From the Department of Biochemistry, State University of New York,
Buffalo, New York 14214-3000
Regulated proteolysis has been postulated to be
critical for proper control of cell functions. Muscle development, in
particular, involves a great deal of structural adaptation and
remodeling mediated by proteases. The transcription factor YY1
represses muscle-restricted expression of the sarcomeric
-actin
genes. Consistent with this repressor function of YY1, the nuclear
regulator is down-regulated at the protein level during skeletal as
well as cardiac muscle cell differentiation. However, the YY1 message remains relatively unaltered throughout the myoblast-myotube
transition, implicating a post-translational regulatory mechanism. We
show that YY1 can be a substrate for cleavage by the calcium-activated neutral protease calpain II (m-calpain) and the 26 S proteasome. The
calcium ionophore A23187 destabilized YY1 in cultured myoblasts, and
the decrease in YY1 protein levels could be prevented by calpain inhibitor II and calpeptin. Treatment with the proteasome inhibitors MG132 and lactacystin resulted in the stabilization of YY1 protein, which is consistent with the finding that YY1 is readily
polyubiquitinated in reticulocyte lysates. We further show that
proteolytic targeting by calpain II and the proteasome involves
different structural elements of YY1. This study thus illustrates two
proteolytic pathways through which the transcriptional regulator can be
differentially targeted under different cell growth conditions.
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INTRODUCTION |
Many cellular processes are known to be controlled by short-lived
proteins, including products of the proto-oncogenes, cell cycle
regulators, and developmentally regulated transcription factors (1-3).
The fast turnover of these regulatory proteins reflects a metabolic
requirement for rapidly changing their concentrations and is
presumably mediated by a complex interplay among various proteases and
protease inhibitors. Selective degradation of transcriptional activators and repressors, in particular, may provide efficient regulatory mechanisms contributing to the rapid shut-off and turn-on of
gene activity, respectively. The recent study of the pleiotropic transcription factor NF-
B has revealed that the ubiquitin-proteasome pathway can function not only in the complete degradation of proteins but also in the regulated processing of precursors into active transcription factors (4, 5). Calpains represent the other major class
of nonlysosomal proteases functioning in a
calcium-dependent fashion (6, 7). Interestingly, both the
proteasome and calpains have been found to play a regulatory role in
the function and/or stability of c-Fos and the tumor suppressor protein
p53 (8-11). Thus, rapid degradation or processing of specific
transcription factors can underlie a wide range of dynamic cellular and
developmental processes.
Muscle development involves a great deal of structural adaptation and
remodeling mediated by induced protein synthesis and degradation
(12-14). Although protein turnover must be highly selective if it is
to be developmentally useful, little is known concerning the regulatory
mechanisms responsible for protein targeting and subsequent degradation
during development. The expression of a lysosomal cysteine protease
family, cathepsins, was found to increase during muscle differentiation
(15). Myoblast fusions in chick and rats were both shown to require
metalloendoprotease activity (16). Consistent with the observed calcium
influx during myoblast membrane fusion (17), the activity of the
calcium-activated neutral protease (calpain) is up-regulated during and
required for myogenesis (14, 18). These findings suggest that temporal regulation of proteolytic events plays an important role in muscle development. In most cases, however, the endogenous protein substrates proteolyzed during differentiation have not been characterized, and the
physiological relevance remains to be examined.
We have previously shown that YY1, a C2H2-type
zinc finger DNA-binding protein (19), is capable of simultaneously
activating and repressing the expression of the c-myc
proto-oncogene and the sarcomeric
-actin gene, respectively (20). In
chick embryonic myoblast culture, YY1 inhibits muscle-restricted
transcription of the skeletal
-actin gene by excluding SRF, a
positive MADS-box myogenic transcription factor, from the most proximal
serum response element of the actin gene promoter (20, 21). In these
studies, myoblasts rendered incapable of differentiation were found to contain higher levels of YY1 and c-Myc proteins compared with the
differentiated myotubes. Given the established inhibitory effect of
c-myc on myogenic differentiation (22) and the activating effect of YY1 on c-myc (20, 23), the down-regulation of YY1 was postulated to be essential for the expression of the sarcomeric
-actin genes. However, the YY1 down-regulation mechanism during myogenesis remains unclear. Here we report that YY1 is similarly down-regulated during the in vitro differentiation of
cultured rat skeletal myoblasts and ventricular cardiomyocytes. In
contrast to the down-regulation of YY1 protein, the YY1 message remains unaltered throughout the myoblast-myotube transition. We present both
in vivo and in vitro evidence that calpains and
the 26 S proteasome are involved in the in vivo stability of
YY1. This finding illustrates a post-translational mechanism through
which the repressor of myogenic transcription may be selectively
inactivated by developmentally regulated proteolysis to facilitate
muscle development.
