From the Department of Biochemistry, Institute of Medical Biology, University of Tromsø, 9037 Tromsø, Norway
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ABSTRACT |
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The transcription factor Pax6 is required for
normal development of the central nervous system, the eyes, nose, and
pancreas. Here we show that the transactivation domain (TAD) of
zebrafish Pax6 is phosphorylated in vitro by the
mitogen-activated protein kinases (MAPKs) extracellular-signal
regulated kinase (ERK) and p38 kinase but not by Jun N-terminal kinase
(JNK). Three of four putative proline-dependent kinase
phosphorylation sites are phosphorylated in vitro. Of these
sites, the serine 413 (Ser413) is evolutionary conserved
from sea urchin to man. Ser413 is also phosphorylated
in vivo upon activation of ERK or p38 kinase. Substitution
of Ser413 with alanine strongly decreased the
transactivation potential of the Pax6 TAD whereas substitution with
glutamate increased the transactivation. Reporter gene assays with
wild-type and mutant Pax6 revealed that transactivation by the
full-length Pax6 protein from paired domain-binding sites was strongly
enhanced (16-fold) following co-transfection with activated p38 kinase.
This enhancement was largely dependent on the Ser413 site.
ERK activation, however, produced a 3-fold increase in transactivation
which was partly independent of the Ser413 site. These
findings provide a starting point for further studies aimed at
elucidating a post-translational regulation of Pax6 following activation of MAPK signaling pathways.
Pax6 is a member of the paired
box-containing
Pax1 gene family
of transcription factors containing nine human (PAX1-PAX9)
and murine (Pax1-Pax9) family members (1). The paired box
was first discovered in Drosophila as encoding a conserved
125-128 amino acid paired domain unique to this family of
developmental control genes (2). Pax6 was initially cloned
from human (3), mouse (4), zebrafish (5), and quail (6). Subsequently,
the Drosophila eyeless gene was shown to be a
Pax6 homolog and Pax6 homologs have now been
described in other invertebrates such as flatworm, ribbonworm,
Caenorhabditis elegans, squid, sea urchin, and ascidian
(reviewed in Ref. 7) as well as in amphioxus (8). Pax6 is
expressed in the developing central nervous system, the eyes, nose, and
pancreas in higher vertebrates (4, 5, 9, 10) and plays a pivotal role
in the development of these organs (7, 11, 12). Loss of a functional
Pax6 allele results in the Mendelian syndromes aniridia,
Peter's anomaly, and congenital cataracts in man (13) and Small
eye in rodents (14). Pax6 acts high up in the
regulatory hierarchy controlling eye development in both vertebrates
and invertebrates (reviewed in Ref. 15). Eyeless,
ribbonworm-, squid-, ascidian-, zebrafish-, or mouse Pax6
are all able to induce ectopic eyes in Drosophila upon
targeting their expression to different imaginal discs (16-20). We
have recently found that zebrafish contain two Pax6 genes,
Pax6.1 and Pax6.2, which are expressed in both
overlapping and distinct regions during development of the eyes and the
central nervous system. Both these genes are able to induce ectopic
eyes in Drosophila (18).
The paired domain is a bipartite DNA-binding domain containing an N-
and a C-terminal subdomain each with a helix-turn-helix motif (21, 22).
Pax6, like Pax3, Pax4, and Pax7, also harbors a second DNA-binding
domain, the paired-type homeodomain (2, 7, 23). In Pax6 this domain is
separated from the N-terminal located paired domain by a flexible,
acidic linker region (5, 7). The region C-terminal to the homeodomain
is enriched in proline, serine, and threonine residues (PST-rich) and
acts as a transactivation domain (TAD) (24-27).
It has previously been shown that quail Pax6 proteins expressed in the
neuroretina are phosphoproteins and phosphoamino acid analysis revealed
phosphoserine and a minor proportion of phosphothreonine (28). The
activity of many transcription factors is regulated in a rapid and
reversible manner by specific phosphorylation events mediated by
protein kinases acting in signaling cascades initiated by extracellular
stimuli (reviewed in Refs. 29 and 30). Mitogen-activated protein
kinases (MAPKs) are proline-directed serine/threonine protein kinases
activated by heterogenous extracellular stimuli, including growth
factors, hormones, cytokines, antigens, and physical-chemical stimuli
such as oxidative stress, heat shock, osmotic imbalance, and UV light.
MAPK cascades play essential roles in regulating many critical cellular
processes, including cell growth and division, differentiation,
apoptosis, and stress-related responses (reviewed in Refs. 31 and 32).
These cascades are organized into modules of three protein kinases
where a MAPK kinase kinase (e.g. Raf-1) activates a MAPK
kinase (e.g. MEK1) which subsequently activates a MAPK
(e.g. ERK1) (31, 33). Following activation the MAPKs translocate to the nucleus to phosphorylate nuclear substrates. Currently, four distinct MAPK cascades are known in vertebrates. However, more will probably be found since the budding yeast
contains five such cascades that orchestrate responses to different
physiological stimuli (34). The extracellular-signal regulated kinase
(ERK) pathway mainly conveys signals from mitogenic and differentiation stimuli. The same may be true for the most recently discovered ERK5/BMK1 pathway (35) while the Jun N-terminal kinase (JNK) and p38
MAP kinase pathways seems mostly involved in transducing various
stress- and cytokine-triggered signals to the nucleus. In each pathway
several MAPK isoforms have been found with ERK1 and -2 in the ERK
pathway, JNK1-3 in the JNK pathway and p38 Here we report for the first time that a Pax family transcription
factor is phosphorylated in vitro and in vivo by
MAP kinases. We found that three of the four putative MAPK
phosphorylation sites in the Pax6 TAD is phosphorylated by ERK and p38
kinase, but not JNK, in vitro. One of these sites,
Ser413, is conserved in Pax6 proteins from sea urchin to
man and is also phosphorylated in vivo following activation
of ERK or p38 kinase. Transient transfection studies with the Pax6 TAD
fused to the DNA-binding domain of GAL4 and in the context of the
full-length Pax6 protein revealed that mutation of this site to alanine
strongly decreased the transactivating potential. Co-transfection of
Pax6 with constitutively active MEK1 (which activates ERK1 and -2) or
p38 kinase together with constitutively active MKK6b enhanced transcriptional activation by Pax6. However, while the potent p38
kinase-mediated increase in transactivation (16-fold) was largely
dependent on the integrity of the Ser413 phosphorylation
site, ERK activation gave a more modest enhancement of transactivation
(3-fold) which was only reduced by 30% when this site was mutated to
alanine. This suggests that activation of p38 kinase has a more direct
positive effect on transactivation due to phosphorylation of the Pax6
TAD at Ser413 while ERK activation is less efficient and
acting more indirectly.
Cell Culture--
HeLa cells (ATCC CCL 2) were grown in Eagle's
minimum essential medium supplemented with 10% fetal calf serum
(HyClone), nonessential amino acids (Life Technologies, Inc.), 2 mM L-glutamine, penicillin (100 units/ml), and
streptomycin (100 µg/ml). NIH 3T3 fibroblasts (passage 123) were
purchased from the American Type Culture Collection (ATCC CRL 1658) and
cultured in Dulbecco's modified Eagle's medium supplemented with 10%
calf serum (HyClone, Logan, Utah), penicillin (100 units/ml), and 100 µg/ml streptomycin (Life Technologies, Inc.)
Plasmid Constructs--
GAL4-Pax6 fusions were made by cloning
parts of the zebrafish Pax6 coding region in-frame with the DNA-binding
domain of yeast GAL4(1-147) in pSG424 (50). All GAL4-Pax6 fusion
constructs are named according to the included parts of the Pax6
protein with the amino acid positions shown in parentheses.
GAL4-Pax6(174-437) was made by cloning the 1239-base pair
PmlI-XbaI fragment of Pax6 into the
SmaI-XbaI sites of pSG424. The 640-base pair
MboII fragment containing the C-terminal TAD of Pax6 was
inserted into the SmaI site of pSG424 to give
GAL4-Pax6(299-437). GAL4-Pax6(299-395) was made from the 299-437
construct by deleting an internal NdeI-SacI fragment followed by religation. Similarly, GAL4-Pax6(299-374) was
constructed by deleting the SpeI-SacI fragment
from GAL4-Pax6(299-437). To construct GAL4-Pax6(299-352) a
HindIII-SacI fragment was deleted from
GAL4-Pax6(299-437). GAL4-Pax6(353-437) was made by ligation of a
HindIII (made blunt)-XbaI fragment into the
BamHI (made blunt)-XbaI sites of pSG424.
GAL4-Pax6(374-437) was constructed from GAL4-Pax6(299-437) by
deletion of the SpeI (made blunt)-HindIII
fragment and ligation of the rest of the vector to a
HindIII-SacI (made blunt) fragment from pSG424.
