(Received for publication, July 28, 1994; and in revised form, October 19, 1994)
From the
The dihydrolipoyl acetyltransferase (E2) component of
the mammalian pyruvate dehydrogenase complex forms a 60-subunit core in
which E2's inner domain forms a dodecahedron shaped
structure surrounded by its globular outer domains that are connected
to each other and the inner domain by 2-3-kDa mobile hinge
regions. Two of the outer domains are 10 kDa lipoyl domains, an
NH
-terminal one, E2
, and, after the
first hinge region a second one, E2
. The pyruvate
dehydrogenase kinase binds tightly to the lipoyl domain region of the
oligomeric E2 core and phosphorylates and inactivates the
pyruvate dehydrogenase (E1) component. We wished to determine
whether lipoyl domain constructs prepared by recombinant techniques
from a cDNA for human E2 could bind the bovine E1
kinase and, that being the case, to pursue which lipoyl domain the
kinase binds. We also wished to gain insights into how a molecule of
kinase tightly bound to the E2 core can rapidly phosphorylate
20-30 molecules of the pyruvate dehydrogenase (E1)
component which are also bound to an outer domain of the E2
core. We prepared recombinant constructs consisting of the entire
lipoyl domain region or the individual lipoyl domains with or without
the intervening hinge region. Constructs were made and used both as
free lipoyl domains and fused to glutathione S-transferase
(GST). Using GSH-Sepharose to selectively bind GST constructs, tightly
bound kinase was shown to rapidly transfer in a highly preferential way
from intact E2 core to GST constructs containing the E2
domain rather than to ones containing only the E2
domain.
GST
E2
-kinase complexes could be eluted from
GSH-Sepharose with glutathione. Delipoylation of E2
by treatment with lipoamidase eliminated kinase binding
supporting a direct role of the lipoyl prosthetic group in this
association. Transfer to and selective binding of the kinase by E2
but not E2
was also
demonstrated with free constructs using a sucrose gradient procedure to
separate the large E2 core from the various lipoyl domain
constructs. E2
but not E2
increased the activity of resolved kinase by up to 43%. We
conclude that the kinase selectively binds to the inner lipoyl domain
of E2 subunits and that this association involves its lipoyl
prosthetic group. We further suggest that transfer of tightly bound
kinase between E2
domains occurs by a direct
interchange mechanism without formation of free kinase (model
presented). Such interdomain movement would explain how a kinase
molecule can rapidly phosphorylate a large complement of pyruvate
dehydrogenase tetramers which are bound throughout the surface of the E2 oligomer.
The mammalian pyruvate dehydrogenase complex (PDC) ()is a large assembly composed of six components with nine
distinct subunits. Four of these components execute the overall
reaction through a series of steps linked by cofactor-mediated active
site coupling: E1, the pyruvate dehydrogenase component; E2, the dihydrolipoyl acetyltransferase component; E3, the dihydrolipoyl dehydrogenase component; and protein X,
the E3 binding component(1, 2, 3) .
Like E2 subunits, protein X contains a lipoyl domain, and its
prosthetic group participates in the middle 3 of the 5 step reaction
series catalyzed by PDC. Control of the PDC reaction is critically
important for regulating cellular fuel utilization, and PDC activity is
controlled primarily by a highly regulated
phosphorylation-dephosphorylation cycle. This regulatory cycle is
carried out by dedicated kinase and phosphatase components that operate
to reduce and increase the activity of the complex through
phosphorylating and dephosphorylating, respectively, the E1
component.
The E2 component has a central role both in the
organization of the complex and in supporting the function of the
kinase and the phosphatase(1, 4) . Mammalian
PDCE2 subunits are highly segmented structures with
seven structural regions consisting of four globular domains connected
by three extended linker or hinge regions (1, 2, 3) . Sixty of E2's
COOH-terminal domain assemble to form a dodecahedron shaped inner core
structure that is surrounded by the remaining three globular domains
flexibly connected by the three hinge regions. Along with other
components and considerations, the domain structure of E2 is
modeled in Fig. 5and Fig. 7. At the
NH
-terminal end of E2, there are two
10-kDa
lipoyl domains, E2
and E2
connected by a
30 amino acid linker or hinge region (H1). In
all
-keto acid dehydrogenase complexes, one or more lipoyl domain
of the E2 component functions in ferrying the intermediate
forms of their pendant lipoyl group between active sites. The mobile
hinge regions allow a single lipoyl domain to visit not only different
active sites but to be available to several equivalent active sites at
least in the case of the E1 and E2
components(3) . The remaining globular domain, in the exterior
region of the mammalian E2 oligomer, functions in the binding
of the E1 component (5, 6) and is designated
the E2
domain. This small (
46 amino acid)
domain is flanked on its NH
-terminal side by a relatively
large,
36 amino acid, hinge region (H2), that connects it to the E2
domain and on its COOH-terminal side by a
20 amino acid hinge region that connects it and the entire
flexible, exterior structure to the oligomer-forming inner domain of E2. The inner domain of E2 also catalyzes the
acetyltransferase reaction.