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MATERIALS AND METHODS |
Muscle Cell Culture--
Primary skeletal and cardiac muscle
cell cultures were prepared as described (24, 25). In brief, minced
tissues were gently agitated in 15 ml of 0.05% trypsin + 1 mM EDTA at 4 °C overnight. Excessive trypsin solution
was removed following overnight agitation, and tissue fragments further
incubated at 37 °C for 10 min, after which 10 ml of minimal
essential medium (MEM)1
containing 10% horse serum was added to inactivate trypsin.
Collagenase (Worthington) and DNase I (Sigma) were then added to a
final concentration of 1 mg/ml and 0.1 mg/ml, respectively, for another
20 min of incubation. Tissue fragments were then triturated three to
four cycles with the addition of 10 ml of medium for each cycle.
Skeletal myoblasts were plated in MEM + 10% fetal bovine serum on
Primaria culture dishes (Falcon) at a density of 3,120 cells/mm2. Cardiac myocytes were plated in MEM + 10% horse
serum at a density of 1,040 cells/mm2. Cultured mouse C2
and Sol8 myoblasts were maintained in MEM + 10% fetal bovine serum and
50 µg/ml gentamicin. Calpain inhibitor II and calpeptin were
purchased from Calbiochem. MG132 was provided by Cecile Pickart
(Johns Hopkin University). Lactacystin was purchased from E. J. Corey (Harvard University).
Crude Myoblast Extracts--
Approximately 10 million Sol8 cells
were harvested by scraping in phosphate-buffered saline, and cells were
spun down and freeze-thawed once. The cell pellet was resuspended in
0.3 ml of ice-cold extraction buffer (20 mM HEPES, pH 7.4, 2 mM EDTA, 0.1 M NaCl, 10% glycerol, and 1 mM dithiothreitol). Cells were homogenized at 4 °C by
100 strokes in a period of 20 min. The cell lysate was briefly
clarified (5 s), and the supernatant was collected and frozen in
aliquots at
75 °C. This crude protein extract was used as the
source of endogenous calpain.
In Vitro Cleavage Assay--
The in vitro cleavage
assay was done in a total volume of 20 µl. In each reaction, 2.5 µg
of purified bacterially expressed YY1 was used as substrate. Either
Sol8 myoblast lysate (3.3 µg) or purified calpain II (1-2 µg; from
Calbiochem or Sigma) was used as the source of protease. Proteins were
first assembled on ice, and CaCl2 was added to initiate the
cleavage reaction. Cleavage reactions were continued at 37 °C for 5 min for lysate or 20 min for purified calpain II. Reactions were
terminated with SDS-PAGE sample buffer supplemented with
-mercaptoethanol. Samples were boiled for 5 min, electrophoresed by
SDS-PAGE, and then probed by Western blot.
Caseinolysis Assay--
To assess the specificity of protease
inhibitors, a modified nonradioactive caseinolysis assay was used (26).
Reactions were set up in 15 µl containing 10 mM Tris (pH
7.6), 60 mM KCl, 0.1 mM EDTA, 5% glycerol, 5 mM CaCl2, and 60 µg of
-casein. An individual control reaction lacking CaCl2 was also set up
for each protease inhibitor. Reactions were initiated by adding various protease inhibitors and 1 µg of calpain II and allowed to proceed at
room temperature for 60 min. 5 µl of 0.25 M EGTA was
added to stop the reaction, and 5 µl of sample was mixed with 0.2 ml of diluted Bio-Rad protein assay dye. A595 was
measured by a Labsystem Multiscan microplate reader. Calpain activity
was expressed as decrease in A595 in the
presence of calcium.