GAL4-Pax6(396-437) was also derived from GAL4-Pax6(299-437) by
deleting the NdeI (made blunt)-HindIII fragment
followed by ligation of the rest of the vector to a
HindIII-SmaI fragment from pSG424.
GAL4-Pax6(374-395) was made from GAL4-Pax6(374-437) by cutting it
with NdeI and SacI, making the ends blunt and
religating them. GAL4-Pax6(353-374) was derived from
GAL4-Pax6(353-437) by digestion with SpeI and
XbaI, making the ends blunt, and religating them. The
GAL4-Pax6(353-395) construct was made from GAL4-Pax6(353-437) by
deletion of the NdeI-XbaI fragment followed by
religation of the rest of the plasmid. GAL4-Pax6(
Specific PCR primers (G4-P6(299) and pSG424.3'; see Table
I) were used to transfer selected Pax6
constructs from pSG424 to pFA-CMV (Stratagene), where the expression of
the GAL4-Pax6 fusions is driven by a CMV promoter. All constructs were
verified by sequencing and the expression and correct sizes of fusion
proteins following transfection of HeLa cells were verified by Western
blotting.
The GST-Pax6(353-437) construct was made by ligating the
HindIII (made blunt)-EcoRI Pax6 fragment into the
SmaI-EcoRI site of pGEX-3X (Pharmacia).
GST-Pax6(299-437) was constructed by inserting a 640-base pair
EcoRI fragment from GAL4-Pax6(299-437) into the EcoRI site of pGEX-1 (Pharmacia). The Pax6 expression vector
pCI-Pax6 and the reporter vectors pG5E1bTATA-CAT,
pTKG5CAT, and pG5E1bTATA-LUC have been
described previously (18, 51).
In Vitro Mutagenesis--
In vitro mutagenesis was
performed according to the instruction manual for the "Quick-Change
Site-Directed Mutagenesis Kit" (Stratagene). All mutagenized
constructs were checked by sequencing. The specific mutagenesis primers
used are shown in Table I.
In Vitro Phosphorylation Assays--
GST-Pax6 fusion proteins
were purified from Escherichia coli LE392 extracts using
glutathione-agarose beads (Pharmacia). The proteins were not
eluted, but left on the beads and stored at 4 °C in
phosphate-buffered saline containing 1% Triton X-100. To equalize the
amounts of proteins used in the kinase assays the concentrations of GST
fusion proteins were estimated by Coomassie Blue staining after sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The
beads were washed twice with the respective kinase buffers before use
in vitro kinase assays. GST-Pax6-containing agarose beads
(5-20 µl) were mixed with either: 1) 2 µl of 10 × MAPK
buffer (500 mM Tris-Cl, pH 7.5, 100 mM
MgCl2, 10 mM dithiothreitol, 10 mM
EGTA), 2 µl of 1 mM ATP, 0.5 µl of
[ Analyses of in Vivo Phosphorylation of GST- and GAL4-Pax6 Fusion
Proteins--
Subconfluent NIH3T3 cells were transfected with 10 µg
of pCI-GST-Pax6(353-437) or phosphorylation site mutants in 10-cm
diameter cell culture dishes using LipofectAMINE (Life Technologies,
Inc.) according to the instructions of the manufacturer. Following
4 h of incubation with DNA, the cells were washed 3 times in PBS and left for 8 h in Dulbecco's modified Eagle's medium supplied with 0.1% serum. After 3 washes in Tris-buffered saline, pH 7.5, the
cells were left for 30 min in phosphate-free Dulbecco's modified Eagle's medium (ICN) containing 0.1% dialyzed newborn calf serum, 25 mM Hepes, pH 7.5, and 2 mM
L-gluthamine and labeled for 8 h by adding 1 mCi/ml
[32P]orthophosphate (Amersham). Some of the dishes were
stimulated by adding dialyzed serum to 10% for 15 min. The culture
dishes were placed on ice, washed twice in ice-cold PBS, and the cells were harvested by scraping in 1 ml of RIPA buffer (PBS containing 1%
Nonidet P-40, 0.5% deoxycholate, 0.1% SDS with added protease and
phosphatase inhibitors as described previously (53)). Cleared lysates
were mixed with 100 µl of 50% slurry of gluthatione-agarose beads
(Pharmacia) and incubated at 4 °C for 1 h on a rotating wheel.
The beads were then washed 8 times in phosphate-buffered saline
containing 1% Triton X-100 and the final pellet was resuspended in 40 µl of 2 × SDS-PAGE gel loading buffer and boiled for 5 min. The
labeled phosphoproteins were separated on a 10% polyacrylamide gel,
electrotransferred onto a polyvinylidene difluoride (PVDF) membrane
(Millipore), and visualized directly by autoradiography. Quantitation
of the signals was performed using a PhosphorImager (Molecular
Dynamics). Indirect detection of phosphorylation at serine 413 by
monitoring a mobility shift was done by Western blotting using
polyclonal Pax6 (P6C) or GST antibodies as described below.
For [35S]methionine/cysteine labeling of proteins the
cells were left for 30 min in methionine/cysteine-free Dulbecco's
modified Eagle's medium (Sigma) containing 25 mM Hepes, pH
7.5, 2 mM L-glutamine, and 10% dialyzed
newborn calf serum before adding 35S-labeled amino acids
(0.1 mCi/ml; Amersham) for 4 h. GST fusion proteins were purified
as described above. Following purification the samples were split in
two, half of the sample were resuspended in 2 × SDS-PAGE gel
loading buffer and the other half was washed twice in
For phosphorylation shift analyses using Western blot detection
subconfluent cell cultures of NIH3T3 or HeLa cells were transfected with 10 µg of the different expression vectors using LipofectAMINE as
described above for NIH3T3 cells or calcium-phosphate coprecipitation for HeLa cells. After 16 h of serum deprivation (0.1% serum) the cells were stimulated with 10% serum or 100 ng/ml phorbol ester (TPA,
Sigma) for the indicated times. GST fusion protein purification and
SDS-PAGE were performed as described above. Following electrotransfer onto PVDF membranes the proteins were detected using polyclonal anti-GST or anti-Pax6 antibodies (P6C) as described below.
For p38 kinase-mediated in vivo phosphorylation, 10 µg of
GAL4-Pax6(299-437) TAD fusion constructs (wild-type and mutants) in
the pFA-CMV expression vector (Stratagene) were co-transfected with 5 µg of p38 kinase (54) and 5 µg of MKK6b(EE) (55) expression vectors
or pcDNA3-HA (vector control) into NIH-3T3 cells using either
Fugene 6 (Roche Molecular Biochemicals) or LipofectAMINE Plus (Life
Technologies, Inc.). For ERK-mediated in vivo
phosphorylation, 10 µg of GAL4-Pax6 TAD fusion constructs and 5 µg
of expression vector for an activated mutant of MEK1, MEK1(EE) (56), or
the respective vector control was used in co-transfections. In
vivo labeling with [32P]orthophosphate was done
essentially as described above with the exception that the cells were
harvested in HA lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1 mM EGTA, 1% Triton X-100) for experiments with p38 kinase and JNK lysis buffer (25 mM Hepes, pH 7.7, 300 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.15 Triton
X-100) for experiments with activated MEK1. Both lysis buffers
contained the following inhibitors: 20 mM
p-nitrophenyl phosphate, 50 mM sodium fluoride,
50 µM sodium vanadate, 5 mM benzamidine, 0.5 µg/ml leupeptin, 2 µg/ml aprotinin, 20 mM
Western Blot--
HeLa cells were seeded at 4 × 105 cells per 10-cm dish the day before transfection. The
relevant expression constructs were transfected using calcium phosphate
coprecipitation. Ten µg of pCI-Pax6 or the vector control pCI-neo was
co-transfected with 5 µg of expression vector for p38 kinase in
combination with 5 µg of expression vector for MKK6b(EE) or vector
control for the latter or with 5 µg of expression vector for MEK1(EE)
or its vector control (see Fig. 8). In all transfections 1 µg of
pCMV- Transient Transfections, CAT, and Luciferase Assays--
The
conditions used for transfections, extract preparation, and
measurements of CAT activity were as described earlier (60). For
transfections for luciferase assays, 4 × 104 cells
were seeded per well in 6-well dishes the day before transfection. Fresh medium was added to the cells 2 h before the DNA on the day
of the transfection. Whenever the Pax6 expressing constructs contained
a cytomegalovirus (CMV) promoter (pCI-neo and pFA-CMV plasmids), 0.05 µg of pCMV- Gel Mobility Shift Assay--
HeLa cells (4 × 105) were seeded on 10-cm dishes the day before
transfection. A total of 30 µg of each Pax6 expression construct was
transfected to 3 dishes. When other plasmids were included 15 µg of
each were added. The cells were left in medium with 10% serum for 2 days before nuclear extracts were made essentially as described (57)
omitting the dialysis step leaving nuclear proteins in the high-salt
buffer. The protein concentration was determined using the Bio-Rad
assay, and 3 µg of nuclear protein extract was used for each binding
reaction in a buffer containing 10 mM Hepes-potassium
hydroxide, pH 7.9, 30 mM potassium chloride, 4 mM spermidine, 0.1 mM EDTA, 0.25 mM
dithiothreitol, 1 mM sodium phosphate, pH 7.2, 4% Ficoll
400, and 3 µg of poly(dI-dC) (24). The P6CON probe (61) was included
(10,000 cpm) and the binding reactions were left on ice for 20 min
before they were loaded on a 5% polyacrylamide gel and run in 0.5 × Tris borate-EDTA (TBE) buffer for 2 h at 200 V.
