Amino acid deprivation induces translation of branched-chain
-ketoacid dehydrogenase kinase
Christopher B.
Doering1 and
Dean J.
Danner2
2 Department of Genetics, 1 Program in Genetics and
Molecular Biology, Emory University School of Medicine, Atlanta,
Georgia 30322
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ABSTRACT |
Leucine, isoleucine, and
valine are used by cells for protein synthesis or are catabolized into
sources for glucose and lipid production. These branched-chain amino
acids influence proteolysis, hormone release, and cell cycle
progression along with their other metabolic roles. The branched-chain
amino acids play a central role in regulating cellular protein turnover
by reducing autophagy. These essential amino acids are committed to
their catabolic fate by the activity of the branched-chain
-ketoacid
dehydrogenase complex. Activity of the branched-chain
-ketoacid
dehydrogenase complex is regulated by phosphorylation/inactivation of
the
-subunit performed by a complex specific kinase. Here we show
that elimination of the branched-chain amino acids from the medium of
cultured cells results in a two- to threefold increased production of
the branched-chain
-ketoacid dehydrogenase kinase with a decrease in
the activity state of the branched-chain
-ketoacid dehydrogenase complex. The mechanism cells use to increase kinase production under
these conditions involves recruitment of the kinase mRNA into
polyribosomes. Promoter activity and the steady-state concentration of
the mRNA are unchanged by these conditions.
posttranscriptional regulation; polyribosomes; regulation of
catabolism
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INTRODUCTION |
CATABOLIC
STATES, INCLUDING starvation, diabetes, infections, trauma, and
cancer, all decrease plasma amino acid concentrations. In response, the
body accelerates protein degradation in skeletal muscle as a means to
maintain plasma amino acid concentration (36, 42). Amino
acids produced from autophagy can be used directly for energy
production, ketone body formation, gluconeogenesis, or new protein
biosynthesis. If protein breakdown persists, a cachectic condition
ensues (28, 44). Various essential amino acids have been
shown to attenuate protein catabolism (39). The
branched-chain amino acids (BCAA), particularly leucine, are important
in controlling protein turnover in most tissues (2, 4, 22, 30,
34, 35, 42). Several roles of leucine in this process have been
described. Leucine stimulates the phosphorylation of 4E-BP1, causing
its release from eukaryotic initiation factor (eIF)-4E to allow
formation of the eIF-4F initiation complex for protein synthesis
(10). When cells are deprived of leucine, 4E-BP1 remains
bound to eIF-4E and global protein synthesis decreases, whereas
polyribosome fractions are diminished (10, 45). In contrast, leucine starvation results in the L-system transporter protein concentration being increased to promote uptake of
extracellular leucine. The increase in transporter results from changes
in translation rate (24).
Conservation of BCAA by cells is important in cell preservation. Within
cells, the BCAA have two fates, incorporation into protein or
irreversible degradation. Oxidative decarboxylation of the
branched-chain
-ketoacids by branched-chain
-ketoacid dehydrogenase (BCKD) commits these amino acids to their catabolic fate
(5). BCKD is a nuclear encoded multienzyme complex located in the mitochondria of all mammalian cells. In response to changing needs for BCAA, the activity of BCKD is regulated to conserve these
essential amino acids. The activity state of BCKD is decreased when the
E1
-subunit of the complex is phosphorylated by a complex-specific kinase (16). The activity state of BCKD
is the percentage of complex that is unphosphorylated and active relative to the total amount of complex present. The amount of kinase
protein present within a cell varies such that tissues with a low
activity state, such as skeletal muscle, contain high levels of kinase
protein. In contrast, liver and kidney contain less kinase protein and
therefore maintain a high activity state of BCKD (7). When
rats are fed a diet low in protein, the activity state of BCKD is
decreased in liver, presumably to conserve protein (12).
This response was shown to require an increase in kinase protein
(41). Together, these observations suggest that activity state is not simply a balance between the specific activity of the
kinase and its countering phosphatase but more a function of the amount
of kinase protein present at any time. Therefore, decreases in the
activity state of BCKD would necessitate an increased amount of kinase
protein. To further investigate the cellular mechanisms regulating the
BCKD activity state, we determined the effects of BCAA depletion on
BCKD activity and BCKD kinase expression in cultured cells. Here we
show that depriving cells of the BCAAs decreases the BCKD activity
state with a concomitant increase in the amount of kinase protein. The
latter response results from a posttranscriptional event involving
enhanced recruitment of mRNA in the polyribosome fraction.