In Vitro Protein Ubiquitination Assay--
In vitro
ubiquitination of YY1 was carried out using rabbit reticulocyte lysates
(a source of ubiquitination enzymes) purchased from Promega. Reactions
were set up in 30 µl containing 22.5 µl of reticulocyte lysates and
0.1 µg of purified recombinant YY1 and incubated at 37 °C for 2 min. Samples were then processed for 10% SDS-PAGE and Western blot
using an anti-YY1 antibody as described below.
Northern Blot--
Total RNA was isolated using the guanidinium
thiocyanate method. Isolated RNA was dissolved in formamide, mixed with
ethidium bromide (final concentration 0.1 µg/ml), and separated on a
1.2% agarose gel containing 37% formaldehyde. RNA was transferred to nylon membrane (Bio-Rad) and immobilized by UV cross-linking. Membrane
was blocked in prehybridization solution (1% sodium dodecyl sulfate,
10% dextran sulfate, and 1 M NaCl) for 5 h and then
incubated with a random-primed YY1 cDNA probe at 55 °C
overnight. Probed membrane was washed twice with 2× SSC at 55 °C
followed by one wash with 0.2× SSC. Membrane was air dried and
processed for autoradiography.
Western Blot--
Cells were harvested by scraping at the time
points indicated and lysed in TNT (0.2 M Tris, pH 8, 0.2 M NaCl, 0.1% Triton X-100) supplemented with 1 mM dithiothreitol, and lysates were processed for
SDS-PAGE. Proteins were electrotransferred to Immobilon-P membrane. The
YY1 and SRF antibodies were described previously (24). The
chemiluminescence kit (Amersham Pharmacia Biotech) was used to detect
transferred proteins.
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RESULTS |
Developmental Down-regulation of YY1 Protein During Muscle
Development--
Our previous studies using an avian embryonic muscle
system showed that cycling myoblasts contain higher levels of YY1
protein but lower levels of SRF protein than post-mitotic,
differentiated myotubes. To determine whether YY1 and SRF are also
differentially regulated in mammalian cells, we set up primary muscle
cell cultures prepared from newborn rats. Western blots shown in Fig.
1 (top) illustrated that the
YY1 protein levels were the highest in replicating rat myoblasts (24 h)
and were reduced between 24 and 48 h when myoblasts were
progressing through cell fusion. It is possible that the residual YY1
protein detected after 24 h was derived from contaminating
fibroblasts since we found that fibroblasts contain higher levels of
YY1 (21). Fig. 1 (middle) shows that SRF protein levels were
increased during myogenesis and were the highest in fully
differentiated myotubes (96 h). The developmental pattern of YY1
protein was further examined in cultured rat ventricular cardiac
myocytes (Fig. 2, top). The
study indicated that YY1 was similarly down-regulated during in
vitro cardiac differentiation. Cardiac fibroblasts maintained
under the same differentiation medium (Fig. 2, bottom)
exhibited similar YY1 protein contents throughout, suggesting a
down-regulation mechanism unique to muscle cell differentiation.

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Fig. 1.
Down-regulation of YY1 and up-regulation of
SRF protein levels during skeletal muscle cell differentiation in
vitro. Muscle tissue was isolated from the hindlimbs of
2-day-old Sprague-Dawley rats. Myoblasts were preplated to deplete
fibroblasts and plated in MEM containing 10% horse serum (0 h). Cells
were harvested by scraping at the time points indicated. 10 µg of
proteins were loaded in each lane for Western analysis. Immobilon-P
membrane was probed with rabbit serum raised against YY1 (top
panel) or SRF (middle panel). The chemiluminescence kit
(Amersham Pharmacia Biotech) was used to detect protein signals. Total
proteins were stained with Coomassie Blue as loading control
(bottom panel).
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Fig. 2.
Down-regulation of YY1 protein during
ventricular cardiac myocyte differentiation. Cardiac ventricles
were isolated from 2-day-old Sprague-Dawley rats. Cells were preplated
to deplete fibroblasts and plated in MEM containing 10% fetal bovine
serum and 20 µM araC. Cells were maintained in the growth
medium for 3 days in the presence of araC, after which culture medium
was changed to the differentiation medium (see text) without araC (0 h). Myocytes were harvested at the time points indicated, and lysates
were processed for Western analysis. Top panel, cardiac myocyte proteins probed with YY1 antibody (CA); middle
panel, cardiac myocyte proteins stained with Coomassie Blue;
bottom panel, cardiac fibroblast proteins probed with YY1
antibody (FB). Fibroblasts were maintained in
differentiation medium as control.