The Transactivation Domain of Pax6 Includes the Entire C-terminal
PST Domain--
The 139-amino acid long C-terminal transactivating
region of zebrafish Pax6.1 (hereafter referred to as Pax6) is enriched in proline (P), serine (S), and threonine (T) residues comprising 45.4% of the total amino acids in this so called PST domain. The over-representation of these residues imposes a hydrophilic nature to
this domain (63% hydrophilic amino acids), but it is also noteworthy that there is both a significant under-representation of charged residues and a over-representation of methionine residues. The sequence
of this region is extremely conserved among vertebrate Pax6 proteins
with zebrafish Pax6 being 93.6% identical (98.6% similar) to human
PAX6 (see Fig. 1A). If
amphioxus, squid, and sea urchin Pax6 proteins are included in the
comparison, the sequence divergence increases but there is a striking
conservation of two C-terminal sequence motifs (GLISPGVSVP(V/I)QVPG and
YW(P/S)R(L/I)Q in Fig. 1A). Using a combination of different
prediction algorithms we deduced the hypothetical secondary structure
of the Pax6 TAD shown in Fig. 1B. This region seems to
contain only one
In order to define the extent of the Pax6 TAD we performed a deletion
study by fusing parts of the Pax6 TAD (from amino acids 299 to
437) to the yeast GAL4 DNA-binding domain (DBD) and assayed transactivation by co-transfection of HeLa cells using two different reporters. One reporter, pG5E1bTATA-CAT, contained 5 GAL4-binding sites upstream of the TATA box from the minimal promoter
of the adenovirus E1b gene (62) allowing activation of a core promoter from a TATA-proximal position to be measured. The other,
pTKG5CAT (51), contained the same GAL4-binding sites
inserted upstream of the herpes simplex virus thymidine kinase promoter
facilitating measurements of both activation and repression of a more
complex promoter. The CAT activity obtained by the GAL4-Pax6(299-437) construct was set to 100% and the activities of the truncated constructs are given in percent relative to this value. The expression and correct sizes of all the different fusion proteins were verified by
Western blotting of extracts from transfected cells (data not shown).
As can be seen from Fig. 2, deletions of
the TAD from either the N- or C-terminal end dramatically reduced the
transactivation potential. Both reporters produced a similar picture
but the reporter containing the minimal E1b promoter was more sensitive
and we therefore refer to the results obtained with this reporter in the following. Deletion of 55 amino acids from the N-terminal end of
the TAD (retaining positions 353-437) reduced transactivation by 65%.
Further deletion of 23 amino acids (retaining positions 375-437)
yielded a residual activity of only 5% while no significant activity
could be measured when only the C-terminal 42 residues from position
396 to 437 were assayed following fusion to the GAL4 DBD. However,
deletion of the C-terminal 43 amino acids showed that, although
inactive by itself, this region is very important for full activity
since the TAD from position 299 to 395 has lost 80% of the activity of
the full-length TAD. Further deletion of the region between positions
395 and 375 reduced only slightly this residual activity while all
activity was lost upon removal of an additional 23 amino acids from the
C-terminal end (retaining position 299-352). We also measured the
activity of the middle region from position 353 to 395 and the two
halves of this region as well as two internal deletions in this area.
The middle region showed very low activity while the two halves were
devoid of activity. The internal deletion of residues 353 to 374 reduced transactivation by more than 50% while deletion of amino acids
375 to 395, removing one of the two repeat modules, had no effect.
Taken together, these results clearly show that the entire C-terminal
PST-rich region of Pax6 acts as an unusually long TAD with no minimal
activation domain. In contrast to what has been found for the Pax2, -5, and -8 subfamily of Pax proteins (63) there does not seem to exist any
inhibitory regions within the Pax6 TAD.
To assess the relative potency of the transactivation domain of Pax6 we
compared the level of transactivation from the
pG5E1bTATA-CAT reporter of GAL4-Pax6(299-437) to that of
the weak activator GAL4-AH and the extremely strong activator GAL4-VP16
in HeLa cells. From three independent experiments performed in
triplicate we found the Pax6 TAD to be only 2.5-fold weaker than the
VP16 TAD and 40-fold stronger than AH. This clearly indicates the
strong transactivation potential of the isolated Pax6 TAD upon fusion
to the GAL4 DBD. Interestingly, when the region from amino acids 175 to
437, comprising part of the linker region, the homeodomain, and the
entire TAD, was fused to the GAL4 DBD a low activity was measured from
the minimal promoter reporter compared with that of the isolated Pax6 TAD.
The Transactivation Domain of Pax6 Is Phosphorylated in Vitro by
the Mitogen-activated Protein Kinases ERK2 and p38, but Not by
JNK--
It has previously been demonstrated that the quail Pax6
protein is phosphorylated in neuroretina cells mainly on serine
residues but with some phosphorylation of threonine also (28).
Inspection of the sequence of the Pax6 TAD revealed four potential
phosphorylation sites for mitogen-activated protein kinases at
Thr323, Ser376, Thr388, and
Ser413 (indicated with asterisks in Fig. 1). As
an initial experiment a GST fusion protein of Pax6 (amino acids
353-437), expressed and purified from E. coli, was used in
in vitro kinase assays with the activated MAPKs ERK2, p38
kinase, and JNK (Jun N-terminal kinase). Activated ERK2 phosphorylated
the GST-Pax6 fusion protein, whereas GST alone or GST fusions of the
C-terminal TADs of Pax2, Pax3, Pax9a, or Pax9b were not phosphorylated
at all. The same result was obtained with activated p38 kinase.
However, when JNK immunoprecipitated from UV-irradiated NIH 3T3 cells
was used only GST-Jun and neither the Pax6, Pax2, Pax9a nor Pax9b GST
fusions were phosphorylated (data not shown). To map the
phosphorylation site(s) for ERK2 and p38 kinase in the Pax6 TAD,
mutations were introduced at the three most C-terminal candidate sites
described above by substituting the respective serine and threonine
residues with alanine. Upon expression in E. coli the
GST-Pax6(353-437) fusion protein gives a band of 35 kDa representing
the full-length fusion protein and three other bands of lower molecular
masses which are specific proteolytic fragments representing
"deletions" from the C-terminal end of the Pax6 TAD (Fig.
3A, lower panel). This specific fragmentation pattern proved beneficially for mapping of
the sites that are phosphorylated in vitro. Thus, in
vitro phosphorylation of GST-Pax6(353-437) mutant proteins with
ERK2 showed that the Thr388 site is not phosphorylated
since the T388A mutant protein gives the same phosphorylation pattern
as the wild-type (wt) protein (Fig. 3A, lane 4).
Due to the specific band pattern, one can easily see that both the
Ser376 and Ser413 sites are phosphorylated
in vitro. In the S376A mutant protein the lower molecular
weight fragments are not phosphorylated (Fig. 3A, lane
3) whereas they are in the T388A and S413A mutants.
Phosphorylation at Ser413 caused a mobility shift that can
no longer be seen in the S413A mutant, (Fig. 3A, lane
5). Since the Thr323 site was not included in this set
of experiments we prepared GST-Pax6 fusions harboring the entire
C-terminal TAD from amino acid 299 to 437 representing a panel of
single and double mutants as well as a triple mutant including all
three phosphorylation sites (T322A/T323A, T375A/S376A, and S413A)
sites. (For simplicity, we refer to the T322A/T323A and T375A/S376A
mutants as single mutants, since only one putative MAPK phosphorylation
site is mutated in each of them.) Following in vitro
phosphorylation both ERK2 and p38 kinase gave the same phosphorylation
pattern (Fig. 3B). The GST-Pax6(299-437) fusion protein
gives two bands, where the slowest migrating band have the expected
size for the full-length fusion protein (about 41 kDa), while the
faster migrating band is a degradation product containing GST and a
short fragment of the Pax6 TAD encompassing the Thr323
phosphorylation site. As is evident when lanes 3, 8, and
10 are compared with the other lanes in Fig. 3B,
the T322A/T323A mutation prevents phosphorylation of the faster
migrating band showing that the Thr323 site is a
phosphorylation site for both ERK2 and p38 kinase in vitro.
None of the other single site mutations affected the phosphorylation pattern. The two double mutants (T322A/T323A, S413A and T375A/S376A, S413A) both caused a marked decrease in phosphorylation intensities when ERK2 was used for in vitro phosphorylation (Fig.
