From the Department of Biology, the
§ Centre for Research in Mass Spectrometry, and the
¶ Department of Chemistry, York University, Toronto M3J 1P3,
Canada
Received for publication, November 5, 2002, and in revised form, February 12, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The phosphorylation status of the myocyte
enhancer factor 2 (MEF2) transcriptional regulator is a critical
determinant of its tissue-specific functions. However, due to the
complexity of its phosphorylation pattern in vivo, a
systematic inventory of MEF2A phosphorylation sites in mammalian cells
has been difficult to obtain. We employed modern affinity purification
techniques, combined with mass spectrometry, to identify several novel
MEF2 phosphoacceptor sites. These include an evolutionarily conserved KSP motif, which we show is important in regulating the
stability and function of MEF2A. Also, an indirect pathway in which a
protein kinase casein kinase 2 phosphoacceptor site is
phosphorylated by activation of p38 MAPK signaling was
documented. Together, these findings identify several novel
aspects of MEF2 regulation that may prove important in the control of
gene expression in neuronal and muscle cells.
Myocyte enhancer factor 2 (MEF2)1 is a transcriptional
regulatory complex mediating diverse cellular functions in neurons (1, 2), skeletal (reviewed in Ref. 3) and cardiac muscle (4-6), and T
cells (7, 8). It is now well established that MEF2 plays a role in the
differentiation of these cell types as well as functioning in a
protective role against neuronal apoptosis.
To respond to diverse developmental and physiological cues, MEF2 is
structurally organized to receive and respond to multiple signals from
several intracellular signaling pathways (reviewed in Refs. 3
and 9). In this regard, perhaps the best characterized is the p38
MAPK-MEF2 axis, in mammals (10, 11) and in yeast (12), although other
kinase-catalyzed cascades mediated by big MAP kinase (13, 14), protein
kinase C (10), and protein kinase CK2 (15) are known to target
MEF2. Moreover, consistent with its role as a signal sensor, putative
phosphoacceptor motifs in the carboxyl terminal MEF2 transactivation
domain may prove to further modulate MEF2 function in response to
extracellular cues.
Given that MEF2, and the biological processes it regulates, are
intrinsically governed by MEF2 phosphorylation status, we undertook to
systematically document MEF2 phosphorylation patterns in mammalian
cells; previous phosphopeptide mapping studies used in vitro
phosphorylated MEF2 protein. The purpose thus being to detect
physiologically relevant, and possibly novel, in vivo MEF2 phosphorylation sites. To accomplish this we used several
state-of-the-art mass spectrometric techniques to detect
phosphorylation sites from MEF2 expressed in mammalian cells. To this
end, we have made use of a mammalian tandem affinity purification (TAP)
method (16, 17) for low-abundance nuclear transcription factors that
allows purification to homogeneity and provides amounts compatible with mass spectrometric analysis of phosphorylation sites.
In these studies, we have identified two important and novel aspects of
MEF2 regulation. One is a highly conserved phosphoacceptor motif that
regulates MEF2 stability and function. The second is an indirect
pathway of MEF2 regulation by p38 MAPK mediated by the CK2 holoenzyme.
These studies on MEF2 regulation thus identify novel aspects of
functional MEF2 regulation that will serve to modulate MEF2 controlled
gene expression in a variety of cell types.
Materials--
Unless otherwise noted, all chemicals were
obtained from Sigma-Aldrich. DNA-modifying enzymes were purchased from
New England Biolabs (NEB).
Cell Culture and Transfections--
COS7 and HeLa cell cultures
were maintained in Dulbecco's modified Eagle's medium
(Invitrogen) containing penicillin, streptomycin (Invitrogen),
and 10% fetal bovine serum (Atlanta Biologicals). Transfections were
performed using the calcium phosphate precipitation method.
His-tagged Protein Purification--
The coding region for human
MEF2A was cloned into the EcoRI site of pCDNA 3.1/His
(Invitrogen). The coding region for the N-terminal His-MEF2A was then
subcloned into the PstI site of pCDNA4/TO/His
(Invitrogen). Cells were transfected with 30 µg of DNA per 100-mm
dish. Typically, 20 plates of cells were used for a single
purification. A standard manufacturer's protocol was used for
purification using Ni-Agarose resin (Qiagen).
Tandem Affinity Purification--
The coding region for human
MEF2A was ligated into the EcoRI site of pCDNA4/TO/TAP
(described in Ref. 17). Cells were transfected with 30 µg of DNA per
100-mm dish. Typically, 5 plates of cells were used for a single
purification. The details of mammalian TAP-tagged protein purification
are explained in Ref. 17. Briefly, cells were lysed by freeze/thawing.
The lysate was passed over IgG resin (Sigma), and the beads were
washed. Tagged proteins were eluted by cleaving with TEV protease
(Invitrogen) then supplementing with Ca2+ and passed over
calmodulin resin (Stratagene) for a second round of purification.
Proteins were finally eluted using either 2 mM EGTA or SDS
sample buffer and analyzed by SDS-PAGE. Proteins were visualized using
Gelcode Blue (Pierce).