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MATERIALS AND METHODS |
Cell lines and culture conditions.
Ham's F-12-K and RPMI 1640 lacking the BCAA were prepared by Atlanta
Biologicals based on the GIBCO formulas. FBS was dialyzed (6,000-8,000 molecular weight cut off) against three changes of eight volumes of 1× PBS to reduce free amino acid concentrations below
detectable levels (6) and was confirmed in these studies by HPLC analysis (14). When stated, individual BCAA were
supplied to the depleted media at the final concentration defined for
each medium (RPMI 1640: I and L at 3.8 X10
4 M, V at
1.7 × 10
4 M; F-12-K: I at 3 × 10
5 M, and L and V at 1 × 10
4 M; see
Ref. 23). Rat liver cells [clone 9 (C9) from ATCC] were maintained in Ham's F-12-K medium supplemented with 10% FBS at 37°C
in 5% atmospheric CO2. DG75, an EBV-negative human B cell line, and human lymphoblasts (EM280) derived by EBV transformation of
human lymphocytes were maintained in RPMI 1640 supplemented with 15%
FBS at 37°C in 5% atmospheric CO2. During the 5-day
culture in BCAA-depleted medium, C9 cells did not have a medium change, whereas DG75 and EM280 had one change of medium after 3 days. HPLC
analysis of conditioned medium from cells grown in BCAA-depleted media
revealed no detectable BCAA.
Enzyme assays and Western blots.
BCKD activity assays were done as previously described
(32). To inhibit endogenous BCKD kinase activity and fully
activate the complex, cells were incubated with 1 mM
-chloroisocaproate (
-CIC) for 10 min before initiation of
activity measurements. Activity state was estimated by determining BCKD
activity in the presence or absence of
-CIC; BCKD activity without
-CIC was divided by the activity in the presence of
-CIC and
multiplied by 100. Total cellular protein and mitochondrial protein
were quantified using bicinchoninic acid protein assay reagents
(Pierce, Rockford, IL) following the manufacturer's protocol.
Western blots with BCKD kinase specific polyclonal antiserum
were done as described (7). Blot quantification was
performed using an ImageQuant densitometer (Molecular Dynamics).
Nucleic acid analysis.
Total RNA was isolated using Tri-Reagent (Sigma) following the
manufacturer's instructions and was quantified by measuring the
absorbance at 260 nm. The ratios of absorbance at 260 nm to that at 280 nm were consistently 1.7-1.9. For Northern blot analysis, 20 µg
total RNA from each sample were resolved in a 1% agarose-formaldehyde gel at 80 V for 4 h. RNA was transferred to a Hybond
N+ nylon membrane by capillary action overnight in 20×
saline-sodium citrate (SSC). Northern blots were done as described
(1) with the following modifications. Hybridization buffer
contained 5× SSC, 50% formamide, 7% SDS, 5% polyethylene glycol,
and 50 µg denatured salmon sperm DNA/ml. The membrane was
prehybridized for 4 h at 42°C before adding the
[
-32P]dCTP-labeled cDNA probe for overnight incubation
at 42°C. The membrane was washed one time with 2× SSC and 0.5% SDS
at 55°C for 20 min, two times with 0.5× SSC and 0.5% SDS at 65°C
for 20 min, and one time with 0.2× SSC and 0.2% SDS at 65°C for 20 min. Hybridizing components were detected by autoradiography using Biomar Blue film (Marsh Biomedical Products, Rochester, NY). Ethidium bromide staining of the gel and hybridization using radiolabeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18S rRNA as probes
were performed to ensure equal loading. GAPDH has been shown previously
not to change as a result of complete amino acid deprivation (17,
26).
Luciferase assay.