Figure 5:
Model of the domains of an E2
subunit binding an unit of the E1 component by its
B domain and a kinase subunit by its L2 domain. In the upper
section the model depicts hinge connected L1 and L2 domains with
attached lipoyl groups. A major portion of L2 and the inner of its
lipoyl-lysine side chain are positioned under (dashed lines)
and interacting with a kinase subunit (K). The
subunit of E1 interacts with the B domain of E2 and the
subunit of E1 is positioned to serve as a substrate for the
kinase subunit. To emphasize the oligomer forming role of the inner (I)
domain of E2 a second I domain is included but without outer
domains.
Figure 7:
Model of movement of the kinase along the
surface of the E2 core. The reaction steps show the reversible
association of a kinase dimer (labeled K for PK1 or PK2) with one L2
domain (step 1, equilibrium constant K) and then with a
second L2-lipoyl domain (step 2, interaction constant, K
).
``Hand over hand'' movement of the kinase is achieved by
repeated dissociation and association in the K
step only
leading to movement by interchange of L2 domains binding the kinase.
The model incorporates a conformational change in the kinase dimer with
the binding of lipoyl domains that results in negative cooperativity (i.e. a rapid exchange by a weaker K
interaction
while being held by a tighter K
interaction). Although
negative cooperativity is included and favored, it is not absolutely
required (cf. the text and Footnote 8 for the requirements). The
domains of E2 are labeled: oligomer forming inner domain, I; E1 binding domain, B; and the inner, L2, and N-terminal lipoyl
domain, L1. The model shows only five E2
domains
out of sixty in a dodecahedron structure and E1 is not
included for clarity but would be bound to the E2
domain as shown in Fig. 5but as an
structure interacting with two B
domains.
In typical preparations of bovine PDC,
20-30 E1 tetramers
and as few as one molecule of kinase are bound to the E2
core. Though very tightly bound to E2
the kinase can efficiently inactivate the complex (k
= 0.5 s
). Indeed, the phosphorylation
of free E1 by free kinase is severalfold slower than when
these components are bound to the E2 core. We wish to identify
the specific domain of E2 that functions in the binding of the
kinase and to gain insights into how this organized state of the
complex greatly enhances the activity of the E1a kinase. The
dissociation of E1 from the E2 core is
slow(7, 8) , and the kinase has an even tighter
affinity for the E2 core(9) . In early work to address
this question of how an E2-bound kinase molecule rapidly
phosphorylates many E2-bound E1 tetramers, studies
were conducted with very dilute complex which led to a major portion of
the E1 but little kinase dissociating from the E2
core. It was found that the few bound E1 were rapidly
phosphorylated (and PDC activity lost) and, moreover, that
phosphorylation of free E1 tetramers occurred at a rate that
corresponded to that estimated for the rate of association of free E1 with the E2 core(10) . Only with the
knowledge of E2's structure and characterization of the
location and nature of kinase binding to the E2 can the
underlying mechanisms be evaluated.
The E1 kinase has been shown to bind to the lipoyl domain region of E2 in bovine PDC(6, 9, 11) .
Selective removal of the lipoyl prosthetic group was shown to lead to
dissociation of the kinase demonstrating an essential role of this
cofactor in tight binding of the kinase(12) . Although the
kinase binds very tightly to the oligomeric E2 core, it was
found to efficiently transfer to a fragment of bovine E2 that
contained both lipoyl domains(9) . We have suggested that
transfer involves a direct interchange of the kinase (i.e. without any free kinase) between the exterior lipoyl domain
regions of the E2 core(9) . Such movement at the
surface of the assembled E2 core would explain how a kinase
molecule is able to both bind tightly to E2 core and still
phosphorylate 20-30 E1 tetramers that are also bound to
the E2
domain at the surface of the E2
core.
