid substitution, and no
difference in pI from wild-type is observed. Finally, the Asp
Asn replacement in awd
causes an
80% reduction of enzyme activity and accumulation of an acidic form. We
suggest that this change interferes with the transfer of phosphate from
His
to nucleoside diphosphates leading to the
accumulation of hexamers in which all of the subunits are
phosphorylated, analogous to the awd
Glu
Lys revertant. An alteration of the side
chain interaction between Ser
with the active site
His
might occur because of the Asp
Asn substitution. This could explain why an acidic isoelectric form
accumulates from the Asp
Asn revertant (Fig. 8) despite the loss of an acidic residue (Fig. 7).
The accumulation of hexamers in which all of the subunits are
phosphorylated is never observed in AWD/NDP kinase extracted from
wild-type or awd
larvae and is only observed in
purified AWD/NDP kinase that is incubated with extremely high
concentrations of nucleoside triphosphates (Fig. 3).
Three
different hypotheses have been presented to account for the conditional
dominant lethality of the awd mutation in a prune background. The first was presented by Sturtevant (25) who discovered the phenomenon. He proposed that a
substrate of the protein encoded by the prune gene accumulates
in prune mutants and can be converted into a toxic substance
by the awd
gene product but not by its wild-type
counterpart. The second hypothesis was presented by Teng et al.(22) and elaborated by Ruggieri and McCormick (39) and Hackstein(40) . They proposed that in awd
mutants, NDP kinase is hyperactive and
deregulated. The transcription of awd
is
certainly not deregulated; it is identical to the transcription of
wild-type awd(20) . If it is meant that KPN/NDP kinase
is hyperactive on normal substrates, then this hypothesis is not
supported by fact that NDP kinase specific activity is slightly less in awd
mutant larvae (Table 2). However, if
by hyperactive it is meant that in awd
mutants,
KPN/NDP kinase has altered substrate specificity then this hypothesis
becomes identical to Sturtevant's hypothesis. The third
hypothesis was presented by Lascu et al.(21) and
modified by Chiadmi et al.(13) . They hypothesized
that the neomorphic function of the mutant KPN protein results from
deleterious effects of monomeric (or dimeric) NDP kinase that is
proposed to accumulate in awd
larvae. This
hypothesis was based on their observations that KPN/NDP kinase purified
from awd
homozygotes binds to normal substrates
only slightly differently than wild-type protein, but that this mutant
form of AWD/NDP kinase is more thermolabile and fails to renature after
denaturation in urea. Their hypothesis is consistent with the idea
based on the x-ray structure that the Kpn loop is involved in subunit
interactions(12) . However, there is no evidence of
accumulation of monomeric NDP kinase either in viable awd
homozygous larvae or in prune/Killer of
prune lethal larvae (this paper).
Our hypothesis for the
conditional dominant lethality of awd (Fig. 9) is similar to the one originally proposed by
Sturtevant in 1956 and is based on the data presented here implying
that revertants we have recovered affect the enzymatic function of
KPN/NDP kinase. We propose that the mechanism of prune/Killer of
prune lethality depends on altered substrate specificity of
AWD/NDP kinase subunits carrying the Pro
Ser amino
acid substitution caused by the awd
mutation. As
with other NDP kinases(1) , wild-type AWD/NDP kinase has a
broad range of substrate specificity. The Kpn loop defined by Dumas et al.(12) positions the nucleotide binding cleft of
the enzyme and indeed moves upon substrate binding(11) . Our
model is that the Pro
Ser substitution within the
Kpn loop alters the nucleotide binding cleft, allowing an even broader
range of substrate specificity. In a prune
background no accumulation of potentially harmful abnormal
substrates occurs. However, according to our hypothesis, in a loss of
function prune mutant, a substrate accumulates that can be
phosphorylated by KPN mutant subunits of NDP kinase but cannot be acted
upon by wild-type AWD/NDP kinase. The inability of wild-type NDP kinase
to act upon this hypothetical substrate explains why loss of prune function is normally not lethal. The dark eye color of prune mutant adults is due to loss of pteridine eye pigments which are
derived from GTP. Hackstein (40) has reported altered
metabolism of guanosine injected into prune mutants. One of
these metabolites could represent the hypothetical substrate we propose
accumulates in prune mutants. According to this
interpretation, prune/Killer of prune lethality is due to
harmful effects caused by the phosphorylation of a molecule that
accumulates in prune mutants. Ultimate proof of this
hypothesis awaits determination of the enzymatic activity of the prune gene product and the identities of its normal
substrates.
Figure 9:
Model
of lethal interaction of prune and awd. A, wild type. The product
of prune converts substrate X to Y; the product of awd converts NDPs to NTPs. B, awd
mutant. The product of prune converts substrate X
to Y; the product of awd converts NDPs to NTPs. C, prune mutant. Substrate X accumulates, because it cannot be
converted to Y; the product of awd converts NDPs to NTPs. D, prune;awd
/awd
double mutant. Substrate X accumulates because it cannot be
converted to Y; the product of awd converts NDPs to NTPs and
phosphorylates substrate X converting it to a toxic
substance.