The luciferase reporter gene assays were done in C9 cells using
rat BCKD kinase promoter constructs (20) driving firefly luciferase production. Three different promoter regions were tested that contained nucleotides upstream of the AUG codon to positions
58,
128, and
449. Cells were transfected by FuGENE 6 (Boehringer Mannhiem) using a ratio of 50:1 test vector to
pRL-cytomegalovirus control renilla luciferase plasmid. After
8 h in complete medium, cells were changed to fresh media either
with or without the BCAA and were cultured for 4 days. Cells were
harvested, and luciferase activities were measured using the Dual
Luciferase Assay system (Promega) according to the manufacturer's
protocol. Luminescence was determined with a Turner model TD20/20
luminometer. Firefly luciferase values were corrected for transfection
efficiency by dividing by the corresponding renilla luciferase activity.
Polyribosome analysis.
Preparation of polyribosomes for analysis of BCKD kinase mRNA
translation was done essentially as described (9). The
EM280 cell line was cultured for 5 days with or without BCAA in the medium. In an attempt to obtain similar cell numbers at the end of 5 days, only 30 × 106 cells were seeded in +BCAA
medium, whereas 70 × 106 cells were seeded in
BCAA
medium. At the end of 5 days, all cells were washed with 1× PBS and
resuspended in fresh medium containing 100 µg cycloheximide for
15-20 min. Cells were then transferred to a buffer containing 75 mM NaCl, 4 mM MgCl2, 20 mM Tris · HCl, pH 7.5, and
0.75% Triton X-100 and were allowed to lyse for 3-5 min on ice.
The solution was clarified by centrifugation at 10,000 g for
10 min, and the total supernatant was used for polyribosome
fractionation. Fractionation was on a 15-45% linear gradient of
sucrose in 80 mM NaCl, 10 mM Tris · HCl, pH 7.5, and 5 mM
MgCl2 by centrifugation at 39,000 g for 90 min
in an SW41 Ti rotor. Fractions were collected in 0.5-ml aliquots, and
RNA profiles were determined by monitoring absorbance at 254 nm. Total RNA was prepared from 350 µl of each fraction using TriReagent (Sigma). The RNA was resuspended in 50 µl of 0.5% SDS and 1 mM EDTA,
added to 150 µl RNA denaturing solution, and heated at 65°C for 15 min (21). Denatured RNA was loaded on a slot blot fitted with a Hybond N+ membrane. Membrane hybridization was done
as for the Northern blot methods described in Nucleic acid
analysis using both BCKD kinase and GAPDH as probes.
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RESULTS |
Activity state and BCAA amino acid metabolism.
Cells cultured in medium lacking the BCAA respond by decreasing the
activity state of the BCKD complex. This response was demonstrated with
hepatocytes and lymphoblasts (Table 1).
After each cell type was incubated in BCAA-depleted media for 5 days, the activity state of BCKD was reduced by at least 50% relative to the
active state in the same cells cultured in complete medium. As shown
for DG75 cells, this response was time dependent (Fig. 1A). Total BCKD activity in
the cells grown in BCAA-depleted medium was the same as in cells in
complete medium, even when cells were cultured in this medium for up to
9 days. The values of 13.4 ± 3.9 pmol
CO2 · mg
protein
1 · min
1 for cells grown for
9 days in the absence of BCAA and 17.7 ± 3.6 for those grown in
complete medium, for this same time, were not significantly different
(P = 0.325). Beyond 3 days in BCAA-free medium cell
growth halts (Fig. 1B), presumably due to G1 cell cycle
arrest caused by isoleucine depletion (13, 27, 48). The
change in BCKD activity and growth at 5 days was not exclusively due to
cell death. Trypan blue exclusion analysis showed that >75% of the
cells were living (Fig. 1C), and the cells grew normally when returned to complete medium after this time (data not shown). Assuming the slight increase observed in cell death could help to
replenish the depleted media with BCAA, we performed HPLC analysis on
the conditioned media after the 5-day culture period (Table 2). The BCAA-deprived media contained no
detectable BCAA by HPLC, whereas the control media contained BCAA
concentrations similar to fresh media. HPLC also failed to detect
intracellular free BCAA in these same cells cultured for 5 days in
BCAA-deprived media.
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Table 1.
Activity state of BCKD complex in cells grown 5 days in the
presence or absence of branched-chain amino acids in the culture
medium
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Fig. 1.