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Constant Expression of YY1 mRNA from Cycling Myoblasts to
Post-mitotic Myotubes--
Although YY1 is presumed to be expressed in
many tissues where it may assume a constitutive rather than a
regulatory role, our studies clearly show that YY1 protein is
down-regulated during skeletal as well as ventricular cardiac muscle
cell differentiation. Since changes in YY1 protein content could be
effected at multiple levels, we sought to examine the kinetics of YY1
mRNA synthesis. The temporal pattern of YY1 expression during
skeletal muscle cell differentiation was analyzed by Northern
hybridization in Fig. 3, which shows that
YY1 mRNA levels did not significantly change from cycling myoblasts
(24 h) to post-mitotic myotubes (72 h). Consistent with our finding,
the YY1 mRNA levels were found to remain relatively constant during
retinoic acid-induced F9 EC cell differentiation (27). Thus, the
down-regulation of YY1 during muscle cell differentiation is most
likely effected at the protein level.

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Fig. 3.
The steady state levels of YY1 transcripts
remain constant during muscle cell differentiation. Total RNA was
isolated from 24-, 48-, and 72-hr time points. 10 µg of total RNA was
loaded in each lane. Mouse YY1 cDNA was used as probe in Northern
hybridization. Top panel, autoradiograph of probed membrane;
bottom panel, total RNA on membrane stained by ethidium
bromide (0.1 µg/ml) after an overnight capillary transfer.
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Calpain II-mediated Proteolytic Cleavage of YY1 in Crude Myoblast
Extracts--
The findings demonstrated above prompted us to examine
whether YY1 might be down-regulated by a protease-mediated mechanism during myogenesis. Calpains were first demonstrated in skeletal muscle
homogenates (28), and its activity was found to be up-regulated and
essential during myogenic differentiation. Of particular note is the
down-regulation of YY1 protein during myoblast fusion (24-48 h; Fig.
1, top), which has long been known to be mediated by
extracellular calcium influx (17). To examine the potential involvement
of the Ca2+-calpain system in the degradation of YY1, we
set up an in vitro calcium-dependent proteolysis
assay using crude myoblast extracts as the source of endogenous
calpain. YY1 and its degradation derivatives were detected by YY1
antibody. Fig. 4 shows that no
degradation of YY1 was evident upon incubation of YY1 with the crude
extract or with the addition of 10-100 µM
Ca2+ (Fig. 4, left panel). Addition of 1 mM Ca2+, on the other hand, resulted in the
appearance of a 40-kDa cleavage product recognized by the YY1 antibody.
This finding indicates that YY1 may be a substrate of endogenous
calpain II (or m-calpain), which requires millimolar levels of calcium
ions for catalytic activity as opposed to calpain I (or µ-calpain),
which requires micromolar levels of calcium ions. We further performed
the assay using purified calpain II. Fig. 4 (right panel)
again shows the appearance of the 40-kDa YY1 cleavage product triggered
by the purified calpain II in the presence of 1 mM
Ca2+. As predicted, calpain II caused no cleavage of YY1 in
the absence of Ca2+ or at lower Ca2+
concentrations.

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Fig. 4.
Calcium-dependent proteolysis of
YY1 by endogenous and purified calpain II. In vitro cleavage
assays were done in a total volume of 20 µl. In each reaction, 2.5 µg of purified bacterially expressed YY1 (20) was used as a
substrate. 3.3 µg of Sol8 myoblast lysate (left half) and
2 µg of purified calpain II (right half) were used as the
source of protease. Proteins were assembled on ice, and
CaCl2 was added to initiate the reaction. Reactions were carried out at 37 °C for 5 min (crude lysate) or 10 min (pure calpain II) and were terminated with SDS-PAGE sample buffer
supplemented with -mercaptoethanol. Samples were boiled for 5 min,
electrophoresed through a 10% gel, and processed for Western analysis.
The full-length YY1 and the cleavage product are indicated by and
, respectively.