In-gel Trypsin Digest--
Protein bands were excised, cut into
1 mm3 pieces, and washed 3 times with 50% acetonitrile/25
mM ammonium bicarbonate for 15 min with shaking. Gel pieces
were incubated with 50 mM ammonium bicarbonate + 10 mM dithiothreitol for 30 min at 50 °C, washed with
acetonitrile, then incubated with 50 mM ammonium
bicarbonate + 55 mM iodoacetamide (freshly made) for 20 min
in the dark. The gel pieces were washed with acetonitrile, air dried,
and rehydrated with 12.5 ng/µl trypsin (Promega, sequencing grade) in
50 mM ammonium bicarbonate, then incubated at 37 °C
overnight. Peptides were extracted once using 3% formic acid, 1 min at
80 °C, followed by 20 min of shaking and 1 min of centrifugation.
Mass Spectrometry--
Peptides were concentrated prior to
analysis using µZipTipsTM (Millipore) according to
the manufacturer's protocol. Peptides were eluted directly onto a
steel target using 10 mg/ml Western Blot Assay--
The MEF2A antibody was generated in
rabbits using full-length His-MEF2A purified from bacterial cells. The
serum was purified against an immobilized MEF2A column (CnBr-activated
agarose, Amersham Biosciences) and IgG was concentrated using a
protein A-Sepharose column (Amersham Biosciences). Total protein was
separated by 10% SDS-PAGE, transferred (Semi-Dry, Hoefer) to
nitrocellulose (Osmonics), blocked with 5% milk in PBS, and probed
with Northern Blot Assay--
Total RNA was isolated with Trizol
reagent (Invitrogen) and resolved on a 1% agarose gel in the presence
of 6.2% formaldehyde and then blotted on to Nytran membrane
(Schleicher & Schuell) in 20× SSC. The complete cDNA (2.9 kb) of
MEF2A, excised from pMT2 vector with EcoRI, and a 316-bp
glyceraldehyde 3-phosphate dehydrogenase fragment, derived from
exons 5-8 (Ambion) and excised with SacI and
HindIII, were used as probes for MEF2A and glyceraldehyde 3-phosphate dehydrogenase, respectively. The probes were labeled with
32P using Random Primers DNA Labeling System (Invitrogen)
and purified on MicroSpinTM G-25 Columns (Amersham
Biosciences). Prehybridization (2 h) and hybridization (overnight) were
carried out in 50% deionized formamide, 5× Denhardt's, 5× SSC, 5 mM EDTA, 0.1% SDS, and 200 µg/ml salmon sperm DNA.
Membranes were washed and signals were visualized by exposure to film
(BioMax MR, Kodak). Between labeling with MEF2A and glyceraldehyde
3-phosphate dehydrogenase, the blots were stripped for 2 h in 50%
deionized formamide, 0.1% SSC, 0.1% SDS preheated to 68 °C.
GAL4 Luciferase Assay--
COS7 cells were seeded at 250,000 cells/well in a 6-well dish the day before transfection. Cells were
transfected with GAL4 luciferase (0.5 µg),
pSV40- Site-directed Mutagenesis--
The GAL4-MEF2A S255A and S255D
mutations were generated using the QuikChange kit (Stratagene)
according to the manufacturer's instructions. The primers used were:
S255A-GTC ATG CCT ACA AAG GCT CCC CCT CCA CCA G and S255D-GTC ATG CCT
ACA AAG GAT CCC CCT CCA CCA G.
Expression and Purification of MEF2A from Mammalian Cells--
Two
purification strategies, His6 and TAP, were employed
for obtaining suitable material to probe the phosphorylation status of
MEF2A in mammalian cell culture. The His6-MEF2A was
expressed in HeLa followed by purification using Ni2+
affinity beads. This strategy was capable of purifying amounts of MEF2A
that could be visualized using Coomassie Blue staining (Fig.
1A). More importantly, the
purified MEF2A was found in two distinct bands, indicating that a
fraction of it had been post-translationally modified. This pattern of
mobility is in agreement with previous detection of MEF2A from HeLa
(18), COS7, and muscle cell cultures (19) by Western immunoblotting,
and is in sharp contrast to the single band obtained when MEF2A is
purified from bacterial cells (data not shown). Purified MEF2A protein
was subsequently used to characterize its endogenous phosphorylation
status.
To investigate the effect of p38 MAPK on the phosphorylation status of
MEF2A, we employed an additional purification strategy utilizing the
TAP scheme. TAP-MEF2A was expressed in COS7 cells in the absence or
presence of expression vectors for p38 MAPK and a constitutively active
upstream regulator, MKK6. The TAP scheme purified MEF2A to near
homogeneity, allowing us to use Coomassie Blue staining to reveal the
sizable shift in mobility of MEF2A caused by p38 MAPK (Fig.
1B). This shift has previously only been detected by
immunoblotting or radiolabelling. This purification was used to probe
for novel p38 phosphorylation sites in MEF2A.