A: branched-chain amino acid (BCAA) depletion
affects branched-chain -ketoacid (BCKD) activity state and cell
growth but not cell viability. DG75 cells were cultured in RPMI 1640 media supplemented with dialyzed FBS (10%) either with (solid line and
) or without (broken line and ) the
BCAA in this media. After the indicated days in culture, ~2
×106 cells were collected and assayed for BCKD activity.
The activity state values for days 3, 5, and
7 are as follows: 100, 93, 63 (+BCAA); 55, 17, 8 ( BCAA).
Each data point is the mean of 3 determinations in 1 of 3 independent
experiments and is representative of each. B: 10 µl of
DG75 cells were removed from culture and counted at each time point
using a hemocytometer. Each data point is the mean of 4 independent
determinations. C: DG75 cells were cultured as above. At
each time point, cells were assessed for cell viability by trypan blue
exclusion analysis. Viable cells were identified by dye exclusion and
divided by the number of total cells to determine percent viability.
Each experiment is the mean of 4 independent determinations.
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Effects on BCKD kinase protein levels.
During BCAA deprivation, there was an increase in the BCKD kinase
protein concentration present in the mitochondria. The amount of kinase
protein in EM280, DG75, and C9 cells cultured without BCAA was markedly
increased over that found in cells grown in complete medium (Fig.
2A). As demonstrated with the
C9 cells (Fig. 2B) by quantitative Western blot analysis
(31), the kinase protein level increased two- to
threefold. Deprivation of leucine alone over this same time period did
not result in elevated BCKD kinase protein levels above that found in
control medium (data not shown).

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Fig. 2.
Western blot for mitochondrial BCKD kinase reveals an
increase in BCKD kinase upon BCAA depletion. A: lymphoblasts
[DG75 and EM280 (280) cell lines] were grown in
suspension culture for 5 days in media with (+) or without ( ) BCAA.
Mitochondrial protein (20 µg) was resolved on 10% SDS-PAGE and
processed for Western blot with BCKD kinase antisera. B: C9
rat hepatocytes were treated as above for Western blot analysis. The
amount of mitochondrial protein loaded on the gel is indicated at
bottom. All blots were reprobed with BCKD complex antisera
and were observed to contain similar levels of each subunit, attesting
to equal loading of the samples (data not shown).
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Measurements of promoter activity and steady-state mRNA.
Synthesis of proteins depends on the sequential activity of gene
transcription, RNA stability, and translation. Increased production of
a protein can be the result of an altered rate of any of these
processes. Two experimental approaches show that neither increased
promoter activity nor RNA stability contributed to the observed
increase in kinase protein. First, luciferase reporter assays with the
kinase promoter driving production of luciferase showed no significant
difference between cells grown in the two culture conditions (Fig.
3). BCKD kinase basal promoter activity
requires only the first 58 bp upstream of the start site (21), and lengthening this to include an additional 391 bp
upstream did not alter the response. Second, Northern blots also did
not show an increase in steady-state mRNA concentration for the kinase transcripts between control and BCAA-deprived cells at days
1 or 5 (Fig.
4A). Densitometry
quantification of data from three independent experiments consistently
showed that the steady-state BCKD kinase mRNA concentration did not
increase with 5 days of BCAA deprivation (Fig.
4B). In fact, a slight decrease in BCKD kinase
mRNA was observed after 5 days of culture in BCAA-depleted medium
compared with cells grown in complete medium. This is consistent with
the finding that total RNA harvested per cell is almost 30% lower in
BCAA-deprived cells vs. control cells (data not shown).

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Fig. 3.
Luciferase activity driven by the rat BCKD kinase
promoter remains unchanged after BCAA depletion. Hepatocytes were
transfected with a plasmid containing 1 of 3 different lengths of
promoter region DNA ( 449, 128, or 58) linked to the firefly
luciferase reporter gene and a control renilla luciferase plasmid.
Cells were maintained for 4 days in medium with (filled bars) or
without (open bars) BCAA before measuring luciferase activity. Results
are means ± SD of 6 independent determinations.
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Fig. 4.