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Intracellular Stabilization of YY1 by Inhibitors of Calpains and
the Proteasome--
We went on to determine whether YY1 protein might
be stabilized in cells treated with specific calpain inhibitors.
Primary myoblasts were used initially to examine the effect of two
specific calpain inhibitors, calpeptin and calpain inhibitor II (29), on the intracellular level of YY1. However, the primary myoblasts were
found to be exquisitely sensitive to the inhibitors, and the treatment
resulted in a population consisting largely of fibroblasts (data not
shown). This finding presumably indicates a vital role for calpains in
maintaining the viability of cultured primary myoblasts. Sol8 myoblasts
(30) were thus used in the subsequent inhibitor experiments. A panel of
cell-permeable protease inhibitors, calpain inhibitor II (29), TLCK
(inhibitor of trypsin-like serine proteases) (31), and MG132 (inhibitor
of the 26 S proteasome) (32) were first examined for their inhibitory
effects on calpain II activity using a quantitative in vitro
assay described previously (26). Fig. 5
shows that whereas TLCK did not appreciably affect calpain II activity
as expected, calpain inhibitor II and MG132, which are similar
tripeptide aldehydes consisting of leucine-leucine-methioninal and
leucine-leucine-leucinal, respectively, each exhibited a comparable potent dose-dependent inhibition of the caseinolytic
activity of calpain II. Thus, MG132 appears to be capable of blocking
the activities of both calpains and the proteasome.

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Fig. 5.
Comparison of three cell-permeable peptide
inhibitors in caseinolysis assay. The assay using -casein as
substrate was as described previously (26). Reactions were set up in 15 µl containing 60 µg of -casein with or without CaCl2
as detailed in the text. Reactions were initiated by adding protease
inhibitors and 1 µg of calpain II (Sigma) and allowed to proceed at
room temperature for 60 min. Reactions were terminated with EGTA, and 5 µl of the sample was mixed with 0.2 ml of diluted Bio-Rad protein assay dye. Calpain activity was expressed as decrease in
A595 in the presence of calcium. Points are
averages of two separate assays and represent mean ± S.D.
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We went on to determine whether the inhibitors might stabilize YY1
protein in the cell. Sol8 myoblasts were treated with the inhibitors,
and Western blots were performed to examine the effect on YY1 protein
levels. Inhibitor concentrations for the treatment were optimized such
that minimal loss of cell viability was observed after treatment. Fig.
6A shows that whereas YY1
protein levels were not significantly altered by TLCK (54 µM) or calpain inhibitor II (40 µM),
incubation of Sol8 myoblasts with 8 µM MG132 consistently increased YY1 protein content at least 2-fold. Prolonged treatment or
treatment with higher doses of MG132 led to a rapid loss of myoblast
viability (data not shown). Since autoproteolytic activation and
catalytic activity of calpains require elevated calcium levels in
myoblasts, an event typically associated with myoblast fusion during
myogenesis (17), an effect of the calpain inhibitor on YY1 protein
stability may require elevated calcium levels in the cell. To provide
evidence along this line, intracellular calpains were stimulated by
treating myoblasts with the calcium ionophore A23187, which has been
shown to promote myogenesis (14) and activate calpains (33). Fig.
6B shows that YY1 protein levels were clearly reduced by
A23187 in treated Sol8 myoblasts. Calpain inhibitor II as well as MG132
were both able to stabilize YY1 in A23187-treated Sol8 myoblasts.
Together, these in vitro and in vivo results
strongly suggest the involvement of the Ca2+-calpain
circuit in signal-mediated intracellular proteolysis of YY1.

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Fig. 6.
Involvement of calpains and the proteasome in
the intracellular stability of YY1. Left, Sol8 myoblasts
were trypsinized, and 2 million cells in 1 ml of medium were incubated
with calpain inhibitor II (40 µM), TLCK (54 µM), or MG132 (8 µM) in 1-ml microtubes at
37 °C for 3 h. Tubes were inverted several times every 30 min. Cells were spun down, lysed, and boiled in 0.1 ml of SDS-PAGE sample
buffer for Western analysis. Right, Sol8 myoblasts were treated with 5 µM A23187 in the absence and presence of
protease inhibitors for 3 h. Samples were processed for Western
analysis using YY1 antibody.