Identification of MEF2A--
Proteins were identified by either
tryptic mass fingerprinting or CID fragmentation. The MALDI-TOF
spectrum for the tryptic digest of MEF2A (Fig.
2A) contains a number of peaks
whose masses match the expected mass of peptides from MEF2A.
This confidently identifies the protein as MEF2A. Further confirmation
was obtained, using a nanospray-QStar instrument, by CID fragmentation
of selected peptides. The fragmentation products reveal short sequences
and characteristic ions (Fig. 2B) that could only be derived
from specific MEF2A peptides. These two types of analyses were
subsequently used for identification of phosphopeptides.
We used a broad range of techniques to characterize the phosphorylation
status of MEF2A in mammalian cell culture. This included comparative
analysis between tryptic mass fingerprints, characteristic 80 Da shifts
in mass caused by phosphorylation, CID fragmentation, immobilized
metal-affinity capture of phosphopeptides, and sequence homology. The
details of these analyses will be discussed below.
A Novel, Endogenous Phosphorylation Site in MEF2A--
The
MALDI-TOF spectra for tryptic mass fingerprints of MEF2A expressed in
mammalian cells were very similar to MEF2A expressed in bacterial cells
with one notable exception. A peak at mass 1437.69 Da, corresponding to
the amino acids 255-269 (SPPPPGGGNLGMNSR), was significantly lower
when MEF2A was expressed and purified from mammalian cells (Fig.
2A), whereas it was prominent in MEF2A expressed in
bacterial cells. This prompted us to investigate what was the cause of
this difference. A post-translational modification, such as
phosphorylation, was a likely candidate; however, no peak was detected
at 80, 160, or 240 Da higher in mass. Careful inspection of the region
near 2000 m/z (Fig. 2C) revealed a
small peak with an m/z value of 1994.02, corresponding to a MEF2A peptide with one missed trypsin cleavage,
amino acids 250-269 (VMPTKSPPPPGGGNLGMNSR), and a peak 79.94 Da higher
in mass (2073.96). Phosphate incorporation adds 79.97 Da. These data
indicate that a measurable fraction of the MEF2A protein pool in
mammalian cells is phosphorylated at Ser-255. This sample was then
analyzed on a nanospray-QStar instrument to obtain CID fragmentation
data (Fig. 2D). Fragmentation of the potential
phosphopeptide revealed a clear set of y fragments identifying a stretch of 4 proline residues. Additionally, the fragment
at m/z 1419.76 can only be explained by a
y15 fragment that has lost
H3PO4 (mass loss of 98 Da). These fragments and others confidently identify this peptide as MEF2A 250-269
phosphorylated at Ser-255.
To ascertain the potential importance of this phosphorylation site we
compared the sequence of human MEF2A with the other MEF2 family members
from various organisms (Fig. 3). The
level of sequence homology for this region is remarkable and confirms the high degree of evolutionary conservation of the KSPP
phosphorylation motif. The lack of conservation at Thr-253 is in
agreement with the fragmentation data, which demonstrates the
phosphorylation of Ser-255, not Thr-253.
Phosphorylation of Ser-255 Targets MEF2A for
Degradation--
Because Ser-255 is in the MEF2 C-terminal
transactivation domain, we assessed the function at this site in a
GAL4-based transcriptional response assay by generating mutations of
Ser-255 in a GAL4-MEF2A (residues 99-507) fusion protein. These
include Ser-255 to aspartic acid (S255D) and Ser-255 to alanine (S255A)
mutations. A GAL4 luciferase assay system in COS7 cells was used to
determine the activity of these proteins (Fig.
4C). The activity of the S255D mutation was markedly lower than that of either the wild type GAL4-MEF2A or the GAL4-MEF2A (S255A) mutation. A Western blot for MEF2A
on cells transfected with these expression vectors indicates decreased
full-length protein levels for GAL4-MEF2A (S255D) and evidence of
degradation products (Fig. 4B). A Northern blot on these
samples demonstrates that transcript expression levels can not account
for the differences in GAL4-MEF2A protein levels indicated in Fig.
4B, thus indicating that the decreased protein levels for
GAL4-MEF2A (S255D) are likely due to instability of the protein. This
is further supported by the detection of at least one large proteolytic
fragment by Western analysis (Fig. 4B).
Identification of Novel p38 MAPK Sites in MEF2A--
Next, we
re-visited the p38 MAPK-catalyzed phosphorylation of MEF2A, because p38
MAPK is a potent physiological activator of MEF2 transactivation
potential (10, 11). Thr-312 and Thr-319 are known phosphorylation sites
important for the increased transcriptional activation of MEF2A by p38
MAPK. Ser-453 and Ser-479 are phosphorylated in vitro but
were not important functionally (11). However, previous results
indicate that the in vivo phosphorylation of MEF2A by p38
MAPK is possibly more complicated than previously reported (10). A
two-dimensional phosphopeptide map of MEF2A expressed in COS7 cells
with p38 MAPK and MKK6 showed a very complex pattern of phosphorylation
(10) that was different from phosphorylation data derived from in
vitro analysis (11). Because this could have important regulatory
consequences, we have used mass spectrometry to further dissect and
characterize p38 MAPK phosphorylation of MEF2A in mammalian cells.