Northern blot reveals no increase in BCKD kinase
steady-state mRNA levels after BCAA depletion. Total RNA from C9 rat
hepatocytes (20 µg) cultured for 1 or 5 days in medium with (+) or
without ( ) BCAA was resolved on 1% agarose and blotted to a nylon
membrane. Hybridization was performed with a radiolabeled mouse BCKD
kinase cDNA. Hybridization with similarly radiolabeled rat
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA or 18S rRNA was
used to assess differences in lane loading. A: ratios of
BCKD kinase to GAPDH as determined by densitometry: day 1,
0.27 (+) and 0.34 ( ); day 5, 0.44 (+) and 0.13 ( ). Gel
is representative of 3 independent experiments. B:
densitometric quantitation of BCKD kinase and 18S RNA from 3 independent experiments separate from that shown in A. The
ratio of BCKD kinase RNA to 18S rRNA is shown for cells grown in medium
with (filled bars) or without (open bars) BCAA for 5 days
(n = 3 experiments).
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Alteration of translation state.
To determine if the translation rate of BCKD kinase mRNA is increased,
polyribosomes were isolated from cells grown in the presence and
absence of the BCAA for 5 days. The EM280 cell line was used for these
experiments due to ease of culturing large cell numbers and the fact
that they showed the greatest response observed by Western blot (Fig.
2A). As seen in Fig.
5A, the profiles of the
polyribosome traces from each experimental condition were not
substantially different except for the fact that less total RNA was
present in the fractions from the BCAA-depleted cells. Hybridization to
total RNA prepared from these polyribosome fractions with BCKD kinase
and GAPDH-radiolabeled probes demonstrates that more of the BCKD kinase
mRNA is found in the higher-order polyribosome (heptasome and larger)
fractions (11-13, 15) from cells grown in the
BCAA-deprived medium (Fig. 5B and Table
3). BCKD kinase transcript was not
detected in fractions that did not contain ribosomes
(fractions 1-4) from cells grown in either
media.

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Fig. 5.
Analysis of polyribosome fractions from cells grown for 5 days in the absence or presence of BCAA in the culture medium reveals
increased BCKD kinase mRNA associated with higher-order polysomes.
EM280 cells were cultured in the presence or absence of BCAA for 5 days. A: cells were harvested, and the cytosol was
fractionated over a sucrose gradient monitored for absorbance at 254 nm
(A254). B: total RNA was isolated from the
resulting fractions and slot blotted to a nylon membrane with
hybridization performed as in Fig. 4. Due to the small amount of RNA
obtained from each fraction, all RNA obtained was loaded without
quantitation. To compensate for differences in RNA content between
fractions, all data are presented as the ratio of densitometry values
for BCKD kinase mRNA with that for GAPDH mRNA in each fraction. Solid
bars, ratios obtained when EM280 cells were grown in the presence of
BCAA for 5 days; open bars, ratios when cells were grown in the absence
of BCAA for 5 days. Results are representative of 3 independent
experiments.
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Table 3.
BCKD kinase mRNA distribution among polyribosome fractions from
lymphoblasts cultured 5 days in the presence or absence of BCAA
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Examples of translational control often involve the nucleotide sequence
and/or secondary structure of the 5'-untranslated region (UTR) of the
specific mRNA (18, 37). In an attempt to assess
functionality of the 5'-UTR, the cDNA for mouse BCKD kinase was
engineered to contain either 107 or 21 nt of 5'-UTR along with the
first 70 of 308 nt in the 3'-UTR. When the constructs were subjected to
in vitro transcription/translation (TNT; Promega), the amount of
immunodetectable kinase protein produced from the 107-base 5'-UTR
construct was not different from the amount synthesized from the
21-base construct (data not shown).
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DISCUSSION |
BCAA concentrations modulate many cellular processes, including
protein synthesis, proteolysis, and hormonal responses whether measured
in whole animals or cultured cells (19, 34, 38, 42).
Although a wide range of cellular functions are influenced by BCAA
concentration, the mechanisms that regulate the catabolism of these
essential amino acids are poorly understood. To determine the effect of
BCAA deprivation on BCAA degradation, we measured BCKD activity state
and BCKD kinase levels in three types of cells in culture and defined
the mechanism these cells used to achieve this regulation.