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YY1 Is Also a Substrate of the 26 S Proteasome--
That MG132, a
well known inhibitor of the 26 S proteasome, was able to stabilize YY1
in myoblasts suggests that YY1 is likely to be a substrate of the
proteasome as well. However, the results shown here together with those
of others (29, 34) indicate that MG132 can inhibit both calpains and
the proteasome. To resolve this issue, we further examined the effect
of lactacystin, which is highly specific for the proteasome (35). We
have shown previously that a putative polyubiquitinated YY1 species,
which could not be demonstrated in actively growing myoblasts, could be
detected in myoblasts deprived of serum (36). Thus, myoblasts
maintained in growth medium as well as serum-free medium were treated
with lactacystin for 30 h, and YY1 protein levels were examined by Western analysis. Fig. 7 shows that
whereas lactacystin did not have a significant effect on the stability
of YY1 in growing myoblasts (panel A), it increased YY1
protein levels severalfold in serum-starved myoblasts (panel
B). To further provide biochemical evidence that YY1 is indeed a
ubiquitination substrate, we carried out an in vitro
experiment using rabbit reticulocyte lysates as an established source
of ubiquitination enzymes (5). In the experiment presented in Fig.
8, purified YY1 was first incubated with
reticulocyte lysates, and ubiquitination products were separated by
SDS-PAGE and probed with YY1 antibody. A ladder of protein bands
exhibiting slower mobilities relative to the free YY1 protein could be
detected, which should correspond to YY1 proteins ligated to one or
more ubiquitin moieties. The YY1 C-terminal truncation mutant N200, on
the other hand, was not appreciably tagged by ubiquitin as indicated by
the absence of ubiquitin conjugate ladders (Fig. 8). We thus conclude
that amino acid sequences located within the zinc finger domains of YY1
contain recognition/degradation signals for the
ubiquitin-dependent proteolytic pathway.

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Fig. 7.
Lactacystin stabilizes YY1 in serum-starved
myoblasts. Sol8 myoblasts (4 × 105) were plated
on 60-mm dishes in medium containing 10% fetal bovine serum.
Panel A, lactacystin (16 µM) was added to the
medium 16 h after plating, and both control and treated cells were
maintained in the growth medium for 30 h before harvesting for
Western analysis using YY1 antibody. Panel B, myoblasts were
switched to serum-free medium containing 5 µg/ml insulin, 1 µg/ml
transferrin, and 5 ng/ml sodium selenite 16 h after plating.
Lactacystin was added at the time of the medium change, and both
control and treated cells were maintained in the medium for 30 h
before harvesting. 100 µg of proteins were applied in each lane.
Coomassie Blue-stained proteins are shown on the bottom as
loading control.
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Fig. 8.
YY1 is readily ubiquitinated in rabbit
reticulocyte lysates. 0.1 µg of YY1 (WT) and the
C-terminal deletion mutant (N200) were each incubated with
reticulocyte lysates as described in the text. Samples were processed
by 10% SDS-PAGE followed by Western analysis using YY1 antibody. The
position of the free YY1 protein is denoted by .
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Targeting by Calpains and the Proteasome Involve Different Domains
of YY1--
That the C-terminal half of YY1, which is rich in PEST
residues (Pro, Glu, Ser, and Thr), is required for protein
ubiquitination is consistent with the notion that short lived proteins
may be proteolytically targeted through these sequences (37). However, there is some controversy as to whether the PEST domain of
PEST-containing proteins may be required for the action of calpains (1,
38, 39). Inspection of cleavage sites among the substrates of calpains reveals no obvious consensus sequence motif. It is also of interest to
determine whether YY1 may be differentially targeted by the proteasome
and calpains. Using various YY1 deletion mutants constructed previously
(24), we found that neither the zinc finger nor the PEST domain, both
located at the C-terminal half of YY1, was required for calpain action
(Fig. 9). Notably, the deletion mutant
N152 but not N170 exhibited resistance to calpain cleavage, suggesting that the 20-amino acid domain missing in N170 might play a signaling role in calpain susceptibility of YY1. Our findings indicate that different structural determinants are involved in calpain- and proteasome-mediated proteolysis.

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Fig. 9.