The kinase is composed of two subunits. One has been
identified as a catalytic subunit(13) , and we have designated
it as K; the other subunit was considered to be a
noncatalytic subunit(13, 14) and has been designated
K
by this laboratory. (
)The amino acid sequence
of the catalytic subunit of the rat heart pyruvate dehydrogenase kinase
was recently determined from its full-length cDNA(15) . An
exciting conclusion was that the K
subunit is a relative of
the bacterial histidine kinase family. Recently, Popov et al.(16) have cloned and expressed the second kinase subunit
of rat heart PDC. They demonstrated that it has a distinct subunit but
related sequence to the K
subunit and that it was
catalytically active although with a lower specific activity than that
of the K
subunit. The latter observation needs to be
reconciled with not losing kinase activity upon cleaving this second
subunit(11, 13) . Nevertheless, we will now change our
designation of the kinase subunits and refer to K
as
pyruvate dehydrogenase kinase 1 (PDK1) and to the other kinase subunit
as pyruvate dehydrogenase kinase 2 (PDK2). In the case of complexes and
subcomplexes containing both, we will reduce this to K1 and K2 for the
sake of clarity, e.g.E2-X-K1K2 subcomplex. PDK2 is
present in bovine preparations of bovine complex at variable levels and
often decreases in resolution processes that first leave the kinase
bound to E2 core and then involve removal of the kinase from
the E2 core (e.g. 9, 17). The severalfold activation
of the kinase occurs upon its binding to the assembled E2
core, and this only requires PDK1(17) . No known functions of
the kinase are lost when PDK2 is proteolized (11, 13, 18) or at very low levels in kinase
preparations.
Recently, we have prepared a variety of constructs of
the lipoyl domain region of the human PDC-E2. ()These were expressed in Escherichia coli as
fusion proteins attached to glutathione S-transferase (GST)
and as free lipoyl domains selectively released from GST by thombin
treatment. When expression was performed with lipoate supplementation,
the lipoyl domains were fully lipoylated. We have made the individual
domains E2
and E2
and those
domains with the connecting H1 hinge region attached, E2
and E2
,
and also made the bilipoyl domain structure, E2
. Several E2 subunits
of bacterial PDCs contain two or three lipoyl domains; however, no
roles have been described that are performed in a specific or even
highly preferential manner by one of the lipoyl domains in an E2. We have established that the bovine E1 kinase
binds to human lipoyl domains with the L2 domain serving as a highly
preferential binding site. We have demonstrated efficient movement of
the kinase between the oligomeric E2 core and lipoyl domain
constructs containing the L2 domain. Additionally, we have
characterized the effects of the individual and bilipoyl domain
constructs on kinase activity.
In Transfer Procedure 2, GST-lipoyl domain constructs were pre-loaded onto GSH-Sepharose columns. In some experiments this was preceded by a procedure to delipoylate a portion of the constructs. In that case, 750-pmol samples of GST constructs were incubated with or without 0.35 µg of lipoamidase for 8 h at 30 °C. Parallel studies demonstrated the lipoamidase treatment completely removed the capacity of the GST constructs to undergo reductive acetylation, but this could be restored, after phenylmethylsulfonyl fluoride inactivation of lipoamidase, by treatment with E. coli lipoyl protein ligase to reintroduce lipoyl groups (data not shown). To each tube, 30 µl of GSH-Sepharose pre-equilibrated in transfer buffer was added and incubated 10 min at 23 °C followed by quantitative transfer to a microcolumn and washing with 30 bed volumes of transfer buffer and removal all but 5 µl of excess buffer. Then 4 µg of E2-X-K1K2 subcomplex in 20 µl was added for the indicated times (typically 30 or 60 s) followed by elution steps and analyses as described above.
The transacetylase activity of E2 was measured by monitoring the increase in acetyl-dihydrolipoamide production by recording the increase in absorbance at 232 nm(9) . Reaction mixtures contained in 1-ml cuvette at 30 °C: 30 mM Tris-HCl, pH 7.5, 1.0 mM dihydrolipoamide, 2 mM acetylphosphate, 5 µM CoA, 50 µM cysteine, and 2 units of phosphotransacetylase. After the 232 absorbance became constant (by 10 s) due to conversion of CoA to acetyl-CoA by the phosphotransacetylase reaction, the E2 source was added and the increase in absorbance with time recorded.