The tryptic mass fingerprint of MEF2A, co-expressed with p38 MAPK and
MKK6, was compared with the lower-mobility band from cells expressing
MEF2A alone (Fig. 5). This simple
comparison reveals a number of phosphorylated peptides, the evidence
for which will now be considered individually.
The peptide of mass 4223.05 (MEF2A 190-233, note that the fourth
carbon isotope, 4226.06, is labeled in Fig. 5) has one methionine residue and two potential p38 MAPK phosphorylation sites. Methionine residues are often partially oxidized due to exposure to air. In the
p38 MAPK-treated sample there is a peak at a mass 80 Da higher,
indicating phosphorylation. This peak is not present in the sample from
cells expressing MEF2A alone. Furthermore, the phosphorylated and
non-phosphorylated peptides both show evidence of an oxidized form at a
mass 16 Da higher from each respective peak. This confirms that this
peptide is the phosphorylated form of MEF2A 190-233.
A similar analysis reveals a phosphorylated peptide at mass 2714.32 (MEF2A 475-498). Again, this peptide is 80 Da higher than the
non-phosphorylated peptide and is absent from the non-treated sample.
Interestingly, in the sample from cells expressing MEF2A alone, the
non-phosphorylated peptide is also absent. Occasionally, phosphopeptides lose their phosphate group, either in solution or
during ionization. Because this peptide is actually a missed tryptic
cleavage of Arg-492, there is the possibility that phosphorylation of
this peptide interfered with the trypsin digestion, prior to the loss
of phosphate.
The MEF2A 95-114 peptide has a mass of 2247.99 whereas its
phosphorylated form is found at 2327.96. The intensity of the latter peak is quite high compared with the former peak. However, it is
difficult to quantify these peptides based on peak intensity, as the
energy required to ionize a phosphopeptide may differ from its
non-phosphorylated form.
In addition to peptide mass fingerprinting, we employed CID
fragmentation to sequence putative phosphopeptides. Furthermore, the
efficiency of nanospray ionization employed in this instrument differs
from that of the MALDI source used for peptide mass fingerprinting. This form of ionization was particularly suitable for detecting the
MEF2A phosphopeptide 404-413 (doubly charged m/z
of 617.28). Subsequent CID fragmentation of this peptide confirmed the
presence of phosphorylation (loss of H3PO4 from
the precursor ion and several fragment ions) at Ser-408 (Fig.
6).
A p38 MAPK-induced Phosphopeptide with no MAPK Consensus--
Of
particular interest is the MEF2A peptide 283-300. This region of MEF2A
is alternatively spliced, and this particular splice variant is
abundant in muscle and nerve cells. Phosphorylation of this peptide is
indicated by the 80 Da shift in mass to 2182.95; however, this peptide
does not contain a proline-directed MAPK consensus site (Fig.
7A). Further evidence that
this peptide is phosphorylated is provided by the oxidation of the two
methionine residues in this peptide. The phosphorylated peptide shows
evidence of two oxidized methionine residues, in agreement with the two oxidized methionine residues seen for the non-phosphorylated peptide. Additional evidence that at least a fraction of this peptide is phosphorylated is provided by the use of a copper immobilized metal-affinity capture purification. MEF2A tryptic peptides were fractionated using a copper(II)-sulfate-treated metal chelating ZipTipTM. Phosphorylated peptides have a higher affinity
for Cu2+ than non-phosphorylated peptides. The selectively
bound peptides were eluted, concentrated on a C-18
ZipTipTM, and analyzed by MALDI-TOF. The presence of the
m/z 2182.92 peptide in this eluate confirms the
phosphorylation status of the MEF2A 283-300 peptide (Fig.
7B). Sequence analysis of this peptide reveals a strong
consensus motif that is targeted by protein kinase CK2. Because
phosphorylation of this peptide is dependent on p38 MAPK activation,
cross-talk between p38 and protein kinase CK2 in MEF2A phosphorylation
is implicated by these data (see "Discussion").
In these studies we have used state-of-the-art mass spectrometric
techniques to asses the in vivo phosphorylation pattern of
the MEF2A transcriptional regulator. This approach has yielded novel
information concerning the regulation of MEF2: first, the identification of a novel phosphoacceptor site that regulates MEF2
stability; second, the identification of previously uncharacterized p38
MAPK phosphoacceptor sites; and third, the discovery of a protein kinase CK2 consensus phosphoacceptor site that requires p38
MAPK activity to be phosphorylated. These studies constitute several
novel aspects of MEF2 regulation by reversible phosphorylation.
Phosphorylation of Ser-255 in MEF2A by endogenous kinases is detected
in several cell types (C2C12, COS, and HeLa), and this phosphorylation
is independent of the activity of p38 MAPK (Fig. 5). This sequence is
highly conserved in members of the MEF2 family across numerous species,
highlighting the importance of this site in the regulation of MEF2
proteins. The conserved sequence consists of a KSP motif, which is a
known phosphoacceptor site for several kinases. This includes MAPK
members (ERK1, ERK2, and stress-activated protein kinase) (20,
21), cdc2-like kinases (including CDK5) (22, 23), and glycogen
synthase kinase 3 (24). The presence of these proteins in both
muscle and nerve cells is suggestive of a link between these pathways
and the functional consequences of phosphorylation of this site in
MEF2.