3B, lanes 8 and 9). For the p38
kinase, however, only the T322A/T323A, S413A double mutation lead to a
decreased phosphorylation indicating that the Thr323 site
is more important for phosphorylation by p38 kinase than the
Ser376 site. For both ERK2 and p38 kinase the triple
mutation (Fig. 3B, lane 10) prevented
phosphorylation completely. This proves that Thr323,
Ser376, and Ser413 are the only phosphorylation
sites used in vitro by ERK2 and p38 kinases in the
C-terminal TAD (amino acids 299-437) of zebrafish Pax6.
Pax6 Is Phosphorylated in Vivo by ERK and p38 MAPKs--
As
described above, three of the four potential phosphorylation sites for
proline-dependent kinases in the TAD of Pax6 are phosphorylated by ERK2 and p38 kinase in vitro. To see if
the Pax6 TAD could be phosphorylated in vivo, NIH 3T3 cells
were transfected with a eucaryotic expression vector for
GST-Pax6(353-437) and in vivo labeled with
[32P]orthophosphate following serum starvation. The cells
were harvested after stimulation with 10% serum, to activate ERK1 and
-2, and the GST-Pax6 fusion protein was purified from the cell lysate by the use of glutathione-agarose beads. There is some phosphorylation of the fusion protein in serum-starved cells, but addition of serum
enhanced this phosphorylation 2.1-fold (Fig.
4A). The membrane was stained
with an antibody raised against the Pax6 TAD to control equal loading
of the lanes. We then utilized the finding that phosphorylation at the
evolutionary conserved Ser413 site causes a mobility shift
of the GST-Pax6(353-437) fusion protein to study the time course of
phosphorylation of this site following serum stimulation. As monitored
by Western blotting, Ser413 is phosphorylated after 15 min
of serum stimulation with a slight increase at 60 min followed by a
decrease 4 h after addition of serum (Fig. 4B). Thus,
the kinetics of phosphorylation of the Ser413 site of the
Pax6 TAD is similar to the kinetics of ERK activation. Using in
vivo labeling with [32P]orthophosphate followed by
serum stimulation of NIH 3T3 cells we confirmed the in vitro
data that suggested the Ser413 site as the cause of the
phosphorylation-induced mobility shift. When the Ser376
site is mutated the lower migrating band disappears, while the shifted
band disappears for the S413A mutant protein (Fig. 4C, upper panel). The weak phosphorylation of the T388A mutant
in this experiment is due to less loading of this protein on the gel as
revealed by Western blotting with the anti-Pax6 antibody (Fig.
4C, lower panel). The results of this in
vivo phosphorylation experiment clearly suggest that the
Ser413 site is contributing much more than the
Ser376 site to the total phosphorylation of
GST-Pax6(353-437). As a final proof that the mobility shift observed
for the GST-Pax6(353-437) fusion protein is indeed due to
phosphorylation of Ser413 we treated
35S-labeled GST-Pax6 fusion proteins purified from
serum-stimulated NIH 3T3 cells with
To further confirm that the Ser413 site is phosphorylated
in vivo by ERK1 and -2 we stimulated HeLa cells transfected
with expression vectors for GST-Pax6(353-437) wt and S413A mutant
proteins with the phorbol ester TPA for 15 min. TPA is a rather
specific inducer of the MEK-ERK pathway in these cells. We also
employed a specific inhibitor of MEK, PD 98059 (64), to determine if
MEK-induced activation of ERK is required for phosphorylation of
Ser413 following stimulation with TPA. As seen from the
Western blot in Fig. 5A, TPA
induced the mobility shift indicative of phosphorylation of
Ser413 while pretreatment with PD 98059 abolished the
TPA-induced phosphorylation of Ser413. To confirm that TPA
leads to ERK activation under these conditions we determined the
phosphorylation of a GST-ElkC fusion protein following
immunoprecipitation of ERK. Thus, TPA treatment (100 ng/ml) for 15 min
induced ERK activation to the same extent as 10% serum (Fig.
5B). These results show that the Ser413 site is
phosphorylated upon activation of ERK MAPKs in serum- and
TPA-stimulated cells. Furthermore, the Ser376 is also
phosphorylated following serum stimulation but not as efficiently as
the Ser413 site.
The above mentioned in vivo phosphorylation experiments were
conducted with GST fusion proteins that do not contain a nuclear localization signal. Immunostaining of transfected cells revealed that
the fusion proteins were located in the cytoplasm (data not shown). To
include the entire Pax6 TAD and to study in vivo
phosphorylation of nuclear localized fusion proteins we constructed
expression vectors where wt and mutant Pax6 TAD (299-437) were fused
in-frame with the GAL4 DBD. We then asked whether activation of ERK and p38 kinase could lead to phosphorylation of the Pax6 TAD in the nucleus
of transfected cells. Thus, in one set of experiments the GAL4-Pax6
fusion constructs were co-transfected with expression plasmids for
activated MEK1. NIH-3T3 cells were transfected, deprived of serum, and
in vivo labeled with [32P]orthophosphate
before harvesting and immunoprecipitation with antibodies against the
GAL4 DBD. As seen from lane 1 in Fig.
6A, the GAL4 DBD is
phosphorylated upon co-transfection with activated MEK1. We found that
there are two putative MAP kinase sites in the N-terminal region of the
GAL4 DBD. However, the wt GAL4-Pax6 TAD fusion protein is clearly
phosphorylated and migrates as a doublet due to the mobility shift
induced by phosphorylation of the Ser413 site (lanes
2 and 3, Fig. 6A). The background level of
phosphorylation in serum-starved NIH 3T3 cells (lane 2) is
increased 2.4-fold (following subtraction of the contribution by
phosphorylation of the GAL4 DBD) upon co-transfection with an
expression vector for activated MEK1 (lane 3). In the S413A
mutant the phosphorylation shift is abolished. In another set of
experiments, GAL4-Pax6 fusion constructs were co-transfected with
expression plasmids for p38 kinase and the constitutively active mutant
upstream kinase, MKK6b(EE). The p38 kinase is inactive by itself and is
activated following co-transfection with an expression vector for
MKK6b(EE) (48). As can be seen from Fig. 6B, the wt Pax6 TAD
is strongly phosphorylated following coexpression of MKK6b(EE) and p38
kinase with all of the labeled protein displaying the mobility shift
indicative of Ser413 phosphorylation. The S413A mutant is
also phosphorylated but does not display the mobility shift seen for
the wt fusion protein (compare lanes 3 and 5). In
the experiment shown twice as much protein was loaded in lane
5 compared with the others. When this is taken into account and
the background due to phosphorylation of the GAL4 DBD is subtracted we
measured a 1.8-2.0-fold reduced phosphorylation in the S413A mutant
compared with the wt. A similar experiment including also the triple
mutant, where all MAPK phosphorylation sites in the TAD are mutated,
showed no increase in the signal above the background due to
phosphorylation of the GAL4 DBD (data not shown). Taken together, the
results shown in Figs. 4-6 show that the Pax6 TAD can be
phosphorylated following activation of both ERK and p38 MAP kinases
in vivo.
Mutation of the Evolutionary Conserved Ser413 Site
Strongly Affects the Transactivating Ability of the Isolated Pax6
TAD--
In order to test whether mutation of the MAPK phosphorylation
sites would affect the transactivating activity of the isolated Pax6
TAD we transfected HeLa and NIH 3T3 cells with GAL4-Pax6(299-437) wt
and mutant expression constructs using pG5E1bTATA-LUC as
the reporter. Following transfection, the cells were left in 10% serum for 24 h before harvesting. As shown in Fig.
7, the T322A/T323A and T375A/S376A
mutations had little or no effect on the transcriptional activity.
However, when these two mutations are combined the transactivation is
reduced by 50% in HeLa cells. In NIH 3T3 cells, this effect is greatly
reduced. In both cell lines the S413A mutant, the two double mutants,
and the triple mutant (all containing the S413A mutation) displayed
markedly decreased transactivation compared with the wt TAD (from 60 to
80% reduction in activity). When the serine in position 413 was
mutated to glutamate (S413E) to mimic the effect of a phosphorylation,
an increase in transcriptional activation was observed. In some
experiments the positive effect of the S413E mutation was even more
dramatic than shown here. Such a difference between Ala and Glu mutants
were not observed for the 376 and 388 sites (data not shown).
Furthermore, we also mutated a putative casein kinase II
phosphorylation site at position 425 both to Ala and Glu without
observing any differences in transactivation. Taken together, this
shows that it is not simply an increase in negative charge that
increases transactivation or loss thereof that decreases it. Rather,
the evolutionary conserved Ser413 residue is clearly
important for full transactivation by the isolated Pax6 TAD, most
likely by serving as an important phosphorylation site.