We evaluated whether the bilipoyl domain
structure separated from GST was effective in binding the kinase.
Increasing concentrations of homogeneous E2 (released from and free of
GST) were incubated with the E2-X-K1K2 subcomplex and followed
by each undergoing fractionation in a micro-sucrose gradient as
described under ``Experimental Procedures.'' The subcomplex
was recovered in a pellet and lower supernatant fraction whereas the E2
was recovered in the upper
supernatant fractions. The portion of kinase activity found in the
upper supernatant fractions increased from 9.3 to 28% as the molar
ratio of the bilipoyl domain structure to E2 subunits was
increased from 1.3 to 7.8. At the same time, there was a decrease in
the kinase activity associated with the pellet fraction. Thus, transfer
of the kinase to the free bilipoyl domain structure was supported but
was somewhat less efficient than transfer to the GST-bilipoyl domain
construct. These initial experiments did not distinguish whether one or
both the lipoyl domains bound the kinase but did indicate the human
construct was effective in binding the kinase.
Figure 1:
Kinase transfer from E2-X-K1K2
to GST-linked E2, E2
, and E2
. Three µg of E2-X-K1K2 was combined with 284 pmol GST-linked E2
(1-98), E2
(120-233), and E2
(1-233) in a final volume
of 20 µl. In accordance with the steps in Transfer Procedure 1 (see
``Experimental Procedures''), the components were combined
and incubated for 25s, mixed with GSH-Sepharose (5 s), and after 30 s,
a 75 µl filtrate fraction was collected, followed by 75 µl
GSH-eluate. Each experiment was conducted in triplicate and kinase
activities for all filtrates and GST eluates were measured in
duplicate. The transacetylation activity and GST activity of all
fractions were measured as described in Experimental Procedures to
establish the distribution of E2 and GST described under
``Results.'' Bars show the range of observed values
and bar heights the average
values.
Figure 2:
SDS-PAGE analysis of GSH eluates. Samples
(5 µl) from 75 µl GSH eluates (Fig. 1) were mixed with
10 µl of SDS-sample buffer and separation conducted with a 10%
polyacrylamide gel with bands when visualized by silver staining. Lane 1 contained 1.5 µg subcomplex and lanes 2-4 show the patterns for eluates of GST-linked E2(1-98), E2
(120-233), and E2
(1-233), respectively,
and lane 5 for the GSH eluate of GST control. The labels on
the right side identify the position of the
GST-construct.
Further studies were done to evaluate kinase binding using
constructs that contained the full H1 hinge region connecting E2 and E2
and an approach
to test binding to GST constructs already bound to the gel matrix
(transfer procedure 2). The GST constructs E2
(1-128), E2
(98-233), and E2
(1-233) were preloaded in
duplicate at equivalent, 750 pM levels, onto 25 µl of
GSH-Sepharose. E2-X-K1K2 (4 µg in 25 µl) was added and
mixed with the loaded gel matrix and incubated for 1 min, and then this
volume was passed through the column, rapidly followed by two 25-µl
washes as described under ``Experimental Procedures.'' There
was no contamination of the initial pass-through fraction with GST
activity using this procedure. The individual GST constructs were then
eluted with GSH as described above. The kinase activity in the GSH
eluates was greater than 33 pmol of
P incorporated
min
for the 98-233 structure and the
1-233 structure but only 4.5 ± 3.13 for the 1-128
structure which was only slightly above the experimental error for the
background. When GST was used, kinase activity was entirely found in
the initial pass-through and none (0 ± 3.1) in GSH eluate. Thus,
we found the same pattern of much greater transfer of the kinase
specifically to E2
constructs but not to the
construct containing only the E2
domain when
these H1-containing structures were preloaded onto GSH-Sepharose.
Similar results were found with these H1-containing constructs in the
case of the nonlipoamidase (-LPA) samples in the transfer
experiments shown in Fig. 4, below. We would note that the
kinase cannot be observed by SDS-PAGE analysis of the GSH eluate when
GST-E2
or GST-E2
were used because these polypeptides comigrated with kinase
bands.