Marten et al. (29) examined the transcription of 19 genes
in cultured hepatocytes for a response to amino acid deprivation. They
found that one-third of the genes investigated responded by decreased
expression, one-third showed increased expression, and one-third did
not respond to the culture conditions. Other investigators have
demonstrated amino acid starvation to alter translation of certain
proteins (37). Observed decreases in protein synthesis
result from a loss of mRNA movement into and dissociation from
polyribosomes (45). These responses were observed in
perfused livers and in cultured hepatocytes and Chinese hamster ovary
cells (37). Further investigations attributed amino acid effects on translation to changes in the phosphorylation state of
specific translation factors (15, 25, 47). Recent reports showed that leucine is the primary amino acid responsible for changing
the phosphorylation state of the translational repressor 4E-BP1 and
thus altering translation (10, 46). In limited cases,
amino acid deprivation results in induction of specific proteins that
tend to be involved in maintenance of cellular amino acid
concentrations (17).
The concentration of free BCAA within a cell is a function of dietary
intake, cell transport, incorporation into protein, and catabolism.
Because degradation of BCAA by BCKD is an irreversible process, shifts
in BCKD activity state represent the major mechanism for regulating
BCAA concentration. In rats, when dietary protein or BCAA are limited,
BCKD activity is suppressed (3, 8, 33). This reduction in
BCKD activity has been attributed to changes in the phosphorylation
state of the complex resulting from increased BCKD kinase activity.
These changes occurred only after the animals were kept on the
protein-deprived diet for 5-12 days. Because BCAA or protein
deprivation in intact animals affects many tissues and metabolic
pathways, we used cultured cells to model only the cellular response.
Within cell culture, the extracellular environment is controlled
easily, and single entities can be varied. Similar to the whole animal
studies, we found that the maximal effect of BCAA deprivation required
at least 5 days. The cells responded to these conditions by decreasing
the BCKD activity state by at least 50% (Table 1). The decrease in
BCKD activity state is in direct response to a two- to threefold
increase in BCKD kinase protein (Fig. 2). No corresponding increase was
observed for BCKD kinase steady-state mRNA levels or kinase promoter
activity, as determined by luciferase reporter activity. These findings do not implicitly demonstrate that there is not some level of change in
mRNA half-life or transcription rate but when taken together argue
against either mechanism being substantially responsible for the
response observed during the course of our study. The increase in BCKD
kinase protein does appear to be the result of increased translation of
the steady-state BCKD kinase mRNA, as evidenced by a shift of these
transcripts into higher-order polyribosomes (Fig. 5 and Table 3). The
experiments also demonstrate that, under both the control and
BCAA-deprived state, all detectable BCKD kinase transcripts are bound
by ribosomes. Therefore, the response is not simply a general shift of
free kinase mRNA to ribosomally bound kinase mRNA but a specific change
in the number of ribosomes bound to each transcript. The >20%
increase in the percentage of kinase transcripts bound to higher-order
polyribosomes appears to account for the two- to threefold increase in
kinase protein over a 5-day period of accumulation.
Because extended times are needed to elicit the response to BCAA
deprivation, it was not possible to use protein synthesis inhibitors to
block the response. The prolonged times also worked against using
transient transfections of the cells to study in vivo roles of the 5'-
and 3'-UTR of this mRNA. Although our results approximate those
measured in protein-deprived rats, the mechanism in rats was attributed
to increased BCKD kinase mRNA concentration (41). This
difference remains to be resolved.
The mechanism these cultured cells use to shift BCKD kinase mRNA into
polyribosomes was not defined. Structural features of the 5'-UTR
regions of mRNAs have been shown to play an important role in
regulation of translation (18). Because the mRNA sequences for human (accession no. AF026548), rat (accession no. M93271), and
mouse (accession no. AF043070) BCKD kinase are available, we performed
qualitative analysis of these for potential regulatory regions. The
length of each appears to be sufficient to contain information for
regulation. The human 5'-UTR contains 273 nucleotides, whereas the rat
and mouse have 121 and 106 bases, respectively. Sequence comparisons
reveal a similarity index of 78.3 for rats and mice and a 48.5 index
between rats and humans, implying conservation and importance of this
region. All contain potential in-frame upstream open reading frames
(uORF) and tracts of pyrimidines and can fold into secondary
structures, as assessed by Lasergene software analysis (Table
4). The inclusion of uORFs has also been
attributed to the instability of mRNAs, but that does not appear to be
a condition observed here, since the steady-state concentration of the
kinase mRNA is not changed (11). Although these putative
features exist, we were not able to demonstrate a change in protein
production by in vitro translation experiments using cDNA constructs
with a deleted 5'-UTR compared with the full-length cDNA. The enriched
environment in reticulocyte lysates for translation is likely to mask
in vivo conditions.