Phosphorylation of the Ser413 Site Positively Modulates
the Transactivating Activity of Pax6--
To see if phosphorylation of
the TAD affected the transactivation potential of the full-length Pax6
protein, HeLa cells were transfected with pCI-Pax6 wt, the
phosphorylation site mutants T322A/T323A, T375A/S376A, S413A, or a
triple mutant containing all three mutations. As a reporter pP6CON-LUC
(18), containing six consensus Pax6 paired domain-binding sites (65)
upstream of the adenovirus E1b minimal promoter, was used.
Co-transfection with an expression vector for activated MEK1 caused a
2.9-fold induction of the transcriptional activity of wt Pax6 compared with co-transfection with the vector control (Fig.
8A). This induction was not
caused by increased levels of Pax6 proteins, since a Western blot of
Pax6 co-transfected with activated MEK1 or the vector control displayed
similar amounts of Pax6 protein. The T322A/T323A and T375A/S376A
mutants showed elevated transcriptional activation potentials compared
with wt Pax6 but where less inducible by activated MEK (2.0- and
1.8-fold, respectively). The S413A mutant demonstrated only half of the
transactivation potential of wt Pax6, but was still induced 2.0-fold by
MEK1(EE). The triple mutant was less active than the wt protein, but
more active than the S413A mutant, and inducible by activated MEK
by a factor of 2.2.
We have found that expression of the activated MEK1 mutant alone is
sufficient to produce maximal activation of a GAL-Elk-1 fusion protein
suggesting that the levels of endogenous ERK1 and -2 are not limiting.
To ensure that activated MEK1 gives maximal activation of Pax6
transactivation we also performed experiments where an expression
vector for ERK1 was co-transfected with activated MEK1. The results
obtained were similar to those obtained with activated MEK1 alone (data
not shown) confirming that the levels of endogenous ERK1 and -2 are not limiting.
When the same set of pCI-Pax6 plasmids were co-transfected with
expression vectors for p38 kinase and MKK6b(EE), wt Pax6 was induced
16-fold compared with transfection with p38 kinase alone (Fig.
8B). The T322A/T323A and T375A/S376A mutants had nearly the
same transcriptional activity as the wt. The S413A mutant showed about
50% of the wt transactivation activity, and was only induced about
6-fold following co-transfection with MKK6b(EE) and p38 kinase. The
triple mutant was only stimulated 2.7-fold indicating some contribution
from the Thr323 and Ser376 sites to the overall
transcriptional activity following activation of p38 kinase. The
Western blot in Fig. 8B demonstrates that equal amounts of
Pax6 protein was expressed in HeLa cells transfected with Pax6 and p38
kinase compared with cells transfected with Pax6, p38 kinase, and
MKK6b(EE). The striking increase in transactivation by Pax6 observed
following activation of p38 kinase is therefore not caused by an
increase in the expression level or stability of the Pax6 protein.
The DNA-binding Affinity of the Paired Domain Is Neither Affected
by Phosphorylation Site Mutations in the TAD nor ERK or p38 Kinase
Activation--
It has previously been shown that mutations in the
homeodomain of Pax3 affect the DNA binding of the paired domain and
vice versa (74-76), and that deletion of C-terminal amino acids
332-416 of quail Pax6 aborts DNA binding (24), suggesting that
structural constraints arising from other parts of the molecule may
modulate the DNA binding by the paired domain. Gel mobility shift
assays were performed to determine if the phosphorylation site
mutations in the TAD affected DNA binding by the paired domain. Nuclear extracts from HeLa cells transfected with the wt and mutant Pax6 expression vectors were used in gel mobility shift assay with the P6CON
probe containing a single consensus Pax6 paired domain-binding site
(Fig. 9A). The steady state
DNA binding efficiency of wt and mutant Pax6 proteins was determined
using a PhosphorImager. The mean result from three independent
experiments performed with two different nuclear extracts showed that
there were no significant differences in DNA binding between the wt and
mutant Pax6 proteins. Next, we wanted to test whether activation of ERK
or p38 kinase would affect DNA binding by the paired domain. Thus, wt
Pax6 was co-transfected with activated MEK1 or p38 kinase and MKK6b(EE) and the appropriate controls into HeLa cells. The cells were
subsequently serum starved before nuclear extracts were made.
Phosphorylation of Pax6 caused by co-transfection of activated MEK1 or
p38 kinase/MKK6b(EE) (Fig. 9A, lanes 8 and
10) does not display any marked effects on DNA binding
compared with the controls (Fig. 9A, lanes 9 and 11). As shown by Western blotting (Fig. 9B), the
nuclear extracts used for gel mobility shift assay contained similar
amounts of Pax6 proteins.
In this report we show that the complete region C-terminal to the
homeodomain of Pax6 is necessary for maximal transcriptional activation. The Pax6 TAD does not contain any internal sequence elements that may inhibit transactivation as found for the Pax2, -5, and -8 subfamily of Pax proteins (63). While this work was in progress
Tang et al. (27) came to a similar conclusion using deletions corresponding to the exons encoding the human PAX6 TAD. Thus,
taken together, these two studies using different deletion constructs
of human and zebrafish Pax6 proteins confirm that Pax6 contains an
unusually long TAD which has been strongly conserved both in primary
sequence and function during vertebrate evolution. The large size of
the TAD suggests that it may provide interaction surfaces for several
cofactors and/or components of the basal transcriptional machinery.
Interestingly, both we and Tang et al. (27) found that the
presence of the homeodomain in GAL4-Pax6 TAD fusions inhibited the
transcriptional activation potential compared with the TAD alone. This
has also been observed with the Pax3 homeodomain in a similar GAL4-Pax3
TAD fusion, where inclusion of the homeodomain reduced the activation
by approximately 20-fold (66). Different models may account for this
behavior. The homeodomain could exert a direct negative effect either
by interfering with TAD function or the reduced activity could reflect a titration of the fusion protein away from the reporter promoter due
to binding of the Pax6 homeodomain to chromosomal sites. However, Chalepakis et al. (67) have shown that the Pax3 homeodomain itself causes repression of a thymidine kinase promoter when fused to
GAL4 making it likely that the homeodomain may recruit a repressor.
Vertebrate Pax6 TADs contain four potential MAPK phosphorylation sites.
We found that three of these sites are phosphorylated in
vitro by both ERK and p38 kinase. JNK was unable to phosphorylate the Pax6 TAD in vitro and the C-terminal TADs of zebrafish
Pax2, Pax9a, and Pax9b were not phosphorylated by any of the three MAP kinases, even though they too contain putative MAPK phosphorylation sites. Furthermore, the murine Pax3 TAD was not phosphorylated by ERK2
or p38 kinase. Of the three sites phosphorylated in the Pax6 TAD the
Ser413 is conserved from sea urchin to man while the other
two sites are conserved in the highly similar vertebrate proteins but
not in amphioxus, squid, and sea urchin. In vivo
phosphorylation experiments using GST-Pax6 TAD wild-type and mutant
proteins demonstrated that the Pax6 TAD is an ERK substrate also
in vivo. Since the two nuclear localization signals of Pax6
reside in the paired domain and directly N-terminal to the homeodomain,
respectively (24), the GST-Pax6 TAD fusions contain no nuclear
localization signals and were only expressed in the cytoplasm. Apart
from demonstrating that the Pax6 TAD is phosphorylated in
vivo following activation of ERKs by serum and phorbol ester,
particularly on the evolutionary conserved Ser413 site, our
results with these fusion proteins are also of interest since the
paired-less isoform of Pax6 (28) is distributed between the nucleus and
cytoplasm with most expression in the cytoplasm (our own observations
and Ref. 24). Using Gal4-Pax6 TAD fusions in co-transfection
experiments we found that both activation of ERKs and p38 kinase lead
to phosphorylation of the Ser413 site in the nuclear compartment.