The extracellular-regulated kinases (ERK1 and ERK2) are abundant in
nerve cells (25) and have been implicated in the myogenic program
(26-30). The activity of ERK is biphasic, with higher activity in
myoblasts and later stage myotubes (26, 28). In the early stages of the
myogenic program, ERK has an inhibitory effect on the expression of
myogenic regulators and muscle-specific proteins (28, 29). In later
stages of differentiation, ERK appears to enhance the formation of
mature myotubes (26, 27) and induce hypertrophy (30). Thus,
phosphorylation-induced degradation of MEF2 proteins by ERK would
explain the early inhibitory effect that ERK has on the myogenic program.
The CDK5 protein is found predominantly in nervous tissue, although it
has also been detected in muscle and other non-neural tissues. It has
been implicated in migration, actin dynamics, microtubule transport,
cell adhesion, axon guidance, synaptic structure and plasticity,
membrane transport, and myogenesis (31). In muscle cells CDK5 can be
detected throughout the myogenic program. The level of expression and
activity of CDK5 is increased during myogenesis. In addition,
proliferating myoblasts show a predominantly cytoplasmic localization
of CDK5, whereas differentiating cells have an increase in nuclear
levels of CDK5 (32). The possibility exists that CDK5 has a role in
regulating the activity of MEF2 proteins by direct phosphorylation,
although how this would pertain to regulation of MEF2 by degradation
would require further investigation.
The ubiquitous phosphorylation of Ser-255, coupled with its role in the
degradation of MEF2, points tantalizingly toward a role for glycogen
synthase kinase 3. Originally thought of as an enzyme solely involved
in glycogen metabolism, this ubiquitous kinase is now known to be
involved in numerous cellular signaling functions (33). Overexpression
of active glycogen synthase kinase 3 induces apoptosis in neuronal
cells by phosphorylation of downstream targets (34). In addition,
glycogen synthase kinase 3 is known to phosphorylate several proteins,
which are subsequently targeted for degradation (35, 36). It is
interesting to note that MEF2 has recently been identified as
protecting neuronal cells from apoptosis (2, 37, 38) and that it is
also targeted for degradation by phosphorylation (39). Whether Ser-255
is the targeted residue that links MEF2 degradation and neuronal
apoptosis will be of great interest.
MEF2A has been convincingly identified as a target for p38 MAPK.
Phosphorylation of MEF2A by p38 MAPK strongly increases the transcriptional activity of MEF2A, and this increase in activity has
been mapped to a couple of key residues (Thr-312 and Thr-319) (10, 11).
However, in vivo phosphorylation of MEF2A by p38 MAPK
produces a complex pattern of phosphorylation that cannot be entirely
explained by these two key residues. The possibility, therefore,
remains that other sites within MEF2A are involved in p38 MAPK
signaling. Although the tryptic phosphopeptide containing Thr-312 and Thr-319 was too large to detect by mass
spectrometry, the data from this study conclusively identify several
new phosphorylation sites, induced by p38 MAPK activity (Fig.
8).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cyano-4-hydroxycinnamic acid in 65%
acetonitrile/0.3% trifluoroacetic acid. To purify phosphopeptides, metal-chelating ZipTipsTM (Millipore) were
used with copper(II)-sulfate according to the manufacturer's
instructions. Peptide fingerprinting was performed on a Voyager DE-STR
(Applied Biosystems) using positive ion reflector mode under optimized
conditions. Fragmentation analysis was performed using a nanospray ion
source on a prototype Q-Star instrument (Sciex). The instrument was
optimized and calibrated daily.
-MEF2A overnight. Blots were washed 3 × 10 min with milk
in PBS, incubated with secondary (1:10,000, Pierce) for 1 h, then
washed 2 × 10 min with milk in PBS + 0.2% Nonidet P-40 and
3 × 10 min in PBS + 0.2% Nonidet P-40. Supersignal West Pico
Chemiluminescence (Pierce) was used for detection.
-galactosidase (0.5 µg), and either GAL4-DBD,
GAL4-MEF2A, GAL4-MEF2A (S255D), or GAL4-MEF2A (S255A) (0.3 µg). Cells
were fed at 24 h and harvested at 48 h post-transfection in
500 µl of 20 mM Tris, pH 7.4, + 0.1% Triton X-100. 100 µl was used to assay
-galactosidase activity as an internal
control of transfection efficiency. 100 µl of lysate was used to
assay luciferase activity (Promega, according to manufacturer's
instructions) on a Berthold luminometer (LB9507).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (53K):
[in a new window]
Fig. 1.