An internal 20-amino acid domain of YY1
mediates calpain susceptibility. The full-length YY1 and six YY1
C-terminal deletion mutants N395, N333, N271, N200, N170, and N152 were
assayed for calpain susceptibility as described in Fig. 4 except that 10 µg of YY1 proteins were used. Cleavages of N395, N333, and N271
are not shown here. No calpain II ( ) and 2 µg of calpain II
(Calbiochem) (+) included in reactions. Proteins were stained with
Coomassie Blue after SDS-PAGE. Cleavage products were denoted by
.
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DISCUSSION |
Several lines of evidence suggest that calpain II plays an
important regulatory role in myogenic differentiation. The increase in
calpain II activity coincides with the onset of myoblast
differentiation, which is marked by cell membrane fusion and calcium
influx (18). Exogenously added calpain II was found to promote myoblast
fusion (40). Studies using calpain inhibitors also showed that
inhibition of the endogenous calpain can suppress myogenesis (14, 41). Although possible roles of calpain II in muscle growth and
differentiation have been postulated (6, 42), the biochemical mechanism
underlying its myogenic function is still unclear. The work presented
here offers a regulatory mechanism for calpain II through proteolytic cleavage of the multifunctional transcription factor YY1, which is
capable of repressing myogenic transcription via its DNA-binding activity (43, 44). A direct in vivo evidence that YY1 can be
proteolyzed by calpains is illustrated here by the A23187 experiment, showing stabilization of YY1 protein by calpain inhibitor II in A23187-treated myoblasts. Consistent with this "proteolytic" effect of A23187, this calcium ionophore was found to activate calpains (33)
and promote myogenesis (17).
The 26 S proteasome system has been extensively studied and found to be
involved in the degradation of several transcriptional regulators such
as NF-
B (4), c-Jun (45), c-Fos (8), p53 (46), and the yeast MAT
2
repressor (2). Our current and previous studies (36) together indicate
that YY1 turnover is mediated by calpains as well as the proteasome. We
found that MG132, which is generally used as an inhibitor of the
proteasome (32), is able to stabilize YY1 in treated myoblasts. MG132
and calpain inhibitor II are both tripeptide aldehydes consisting of
leucine-leucine-leucinal and leucine-leucine-methioninal, respectively. Thus, the ability of MG132 to inhibit calpains as shown here may not be
unexpected. Since MG132 appears to be an inhibitor of both calpains and
the proteasome, it is possible that stabilization of YY1 in
vivo can be best observed under the condition where both
proteolytic pathways are blocked concurrently. This notion is
consistent with the findings here that the stabilization of YY1
in vivo by lactacystin can only be observed in serum-starved myoblasts that exhibit elevated proteasome activities (36). In cellular
processes associated with signal-induced calcium influx (as mimicked by
A23187 treatment), calcium-mediated calpain activation may become a
major proteolytic driving force. This may explain why stabilization of
YY1 by calpain inhibitors can only be observed in A23187-treated
myoblasts.
The majority of short lived proteins are found to possess one or more
domains rich in Pro, Glu, Ser, and Thr residues (PEST domain) (37).
However, the mechanism that PEST regions confer susceptibility to rapid
proteolysis remains unclear. The structural features or signals of
transcription factors that cause them to be rapidly degraded in
vivo remain to be further characterized. We present evidence that
calpains and the proteasome utilize different structural elements for
substrate targeting. Our finding supports the previous notion that PEST
sequences do not influence substrate susceptibility to calpain
proteolysis (38). Proteolytic targeting by the proteasome on the other
hand involves the participation of PEST residues. These differential
targeting mechanisms of calpains and the proteasome presumably provide
elements of specificity necessary for cellular regulation and allow for
a more versatile developmental control module. Whereas the
ubiquitin-dependent pathway is well known for its
housekeeping function, we propose that the proteasome and calpains may
differentially regulate the stability of a protein under different
growth conditions and signaling pathways, as demonstrated by the use of
A23187 and serum starvation here. Like YY1, the stability of c-Fos and
p53 has previously been found to be co-regulated by calpains and the
proteasome (8-11). Aside from the involvement of various proteases in
controlling the level of transcription factors, the eukaryotic
DNA-binding factor AEBP1 has been found to possess a protease activity
(47). Thus, targeting proteases to a transcriptional machinery may
represent a unique feature in gene regulation.