Specific phosphorylation events can regulate the activity of
transcription factors via several different mechanisms involving changes in protein stability, DNA binding activity, subcellular localization, or protein-protein interactions (reviewed in Refs. 30 and
68). We have shown by gel-mobility shift assays and Western blots that
for Pax6 neither the DNA binding activity nor the protein stability are
affected by mutations of phosphorylation sites or activation of ERK or
p38 kinase. Since the full-length Pax6 protein is localized in the
nucleus (data not shown and (24) subcellular localization is not
regulated by phosphorylation of the Pax6 TAD. Both in the contexts of
fusions to the GAL4 DBD and in the full-length Pax6 protein mutation of
the Ser413 site to an alanine greatly reduced the
transactivation potential. This could be due to structural effects not
reflecting any direct role for phosphorylation. However, mutation to a
glutamate increased the activity of the GAL-Pax6 TAD fusion and
activation of p38 kinase strongly enhanced the activity of the wt Pax6
protein whereas the activities of the Ser413 and triple
mutant (where all three MAPK phosphorylation sites in the TAD are
mutated to alanines) showed greatly reduced responses to p38 kinase
activation. The results shown in Fig. 8 suggest that activation of p38
kinase exerts a much more pronounced positive effect on the
transactivation ability of Pax6 than activation of ERKs does (16-fold
compared with about 3-fold). The effect mediated by ERK activation is
also to some extent independent of the Ser413 site
indicating a significant indirect contribution probably through
phosphorylation of other proteins that Pax6 depend on for efficient
transactivation. However, the stimulation of Pax6 transactivation
following activation of p38 kinase is strongly dependent on the
Ser413 site with some contribution also from the two other
phosphorylation sites. In addition, activation of p38 kinase also
reveals a smaller but significant, positive modulation of Pax6
transactivation which is independent of the phosphorylation sites in
the TAD. This is evident since both the Ser413 and the
triple mutant are stimulated by p38 kinase, although much less so than
the wt Pax6 protein. Thus, both ERK and p38 kinase activation display
an indirect mechanism whereby the transactivation potential of Pax6 is
increased 2-3-fold. In addition, p38 kinase, and to a more modest
extent ERK, is able to exert a direct effect which is dependent on the
integrity of the Ser413 site. Thus, direct phosphorylation
of this site seems necessary for Pax6 to achieve full transactivating
activity. Our structure predictions suggest that the Ser413
site is located in a surface exposed turn between to hydrophobic The different efficiencies of ERK and p38 kinase activation in
enhancing the transactivation potential of Pax6 is most readily explained by differences in activity toward the substrate in
vivo. This is illustrated in Fig. 6 where only part of the wt
GAL-Pax6 TAD fusion proteins displays the mobility shift due to
phosphorylation of Ser413 following co-transfection with
activated MEK1 while all of the protein is shifted following activation
of p38 kinase.
This work represents the first report of induced phosphorylation of a
Pax protein. Dörfler and Busslinger (63) stimulated B cell lines
with various interleukins, serum growth factors, or phorbol esters in
an effort to demonstrate a link between transcriptional activation of
Pax5 and signaling pathways, but failed to identify such a link. A
challenge for future studies is to determine the role of
phosphorylation of the Pax6 TAD in more physiologically relevant
settings. Pax6 can potentially receive signals mediated by MAP kinase
cascades in all the tissues where it is expressed during development
and/or in the adult organism. The nature of the external signal could
differ according to the specific tissue, and the final response
elicited by phosphorylation of Pax6 will most likely depend on tissue-
and cell-type specific parameters such as availability of specific
cofactors and other transcription factors acting together with Pax6 on
specific regulatory sites.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-
in the p38 MAPK
pathway (reviewed in Ref. 31). A growing number of transcription
factors have been identified as nuclear targets for MAPK pathways
(reviewed in Ref. 30). Thus, c-Myc (36), Spi-B (37), BCL-6 (38),
Microphthalmia (39), and several ETS-domain transcription factors such
as vertebrate c-Ets1 and -2 (40), C. elegans LIN-1 (41) and
Drosophila Yan and Pointed P2 (42) as well as the C. elegans winged-helix factor LIN-31 (41) are phosphorylated by
ERKs. c-Jun (43, 44) and NFAT4 (45) are targets of JNK, while CHOP (46)
is a nuclear substrate for p38 kinase. This picture is further
complicated by the fact that several transcription factors have been
shown to be the targets of more than one MAPK pathway. Elk-1, a member
of the ternary complex subfamily of ETS proteins, is targeted by all
three pathways, while SAP-1 is activated upon phosphorylation by ERK or
p38 kinase (Ref. 47 and references therein). Furthermore, MEF2C is
phosphorylated by both p38 kinase and ERK5/BMK1 (48) and ATF-2 and ATFa
are targets of both the JNK and p38 kinase pathways (49).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
375-395) was
derived from GAL4-Pax6(299-437) by an internal deletion of the
NdeI-SpeI fragment. GAL4-Pax6(
353-375) was
also made by an internal deletion of GAL4-Pax6(299-437) employing the
HindIII and SpeI sites.
Sequences of oligonucleotides used as polymerase chain reaction primers
for plasmid constructions and site-directed mutagenesis and as
double-stranded binding site probes in gel mobility shift assays
-32P]ATP (100 µCi/µmol), and 1 unit of
recombinant active ERK2 kinase (New England Biolabs) in a total volume
of 20 µl, or with 2) 4 µl of 10 × p38 kinase reaction buffer
(250 mM Hepes, pH 7.5, 100 mM magnesium
acetate), 500 µM ATP, 0.5 µl of
[
-32P]ATP (100 µCi/µmol), and 1 µl (1 µg) of
recombinant active p38 kinase (Stratagene) in a total volume of 40 µl. The reactions were left at 30 °C for 30 min, flicking the
tubes every fifth min. The agarose beads were subsequently washed with
500 µl of phosphate-buffered saline (PBS) before boiling in 25 µl
of 2 × SDS-PAGE gel loading buffer. The proteins were loaded on a
10% SDS-polyacrylamide gel, and run at 20 mA for 1.5-2 h in a Tris glycine buffer. The gel was dried, and exposed for autoradiography. Assays with c-Jun N-terminal kinase (JNK) was performed as described previously (52).
-phosphatase
assay buffer (New England Biolabs) before addition of 1000 units of
-phosphatase (New England Biolabs) in a total volume of 25 µl. The
dephosphorylation was stopped after a 30-min incubation at 30 °C by
adding 10 µl of 5 × SDS-PAGE gel loading buffer. The samples
were boiled and proteins were separated on a 10% SDS-polyacrylamide
gel, electrotransferred onto a PVDF membrane, and visualized by
autoradiography using Kodak BioMax MR film.
-glycerophosphate, 0.5 mM phenylmethyl sulfonate, 0.7 µg/ml pepstatin A. Polyclonal anti-GAL4 DBD antibodies and protein
A/G-Sepharose beads (both from Santa Cruz Biotechnology) were used for
immunoprecipitation of the GAL4-Pax6 fusion proteins. The beads were
washed 3 times with the lysis buffer before boiling in 30 µl of
gel-loading buffer and electrophoresis on a 10% SDS-polyacrylamide
gel. The proteins were blotted onto a PVDF membrane and
32P-labeled proteins were detected by autoradiography.
gal (Stratagene) was included to allow measurement of
-galactosidase activities that were used to normalize for variations
in transfection efficiencies. The cells were harvested after 2 days
either by directly scraping them into 100 µl of 2 × SDS-PAGE
gel loading buffer or into the Dual-Light lysis buffer (Tropix Inc.) to
set aside an aliquot for
-galactosidase activity measurements,
before the remaining was mixed with 5 × SDS gel loading buffer
and boiled. For some blots nuclear extracts prepared as described (57)
were used. When indicated in figure legends the amount of protein
loaded on the gel had been adjusted according to measurements of
-galactosidase activity. Proteins run on a 10% SDS-PAGE gel were
blotted onto a PVDF membrane and blocked overnight at 4 °C in a
buffer consisting of Tris-buffered saline with 0.1% Tween 20 (TBST)
and 5% non-fat dried milk. The following primary antibodies were used:
anti-GST,2 anti-GAL4 DBD
(diluted 1:1.000; Santa Cruz Biotechnologies), serum 14 (28) (diluted
1:5.000), and P6C; affinity purified anti-Pax6 C-terminal antibodies
(58, 59) (diluted 1:800). The primary antibodies were applied for
1 h at room temperature. The membrane was then washed 5-6 times
in the TBST buffer for 30-60 min. The secondary antibodies
(anti-rabbit IgG-AP, Santa Cruz Biotechnology or anti-mouse IgG-AP,
Sigma) were diluted 1:2.000 or 1:20.000, respectively, in the blocking
buffer and left for 1 h at room temperature. The washing described
above was repeated. CDP-Star substrate (Roche Molecular Biochemicals)
was used according to the manufacturers instructions to visualize the
specific protein bands.
gal (Stratagene) was co-transfected for each well as a
control of transfection efficiency. When the expression constructs
harbored a SV40 early region promoter (pSG424 vector), 0.1 µg of
pCH110 (Pharmacia) was co-transfected. After transfection, the cells
were either left in medium with 10% serum for 24 h before being
harvested or kept in 10% serum for 24 h and in 0.1% serum for
about 20 h before harvesting. The cells were washed twice with PBS
in the wells before preparing extracts using the Dual-Light luciferase
and
-galactosidase reporter gene assay system (Tropix Inc.) and
analyzed in a Labsystems Luminoskan RT dual injection luminometer.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helical segment with most of the domain being
occupied by short
-sheets and random coil elements as well as four
turns. The
-sheets are generally hydrophobic suggesting that they
pack into the interior of this domain. The prediction of several
-turns also supports the notion of a perhaps more globular domain
than just a flexible, extended and rather unstructured TAD.