Figure 4: Transfer of the kinase from the E2-X-K1K2 subcomplex to lipoylated and delipoylated GST constructs. Following incubation with (+LPA) or without (-LPA) lipoamidase as described under ``Experimental Procedures,'' 750 pmol of the indicated GST constructs were bound to 25 µl GSH-Sepharose. Using Transfer Procedure 2, 4 µg E2-X-K1K2 was mixed for 60 s with the indicated gel-bound GST-construct (or GST) followed by collecting 75 µl of E2-containing filtrate and then 75 µl of GST-containing GSH eluate. Each experiment was performed in duplicate and the kinase activity of each fraction was measured in duplicate. More than 99.5% of the GST activity was removed from the initial (pass-through) filtrate and >95% of the E2 activity was present in that filtrate.
Figure 3:
Transfer of the kinase from E2-X-K1K2 to GST-free lipoyl domain constructs. On top of each
microsucrose gradient, 50 µl mixture containing 3 nmol (60
µM) of the indicated lipoyl domain construct
[E2(1-98), E2
(1-128), E2
(98-233), E2
(120-233), and E2
(1-233)], 25 µg E2-X-K1K2 subcomplex, and 50 µg bovine serum albumin
(BSA). Centrifugation was conducted at 27,000 rpm in Sorvall HA629
rotor for 180 min at 20 °C. From the top to the bottom of the gradient, the fractions collected (cf. ``Experimental
Procedures'') are labeled S1 to S3 and each redissolved pellet
fraction is labeled with a P. One-eighth the volume of each fraction
was analyzed by SDS-PAGE. in gels containing 15% acrylamide and bands
were visualized by silver staining. Each fraction was assayed for
kinase activity and E2 activity. The S1 and S2 fractions
contained BSA that migrated slightly below E2. The position of
one major and a minor bands from BSA are best observed in S1 lane of
1-98 experiment since the control S1 lane was at the edge of the
gel. A band for E3 does come between the two bands for BSA. A
small portion of the 1-233 construct underwent degradation; the
S1 lanes contain about 0.25 nmol of each construct, this is about 7
µg of the 1-233 construct and silver staining readily detects
0.05 µg.
Analysis of kinase activity in the various fractions of the sucrose
gradients also supported binding to E2 and not to E2
. However, in contrast to the gel
electrophoresis pattern, higher kinase activity was found in E2
(120-233) construct supernatant versus that of the E2
(98-233) or E2
(1-2-233) constructs.
This discrepancy of higher activity but lower band density suggests the
presence of the H1 hinge region may decrease the observed kinase
activity, but further studies are needed to confirm this result.
(Inhibition by E2
, not present in 98-233
construct, was observed (below)). The kinase activities in all the S1
fractions of all the E2
-containing constructs
were stimulated about 2.5-fold when assayed in the presence of 10
µg of kinase-depleted E2-X subcomplex.
We have suggested that the inner hydrophobic portion of the
lipoyl-lysine prosthetic group of E2 directly
contributes to binding of the kinase for two reasons(12) .
First, unlike removal of lipoate, modification of the reactive
dithiolane ring portion of the lipoate did not prevent kinase binding
and secondly suggesting that this reactive portion of the prosthetic
group was not an essential component of the kinase-binding site.
Second, the resolved kinase has a tendency to stick to hydrophobic
surfaces suggesting that it has an exposed site designed to interacts
with a hydrophobic structure. We cannot eliminate the prospect that
delipoylation causes a change in the structure of the L2 domain which
prevents its binding of the kinase. This is not expected due to the
role of the lipoyl-lysine as a swinging arm. Regardless of how removal
of the lipoate prevents kinase binding, unique structural interactions
of the kinase with novel features in the tertiary structure of the L2
domain seem required to explain binding by this domain but not by the
L1 domain which has a highly related sequence (26, 27) with only the COOH-terminal regions showing
substantial sequence and size variation.
This COOH-terminal
portion of L1 and L2 extends beyond the region that can be aligned with
the sequences of lipoyl domains of prokaryotic PDC-E2s or the E2s of other
-keto acid dehydrogenase complexes. We have
suggested that this unique part of the L2 structure may have a
specialized role in interacting with the kinase and/or phosphatase
components of mammalian PDC.