There are inherent properties of BCKD kinase that prohibit using
standard methods to continue examination of potential mRNA regulatory
sequences and to address potential changes in kinase protein turnover
rates that may occur during BCAA deprivation. First, construction of a
tagged kinase protein is complicated by the need for the mitochondrial
targeting peptide at the amino terminal end that is lost after import
into the matrix, and the carboxy terminal end of the protein contains
the catalytic domain. A carboxy terminal V5-His6-tagged
version of the human BCKD kinase is catalytically inactive although it
is correctly imported and processed in cells that are transfected with
plasmid directing its synthesis (unpublished observation). Second, the
presence of BCKD kinase within all tissues containing mitochondria
significantly complicates experiments involving comparisons of kinase
expression from the transgene without a unique marker. To overcome this
obstacle, we are currently developing a model system that is completely
devoid of endogenous BCKD kinase. This system will allow direct
investigation into the role of the potential regulatory regions of the
BCKD kinase mRNA under various experimental conditions, including BCAA
deprivation. Third, the absolute amount of BCKD kinase protein within a
cell is extremely small. Estimates of the fraction of the mitochondrial protein contributed by the BCKD complex put this figure at 0.01% of
total mitochondrial protein. Depending on the tissue source, <1% of
this amount can be attributed to the kinase (40, 43). Conventional methods of studying protein turnover use in vivo labeling
of the protein followed by immunoprecipitation and quantitation based
on the fraction of radiolabel in the sample. The small percentage of
total mitochondrial protein contributing to the kinase and the long
time period needed for the induction of the shift into the polyribosome
fraction makes experiments of this type technically difficult in design
and quantitation. Therefore, we have relied on other analyses to
suggest the mechanism for the observed increase in BCKD kinase, and by
the process of elimination, conclude that the shift of the mRNA into
the higher-order polyribosome fraction implies increased translation.
It remains a possibility that the turnover rate of the kinase is slowed
under the conditions of BCAA starvation and thus contributes to the
observed increase in the concentration of this protein. From our
studies, we can only state that increased translation is occurring and
thus accounts at least in part for the observed increase in
mitochondrial BCKD kinase.
A 50% decrease in BCKD activity state has a substantial effect on BCAA
metabolism within these cells. By decreasing the activity state without
changing total activity, the cells are effectively limiting the
catabolism of BCAA to one-half of the normal rate. This may be enough
to prevent substantial losses in total cellular protein in the absence
of normal cellular leucine concentrations. In our study, total cellular
protein did not decrease in cells grown in the absence of BCAA despite
the fact that there are no detectable BCAA either inside or outside the
cell. In fact, total protein harvested was slightly higher on a per
cell basis in the BCAA-deprived cells (540 vs. 420 ng/cell in control
cells). One possibility to account for the lack of detectable free BCAA
is that all potentially free BCAA are rapidly bound as charged
aminoacyl-tRNAs and are incorporated into newly synthesized proteins.
Irrespective of this model, the ability of cells to adapt and retain
cellular protein levels in the absence of these essential amino acids
reflects the importance of BCKD regulation.
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ACKNOWLEDGEMENTS |
We express gratitude to Elizabeth Jackson for technical assistance.
Special thanks are also given to Dr. S. R. Price for help with the
luciferase assays and editorial comments and to Dr. Y Feng for help
with polyribosome analysis.
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FOOTNOTES |
This work was supported by grants from the Emory University Research
Committee and National Institutes of Health (NIH) Grant DK-38320.
C. B. Doering was supported in part by NIH training Grant
5T32GM-08490. Lymphocyte transformations done in the Clinical Research
Center at Emory University were supported by NIH Grant MO1-RR-00039.
Address for reprint requests and other correspondence: D. J. Danner, Dept. of Genetics, Emory Univ. School of Medicine, 1462 Clifton Rd., Rm. 446, Atlanta, GA 30322 (E-mail:
ddanner{at}emory.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 29 February 2000; accepted in final form 7 June 2000.
 |
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