Purification of MEF2A from mammalian cell
culture. A, His6-tagged MEF2A was
overexpressed in HeLa cells and purified using a Ni2+
affinity beads. The proteins were stained by Coomassie Blue and
identified by peptide mass fingerprinting. MEF2A was identified in two
of the bands, suggesting it had been post-translationally modified.
B, TAP-tagged MEF2A, along with p38 MAPK and its
upstream activator, MKK6, were overexpressed in COS7 cells.
TAP-MEF2A was purified using the TAP scheme (16). The p38 MAPK
caused a significant shift in the mobility of MEF2A as detected by
Coomassie Blue staining.
View larger version (25K):
[in a new window]
Fig. 2.
Identification of MEF2A and a novel
phosphorylation site. A, tryptic peptide mass
fingerprint of His6-MEF2A identifies the presence of MEF2A
and heat shock cognate 71 kDa. B, the identification of
MEF2A was confirmed by sequencing several peptides using CID
fragmentation. C, careful inspection of the region around
2000 m/z reveals two peptides separated by 79.94 Da. This suggests that at least a fraction of the peptide is
phosphorylated. D, the phosphorylated peptide was confirmed
by sequencing using CID fragmentation. The phosphorylated residue is
identified as Ser-255.
View larger version (51K):
[in a new window]
Fig. 3.
Sequence comparison between MEF2A Ser-255
region and numerous MEF2 proteins. The sequence KSPPP is conserved
across species and MEF2 isoforms. This suggests that phosphorylation of
Ser-255 is important for the function of MEF2 proteins.
View larger version (26K):
[in a new window]
Fig. 4.
Phosphorylation of Ser-255 targets MEF2A for
degradation. COS7 cells were transfected with vectors coding for
GAL4 DNA binding domain (Gal4DBD) (lane
1), wild type GAL4DBD-MEF2A (lane 2), or
GAL4DBD-MEF2A with serine mutated to either aspartic acid
(lane 3) or alanine (lane 4). Cells were
harvested, and RNA and protein were isolated. A, Northern
blot analysis of total RNA using a MEF2A-specific probe (top
row) and glyceraldehyde 3-phosphate dehydrogenase-specific probe
(second row). B, Western blot analysis for
MEF2A. Proteolytic fragment of GAL4-MEF2A. C, GAL4
luciferase reporter gene assay. Data are expressed relative to the
-galactosidase corrected luciferase activity for GAL4DBD and are the
mean ± S.E. (n = 6).
View larger version (30K):
[in a new window]
Fig. 5.
Mass spectrometry identifies a number of
novel p38 MAPK phosphorylation sites in MEF2A. Tryptic peptide
maps of MEF2A purified from COS7 cells overexpressing MEF2A alone or
with p38 MAPK and MKK6. The p38 MAPK induces the phosphorylation of a
number of MEF2A peptides. Potential MAPK phosphoacceptor sites are
underlined. Interestingly, peptide (a) does not
contain a typical MAPK phosphoacceptor site. This suggests that this
site is actually phosphorylated by something downstream of p38 MAPK.
b and c contain candidate MAPK sites.
d was detected near the limit of detection for this
instrument. This region has been magnified and the fourth isotope
labeled for clarity.
View larger version (14K):
[in a new window]
Fig. 6.
An additional MEF2A phosphopeptide identified
by CID fragmentation. The doubly charged precursor of
m/z 617.28 was identified as phosphorylated MEF2A
(404-413). Phosphorylation is confirmed by the loss of
H3PO4 from the doubly charged precursor. The
phosphorylation site is identified as Ser-408 by the loss of
H3PO4 from y6 and
y8 fragments and from the internal fragment
(IpSP).
View larger version (18K):
[in a new window]
Fig. 7.
Evidence for p38 MAPK-induced phosphorylation
of MEF2A 283-300. A, the MEF2A 283-300 peptide
contains two methionine residues that are often oxidized upon exposure
to air. The peptide at m/z 2182.95 also shows
evidence of oxidation. This supports the idea that 2182.95 is the
phosphorylated form of MEF2A 283-300. B, immobilized
Cu2+ affinity ZipTipsTM were used to purify
tryptic phosphopeptides from MEF2A. The m/z
2182.92 adds further proof that MEF2A 283-300 is phosphorylated in
cells overexpressing p38 MAPK. The m/z 2102.97 peptide is likely a result of gas-phase dephosphorylation of the
m/z 2182.92 peptide.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (14K):
[in a new window]
Fig. 8.
Summary of phosphorylation sites in
MEF2A. A number of phosphorylation sites have been identified here
or in previous reports (11, 15). These sites and their likely
kinase are indicated. Important features, such as alternative splicing
(87-132 and 288-295) and the p38 docking site, are also
indicated.
Most notable among these p38 MAPK-induced phosphorylation sites is the
confirmation of phosphorylation at a consensus protein kinase CK2 site,
Ser-289. This region of MEF2A (and the homologous region of MEF2C) is
known to be alternatively spliced in different tissues (40, 41),
suggesting a possible tissue-specific functional role for this region.