Fig. 5models the
interaction of a kinase subunit specifically with the L2 domain of E2 and incorporates interaction with the inner part of the
lipoyl-lysine prosthetic group as part of the binding site. The model
also shows one
unit of an E1 tetramer binding via
its
subunit to the B domain of E2 (5) with its
subunit positioned to interact with the kinase.
Figure 6:
Effects of lipoyl domain constructs on the
activity of resolved (E2 free) kinase. Kinase assays were
constructed in 50 µl reaction mixtures with 28.5 µg E1
containing about 31 µunits kinase. (Units are for E2-activated kinase activity; 1 unit = 1 nmol P-phosphate incorporated min
mg
). The indicated GST-free lipoyl domain
constructs were added at the listed concentrations for 60 s just prior
to initiation of activity. Assays were conducted in duplicate with a
reaction time of 60 s. Other conditions were as described under
``Experimental Procedures.''
Besides the
possibility of inhibition by E2 being due to a
specific interaction with the kinase, inhibition might result from
interaction of the lipoyl domain with E1 making E1 a
poorer substrate for the kinase. Both lipoyl domains are substrates of E1 in the second reaction step in the PDC reaction. However,
steady state kinetic studies (
)gave a K
of E1 for E2
(1-98) of 69.4
µM and, even at the highest level, E2
(1-98) was present at only 25 µM in the above study. Thus, it would not be expected that
interaction of E2
with E1 caused the
observed inhibition.
The data in Fig. 6indicate the free E2 enhances kinase
activity much less than the oligomeric E2 core (1.4 fold versus
3.5 fold by intact E2 core under these
assay conditions). Moreover, a much higher concentration of this free
lipoyl domain is required to maximally activate the kinase than would
be expected if binding to this free domain to the kinase was as tight
as to oligomeric E2(9) . The proportion of kinase
transferred from E2 to GST-E2
in
experiments shown in Fig. 1and Fig. 4is substantial as
indicated by the relative bar heights of the hatched (passed though
with E2) versus the solid bars (eluted with the
GST-E2
). The data suggest not only a high level
of kinase is transferred but a higher recovery of activity than would
be expected if GST-E2
gave the same enhancement
of kinase activity as free E2
. Thus, the high
transfer and good recovery of kinase activity indicate that there is
tight binding to the GST-E2
structures and
suggest the possibility of a greater enhancement of kinase activity by
GST-E2
constructs than free lipoyl domains. Since
the GST is a dimer, (
)one interesting reason that there
might be tighter binding of the kinase is the potential capacity to
achieve bifunctional binding by an intramolecular reaction of dimeric
kinase molecules. This would be particularly true in the case of the
GST-E2
and
GST-E2
constructs in which the E2
domain is well separated by mobile connecting
regions from the GST. Further studies are underway to establish the
quaternary structure of the kinase, to estimate the rate of transfer of
the kinase, and to evaluate the influence of changes in the oligomeric
state of various E2
-containing structures on
kinase binding and activity enhancement.
Our data demonstrate the
effectiveness of the human E2-lipoyl domain constructs that we
have prepared in determining the specific domain and specific
requirements for efficient accepting of kinase tightly bound to a large E2 oligomer. Our results demonstrate the great versatility in
using GST-linked constructs as acceptors. Our results indicate that a
combination of the lipoyl prosthetic group and a specific portion of
the structure of E2 creates the kinase binding
site. Some structure in E2
must distinguish it
from E2
and it is unlikely that this simply
involves the presentation of the lipoyl prosthetic group since these
are attached to highly conserved regions. Both E2
and E2
are larger than the lipoyl domains
from bacterial PDC-E2 or the E2 components of other
-keto acid dehydrogenase complexes. Additional structure is
located at the COOH termini of the human E2
and E2
and these parts of the two lipoyl domains have
the greatest differences between them in both sequence and size.
We suggest that the COOH-terminal end of E2
(residues 208-229) is the most likely region contributing
to kinase binding. A weak interaction with E2
may
occur since it inhibits kinase activity (Fig. 6) and the
SDS-PAGE analyses suggested (in contrast to kinase assays) somewhat
greater binding to the bilipoyl domain construct. The model in Fig. 5shows what we have established whereas the model in Fig. 7is both a reasonable and exciting explanation of our
results and will be evaluated further.