Interestingly, all but one of the charged residues are predicted to
reside in
-turn structures on the surface of the protein. The last
charged residue is at the extreme C terminus which is solvent-exposed
in most proteins anyway. The predicted isoelectric point is 5.5-6.2
due to a net negative charge. A short repeat module with 8 out of 12 identical residues is also found within the TAD. This module is
preceded by a putative
-sheet and contains two conserved charged
residues and a predicted turn (Fig. 1B).
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Fig. 1.
Localization of putative phosphorylation
sites for proline-dependent protein kinases and predicted
secondary structure elements in the Pax6 transactivation domain
(TAD). A, sequence alignment of the Pax6 TAD including
sequences from zebrafish (Zpax6.1 and
Zpax6.2)(5, 18), human (Hpax6) (3), quail
(Qpax6) (6), amphioxus (Apax6) (8), squid
(Lpax6) (19), and sea urchin (Spax6) (25).
Putative phosphorylation sites for proline-dependent
protein kinases are indicated with asterisks above the
alignment. Residues displayed on a black background are
identical in all the compared species whereas other residues conserved
in most of the proteins aligned are indicated by two shades of
gray. Dashes indicate gaps introduced to facilitate
optimal alignment. B, predicted secondary structure elements
within the Pax6 TAD displayed together with the primary sequence of the
zebrafish Pax6.1 TAD (positions 299 to 437). Black bars
indicate -sheets and an open bar an
-helix. These
elements were predicted using both the PHD neural network method with a
multialignment as input (72) and the Alexis program of the Seqsee
program suite (73). Vertical arrows indicate turns predicted
using the ProtScale resource at the Expasy server
(http://www.expasy.ch/cgi-bin/protscale.pl). R1 and
R2 denote a short repeat module indicated in bold
together with the two highly conserved C-terminal sequence motifs.
Plus and minus signs above the sequences demark
the basic and acidic residues, respectively. Numbers above
the sequence denote deletion end points for GAL4-Pax6 fusion constructs
(vertical lines) and putative phosphorylation sites for
proline-dependent protein kinases
(asterisks).
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Fig. 2.
The Pax6 TAD includes the entire C-terminal
region and contains no internal inhibitory domains. Different
parts of the C-terminal PST domain of Pax6 were fused to the
DNA-binding domain (amino acids 1-147) of the yeast transcription
factor GAL4 and co-transfected into HeLa cells with reporter plasmids
containing 5 GAL4-binding sites either upstream of the adenovirus E1b
TATA minimal promoter (pG5E1bTATA-CAT) or the thymidine
kinase promoter (pTKG5CAT). A, schematic drawing
of the different constructs. The numbers in
parentheses refer to amino acid positions defining the parts
of the Pax6 protein included in the fusions. The locations of the
paired domain (PD), the homeodomain (HD), and the
TAD are indicated. B and C, deletions from either
the N- or C-terminal part of the TAD lead to a sharp decrease in
transcriptional activity. The results are presented as percent activity
relative to the complete TAD (aa 299-437) which was set to 100%. The
data represent the means of four to seven independent experiments
performed in triplicates for each construct. For all transfections 2 µg of both effector and reporter plasmids, supplemented with 2 µg
of sonicated salmon sperm DNA, were used. The cells were kept in 10%
serum and harvested 48 h post-transfection. GAL4-AH and GAL4-VP16
were included as controls of a weak and a very strong transactivation
domain, respectively.
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Fig. 3.
Three of the four putative phosphorylation
sites in the Pax6 TAD are phosphorylated by ERK2 and p38 kinase
in vitro. A, ERK2 in vitro
kinase assays with glutathione S-transferase-Pax6(353-437)
fusion proteins. A C-terminal fragment of Pax6 (amino acids 353 to 437)
was fused to GST and alanine substitutions at three potential
phosphorylation sites for proline-directed protein kinases (S376A,
T388A, and S413A) were introduced by in vitro mutagenesis.
Purified fusion proteins of GST-Pax6(353-437) and the three different
point mutants were in vitro phosphorylated with ERK2
(top panel). The fusion proteins give a characteristic four
band pattern due to intracellular protease attacks at specific regions
upon overexpression in E. coli (see the Coomassie
Blue-stained gel in the lower panel). The slowest migrating
band is the full-length fusion protein, while the other bands represent
fusion proteins containing specific C-terminal deletions of the Pax6
TAD. The S376A mutation (lane 3) prevents the most rapidly
migrating bands from being phosphorylated, while the T388A mutation has
no effect (lane 4). Only the full-length fusion protein
contains the Ser413 site. In addition, phosphorylation at
this site causes a mobility shift of the full-length fusion protein. As
seen from lane 5 in the top panel the S413A
mutation leads to disappearance of the slowest migrating and most
heavily phosphorylated band. B, ERK2 and p38 kinase in
vitro kinase assays with GST-Pax6(299-437) fusion proteins. To
include the putative phosphorylation site in position 323, GST-Pax6
fusions (wt and the relevant alanine substitution mutants) harboring
the entire TAD (amino acids 299 to 437) were analyzed for
phosphorylation by active, recombinant ERK2 (top panel) and
p38 kinase (middle panel). As seen from the Coomassie
Blue-stained gel (bottom panel) the fusion proteins give two
bands corresponding to the full-length fusion protein and a C-terminal
truncated protein where most of the C-terminal of Pax6 is deleted
retaining the putative T323 phosphorylation site. As is evident from
lanes 3 and 8 in the top and
middle panels both ERK2 and p38 kinase phosphorylate this
site with p38 kinase being most efficient in doing so. Mutations
affecting all three sites (T322A/T323A, T375A/S376A, and S413A)
completely prevent phosphorylation of the Pax6 TAD by ERK2 and p38
kinase (lane 10).
-phosphatase which specifically
dephosphorylates phosphoserine and phosphothreonine residues in
proteins. As shown in Fig. 4D, phosphatase treatment leads
to loss of the shifted band.
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Fig. 4.
The Pax6 TAD displays serum-induced
phosphorylation at Ser413 in vivo with
kinetics similar to the activation of ERK1 and -2. A,
in vivo phosphorylation of the Pax6 TAD is increased
following addition of 10% serum to quiescent NIH 3T3 fibroblasts for
15 min. NIH 3T3 cells transfected with a GST-Pax6(353-437) fusion
protein expression vector were metabolically labeled with
[32P]orthophosphate, stimulated with serum for 15 min and
GST-fusion protein purified as described under "Materials and
Methods." Following SDS-PAGE and transfer to a PVDF membrane
quantitation of phosphorylation was performed using a PhosphorImager
(top panel) and the fusion protein was detected by
chemiluminescence using P6C, an affinity purified antibody raised
against the C-terminal TAD of Pax6 (bottom panel). Note the
specific increase of the upper shifted band due to serum-induced
phosphorylation of the Ser413 site. B,
phosphorylation of Ser413 mimics the kinetics of ERK
activation in serum stimulated NIH 3T3 cells with an increase from 15 to 60 min and a subsequent decrease 4 h following serum addition.
NIH 3T3 cells transfected with the GST-Pax6(353-437) fusion protein
expression vector were deprived of serum for 16 h and stimulated
with 10% serum for the indicated times before extract preparation and
detection of the phosphorylation shift by Western blotting using the
same antibody as in A. C and D, phosphorylation
of Ser413 induces a mobility shift of the
GST-Pax6(353-437) fusion protein. NIH 3T3 cells transfected as above
were grown in 10% serum and labeled with
[32P]orthophosphate (C) or
[35S]methionine/cysteine (D). In C,
the upper panel shows 32P-labeled fusion
proteins following purification on glutathione-agarose beads while the
lower panel is a Western blot of these proteins with the P6C
anti-Pax6 antibody to verify protein loading on the gel. Note that the
upper shifted band is not present for the S413A mutant and that the
weak phosphorylation signals of the T388A mutant protein is due to less
protein being loaded on the gel. D, the mobility shift
induced by phosphorylation of Ser413 is removed by
treatment with a serine-threonine phosphatase. Purified fusion proteins
labeled in vivo with [35S]methionine/cysteine
were either untreated or treated with phosphatase
(
PPase) before SDS-PAGE, electrotransfer to a PVDF
membrane, and autoradiography.
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Fig. 5.
TPA-induced phosphorylation of the
Ser413 site is blocked upon pretreatment with the specific
MEK inhibitor PD 98059. A, HeLa cells transfected with
expression vectors for GST-Pax6(353-437) wt and S413A fusion proteins
were either untreated or pretreated with the MEK inhibitor PD 98059 (50 µM) for 30 min before being stimulated with TPA (100 ng/ml) for 15 min. GST-Pax6 fusion proteins were subsequently purified
on glutathione-agarose beads and analyzed for phosphorylation induced
mobility shift by SDS-PAGE and Western blotting with the P6C antibody
against the Pax6 TAD. B, TPA treatment (100 ng/ml) for 15 min induce ERK-activation in HeLa cells to a similar extent as 10%
serum. Phosphorylation of a GST-ElkC fusion protein purified from
E. coli by ERK immunoprecipitates (upper panel)
using agarose-conjugated polyclonal anti-ERK antibody (Santa Cruz
Biotechnology). Immunoprecipitated ERK was visualized by Western blot
using a monoclonal antibody to ERK2 (Upstate Biotechnology) and
chemiluminescent detection (lower panel).