Its sequence is highly suggestive of a protein kinase CK2 site
((S/T)XX(D/E)) that, until now, had not been
confirmed as a phosphoacceptor site. The data presented here
demonstrates that Ser-289 is likely a target of protein kinase CK2, but
more interestingly, it suggests a link between p38 MAPK activity and the phosphorylation of MEF2A by protein kinase CK2. In fact, a previous
report indicates that the CK2 holoenzyme ( and
subunits) can be
activated specifically by p38 MAPK (42), indicating the possibility
that CK2 and p38 MAPK may cooperatively dock with MEF2A.
MEF2 proteins are involved in a number of different cellular processes
including proliferation, differentiation, apoptosis, and hypertrophy.
These disparate roles for MEF2 are partly explained by the regulated
activation of specific signaling pathways. Here we provide conclusive
evidence for the phosphorylation of MEF2A by a number of
signal-dependant kinases. These data concerning reversible
phosphorylation of MEF2A will be a fundamental aspect of understanding
the diverse function of MEF2 proteins.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Dr. Séraphin for providing us with the yeast TAP-tag vectors.
![]() |
FOOTNOTES |
---|
* This study was supported by grants from the Canadian Institutes of Health Research (CIHR), Natural Science and Engineering Research Council (NSERC), Heart and Stroke Foundation of Canada, MDS Sciex, and York University. Some hardware funding was provided by the Canada Foundation for Innovation (CFI) and the Ontario Innovation Trust (OIT).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.
To whom correspondence should be addressed: York University,
4700 Keele St., 247 Farquharson Bldg., Toronto M3J 1P3, Canada. Tel.: 416-736-2100 (ext. 30389); Fax: 416-736-5698; E-mail:
jmcderm@yorku.ca.
Published, JBC Papers in Press, February 12, 2003, DOI 10.1074/jbc.M211312200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: MEF2, myocyte enhancer factor 2; TAP, tandem affinity purification; MAPK, mitogen-activated protein kinase; MALDI, matrix-assisted laser desorption ionization; TOF, time of flight; ERK, extracellular-regulated kinases; CK2, casein kinase 2; DBD, DNA binding domain; MKK6, MAP kinase kinase; CID, collision-induced dissociation; CDK5, cyclin-dependent kinase 5.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Lin, X., Shah, S., and Bulleit, R. F. (1996) Brain Res. Mol. Brain Res. 42, 307-316[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Mao, Z.,
Bonni, A.,
Xia, F.,
Nadal-Vicens, M.,
and Greenberg, M. E.
(1999)
Science
286,
785-790 |
3. | Naya, F. S., and Olson, E. (1999) Curr. Opin. Cell Biol. 11, 683-688[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Edmondson, D. G.,
Lyons, G. E.,
Martin, J. F.,
and Olson, E. N.
(1994)
Development
120,
1251-1263 |
5. |
Lin, Q.,
Schwarz, J.,
Bucana, C.,
and Olson, E. N.
(1997)
Science
276,
1404-1407 |
6. | Han, J., and Molkentin, J. D. (2000) Trends Cardiovasc. Med. 10, 19-22[CrossRef][Medline] [Order article via Infotrieve] |
7. |
Youn, H. D.,
Sun, L.,
Prywes, R.,
and Liu, J. O.
(1999)
Science
286,
790-793 |
8. | Swanson, B. J., Jack, H. M., and Lyons, G. E. (1998) Mol. Immunol. 35, 445-458[CrossRef][Medline] [Order article via Infotrieve] |
9. | Cox, D. M., Quinn, Z. A., and McDermott, J. C. (2000) Exerc. Sport Sci. Rev. 28, 33-38[Medline] [Order article via Infotrieve] |
10. |
Ornatsky, O. I.,
Cox, D. M.,
Tangirala, P.,
Andreucci, J. J.,
Quinn, Z. A.,
Wrana, J. L.,
Prywes, R., Yu, Y. T.,
and McDermott, J. C.
(1999)
Nucleic Acids Res.
27,
2646-2654 |
11. |
Zhao, M.,
New, L.,
Kravchenko, V. V.,
Kato, Y.,
Gram, H.,
di Padova, F.,
Olson, E. N.,
Ulevitch, R. J.,
and Han, J.
(1999)
Mol. Cell. Biol.
19,
21-30 |
12. | Dodou, E., and Treisman, R. (1997) Mol. Cell. Biol. 17, 1848-1859[Abstract] |
13. |
Kato, Y.,
Kravchenko, V. V.,
Tapping, R. I.,
Han, J.,
Ulevitch, R. J.,
and Lee, J. D.
(1997)
EMBO J.
16,
7054-7066 |
14. |
Yang, C. C.,
Ornatsky, O. I.,
McDermott, J. C.,
Cruz, T. F.,
and Prody, C. A.
(1998)
Nucleic Acids Res.
26,
4771-4777 |
15. |
Molkentin, J. D.,
Li, L.,
and Olson, E. N.
(1996)
J. Biol. Chem.
271,
17199-17204 |
16. | Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M., and Seraphin, B. (1999) Nat. Biotechnol. 17, 1030-1032[CrossRef][Medline] [Order article via Infotrieve] |
17. | Cox, D. M., Du, M., Guo, X., Siu, K. W. Michael, and McDermott, J. C. (2002) BioTechniques 33, 267-270[Medline] [Order article via Infotrieve] |
18. |
Lemercier, C.,
Verdel, A.,
Galloo, B.,
Curtet, S.,
Brocard, M. P.,
and Khochbin, S.
(2000)
J. Biol. Chem.
275,
15594-15599 |
19. |
Quinn, Z. A.,
Yang, C. C.,
Wrana, J. L.,
and McDermott, J. C.
(2001)
Nucleic Acids Res.
29,
732-742 |
20. |
Veeranna,
Amin, N. D.,
Ahn, N. G.,
Jaffe, H.,
Winters, C. A.,
Grant, P.,
and Pant, H. C.
(1998)
J. Neurosci.
18,
4008-4021 |
21. |
Giasson, B. I.,
and Mushynski, W. E.
(1997)
J. Neurosci.
17,
9466-9472 |
22. | Shetty, K. T., Link, W. T., and Pant, H. C. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6844-6848[Abstract] |
23. | Lew, J., and Wang, J. H. (1995) Trends Biochem. Sci. 20, 33-37[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Guan, R. J.,
Khatra, B. S.,
and Cohlberg, J. A.
(1991)
J. Biol. Chem.
266,
8262-8267 |
25. | Fukunaga, K., and Miyamoto, E. (1998) Mol. Neurobiol. 16, 79-95[Medline] [Order article via Infotrieve] |
26. |
Wu, Z.,
Woodring, P. J.,
Bhakta, K. S.,
Tamura, K.,
Wen, F.,
Feramisco, J. R.,
Karin, M.,
Wang, J. Y.,
and Puri, P. L.
(2000)
Mol. Cell. Biol.
20,
3951-3964 |
27. |
Gredinger, E.,
Gerber, A. N.,
Tamir, Y.,
Tapscott, S. J.,
and Bengal, E.
(1998)
J. Biol. Chem.
273,
10436-10444 |
28. |
Bennett, A. M.,
and Tonks, N. K.
(1997)
Science
278,
1288-1291 |
29. |
Coolican, S. A.,
Samuel, D. S.,
Ewton, D. Z.,
McWade, F. J.,
and Florini, J. R.
(1997)
J. Biol. Chem.
272,
6653-6662 |
30. | Musaro, A., McCullagh, K. J., Naya, F. J., Olson, E. N., and Rosenthal, N. (1999) Nature 400, 581-585[CrossRef][Medline] [Order article via Infotrieve] |
31. | Dhavan, R., and Tsai, L. H. (2001) Nat. Rev. Mol. Cell. Biol. 2, 749-759[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Lazaro, J. B.,
Kitzmann, M.,
Poul, M. A.,
Vandromme, M.,
Lamb, N. J.,
and Fernandez, A.
(1997)
J. Cell Sci.
110,
1251-1260 |
33. | Cohen, P., and Frame, S. (2001) Nat. Rev. Mol. Cell. Biol. 2, 769-776[CrossRef][Medline] [Order article via Infotrieve] |
34. |
Pap, M.,
and Cooper, G. M.
(1998)
J. Biol. Chem.
273,
19929-19932 |
35. |
Sears, R.,
Nuckolls, F.,
Haura, E.,
Taya, Y.,
Tamai, K.,
and Nevins, J. R.
(2000)
Genes Dev.
14,
2501-2514 |
36. |
Diehl, J. A.,
Cheng, M.,
Roussel, M. F.,
and Sherr, C. J.
(1998)
Genes Dev.
12,
3499-3511 |
37. |
Okamoto, S.,
Li, Z.,
Ju, C.,
Scholzke, M. N.,
Mathews, E.,
Cui, J.,
Salvesen, G. S.,
Bossy-Wetzel, E.,
and Lipton, S. A.
(2002)
Proc. Natl. Acad. Sci. U. S. A.
99,
3974-3979 |
38. |
Okamoto, S.,
Krainc, D.,
Sherman, K.,
and Lipton, S. A.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
7561-7566 |
39. |
Li, M.,
Linseman, D. A.,
Allen, M. P.,
Meintzer, M. K.,
Wang, X.,
Laessig, T.,
Wierman, M. E.,
and Heidenreich, K. A.
(2001)
J. Neurosci.
21,
6544-6552 |
40. | McDermott, J. C., Cardoso, M. C., Yu, Y. T., Andres, V., Leifer, D., Krainc, D., Lipton, S. A., and Nadal-Ginard, B. (1993) Mol. Cell. Biol. 13, 2564-2577[Abstract] |
41. | Yu, Y. T., Breitbart, R. E., Smoot, L. B., Lee, Y., Mahdavi, V., and Nadal-Ginard, B. (1992) Genes Dev. 6, 1783-1798[Abstract] |
42. |
Sayed, M.,
Kim, S. O.,
Salh, B. S.,
Issinger, O. G.,
and Pelech, S. L.
(2000)
J. Biol. Chem.
275,
16569-16573 |