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Fig. 6.
Activation of ERK or p38 kinase leads to
phosphorylation of the Pax6 TAD in the nucleus. A,
co-transfection of activated MEK1 with the Pax6 TAD fused to the GAL4
DNA-binding domain increases the phosphorylation of the Pax6 TAD. Note
that the mobility shift due to phosphorylation of Ser413
(lanes 2 and 3) is absent when this site is
mutated to alanine (lanes 4 and 5). The
upper panel shows an autoradiography of
32P-labeled GAL4-Pax6(299-437) fusion proteins
immunoprecipitated with and antibody to the GAL4 DNA-binding domain.
The lower panel displays a Western blot of the
immunoprecipitates with the anti-Pax6 TAD antibody. B,
co-transfection of MKK6(EE) and p38 kinase with GAL4-Pax6 TAD
expression constructs reveal nuclear phosphorylation of the Pax6 TAD.
All experiments were performed in NIH 3T3 cells which where labeled
with [32P]orthophosphate for 6 h in 0.1% serum
before preparation of extracts as described under "Materials and
Methods." The Pax6 TAD fusions to the GAL4 DNA-binding domain were
expressed from the FA-CMV vector.
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Fig. 7.
Mutation of the evolutionary conserved
Ser413 site strongly affects the transactivation potential
of the isolated Pax6 TAD. The transactivation potential of the wt
and mutant Pax6 TAD constructs fused to the DNA-binding domain of GAL4
was determined in HeLa (upper panel) and NIH 3T3 cells
(lower panel) growing in 10% serum. Co-transfections were
performed in 6-well cell culture dishes with 0.5 µg of pSG424 of
expression vector for wt GAL4-Pax6(299-437) or the different mutants
together with 0.5 µg of pG5E1bTATA-LUC reporter plasmid.
The cells were harvested for luciferase and -galactosidase activity
assays following growth in 10% serum for 24 h. To normalize for
variations in transfection efficiencies, 0.1 µg of pCH110 was
included in each transfection to allow measurement of
-galactosidase
activities. The ratio between luciferase and
-galactosidase activity
obtained with the wt GAL4-Pax6(299-437) expression vector was set to
100%. For HeLa cells the results represent the mean with standard
deviations of three independent experiments performed in triplicate.
For NIH 3T3 cells the data are the mean with standard deviations of two
independent transfections done in triplicate.
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Fig. 8.
Activation of p38 kinase strongly enhances
the transactivation potential of Pax6 via phosphorylation of
Ser413 while activation of ERKs shows a more unspecific
effect. A, activated MEK1 increases Pax6-mediated
transactivation in a manner only partially dependent on
Ser413 phosphorylation. HeLa cells were transfected with
0.25 µg of expression vector for Pax6 (or phosphorylation site
mutants), 0.5 µg of pP6CON-LUC reporter, 0.5 µg of vector control
or expression vector for activated MEK1. To normalize for variations in
transfection efficiencies, 0.05 µg of pCMV- gal was included in
each transfection to allow measurement of
-galactosidase activities.
Twenty-four h after transfection the cells were incubated in 0.1%
serum for 20 h before harvesting. The data are expressed as the
ratio between luciferase and
-galactosidase activities with the mean
and standard deviations calculated from five independent experiments
performed in triplicate. The Western blot above the graph, performed
with an antibody against the Pax6 TAD (serum 14) (28), shows that
co-transfection with activated MEK1 (lane 3) does not affect
the expression level of Pax6 protein. Measurements of
-galactosidase
activity were used to compensate for differences in transfection
efficiencies so that equal amounts of cell lysate (measured as units of
-galactosidase activity) were loaded on the gel. B,
activation of p38 kinase strongly enhances the transactivation
potential of Pax6 via phosphorylation of Ser413. HeLa cells
were transfected as described in A, except that expression
vector for p38 kinase together with vector control for MKK6b(EE)
(p38) or expression vector for p38 kinase together with
expression vector for MKK6b(EE) (p38 + MKK6b(EE) were used
instead of MEK1(EE) vector control and expression construct. The results represent the mean
with standard deviations of three independent transfections done in
triplicate. The Western blot displayed above the graph was performed
similarly as in A (using the P6C antibody) and shows that
activated p38 kinase does not affect the expression level of
Pax6.
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Fig. 9.
The DNA binding affinity of the Pax6 paired
domain is not affected by mutation of the phosphorylation sites or by
co-transfection with activated MEK1 or p38 kinase. A,
gel mobility shift assay of nuclear extracts from HeLa cells
transfected with expression vectors for Pax6 wt and mutant proteins
(lanes 2-6). Lanes 8-11 contain nuclear
extracts from cells transfected with expression vectors for wt Pax6 in
combination with an expression vector for activated MEK1 (lane
9) or expression vectors for both p38 kinase and MKK6b(EE)
(lane 11) and their respective controls (lanes 8 and 10). For experimental details, see "Materials and
Methods." Nuclear proteins (3 µg) were incubated with the P6CON
probe containing a single consensus Pax6 paired domain-binding site
(61) on ice for 20 min, and run on a 5% polyacrylamide gel for 2 h at 220 V. B, mutations of the phosphorylation sites in the
TAD do not affect the expression level of Pax6 and the Pax6 protein
level is not increased upon co-transfection with activated MEK1 or
activation of p38 kinase. Three µg of nuclear extract was loaded in
each lane of a 10% SDS-polyacrylamide gel and Western blot performed
using the P6C antibody (1:800 dilution). The differences in the number
of detected bands between lanes 1-6 and lanes
7-10 are due to the use of different batches of primary antibody,
membrane type, and detection system. The Western blot shown as
lanes 1-6 was performed as described under "Materials and
Methods," while for the Western blot shown as lanes 7-10
a Hybond membrane (Amersham) and anti-rabbit horseradish
peroxidase-conjugated secondary antibodies (Transduction Laboratories)
were used with the ECL detection system (Amersham). The location of a
Pax6 isoform lacking the paired domain due to expression from an
internal start codon (28) is indicated (Pax6 PD).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-sheets making it likely that phosphorylation of this site may induce a conformational change in the TAD. Such a conformational change
may affect both inter- and intramolecular protein-protein interactions.
Although not proven directly, several studies indicate that there could
be an interaction between the TAD of Pax6 and the DNA-binding domains
(24, 69, 70). We also found that the Pax6 TAD acts much more potently
when fused to the heterologous GAL4 DBD than in its natural context
when assayed on the same promoter containing either five GAL4-binding
sites or six paired domain-binding sites, respectively. Hence,
phosphorylation may relieve an intramolecular inhibition by inducing a
conformational change in the Pax6 TAD. Subsequently, efficient contacts
may be established between the TAD and coactivator(s) and/or components of the basal transcriptional machinery to allow full activation of
transcription. The indirect mechanism observed suggests activation by
ERK and p38 kinase of such cofactors or other proteins involved in
unleashing the potential of the Pax6 TAD. To our knowledge, no reports
on interactions between cofactors/corepressors or general transcription
factors and the TADs of any Pax proteins have been published. The
transcription factor Microphthalmia interacts specifically with the
cofactor CBP/p300 following ERK-mediated phosphorylation of
Ser73 (71). However, preliminary experiments have failed to
reveal a significant stimulation of Pax6-mediated transactivation by CBP (data not shown) suggesting that other factors are involved.
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ACKNOWLEDGEMENTS |
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The skillful technical assistance of Aud Øvervatn is gratefully acknowledged. We are indebted to J. Han for the MKK6b(EE) and p38 kinase expression plasmids, C. J. Marshall for the expression plasmid for MEK1(EE), and S. Saule for Pax6 TAD antiserum 14.
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FOOTNOTES |
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* This work was supported by grants from the Norwegian Cancer Society, the Norwegian Research Council, the Aakre Foundation, and the Blix Foundation (to T. J.).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.
Fellows of the Norwegian Research Council.
§ Fellow of the Norwegian Cancer Society.
¶ To whom correspondence should be addressed: Dept. of Biochemistry, Institute of Medical Biology, University of Tromsø, 9037 Tromsø, Norway. Tel.: 47-776-44720; Fax: 47-776-45350; E-mail: terjej{at}fagmed.uit.no.
2 I. Mikkola, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: Pax, paired box; GST, glutathione S-transferase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; JNK, c-Jun N-terminal kinase; TPA, 12-O-tetradecanoylphorbol-13-acetate; PAGE, polyacrylamide gel electrophoresis; CAT, chloramphenicol acetyltransferase; LUC, luciferase; TAD, transactivation domain; wt, wild-type; DBD, DNA-binding domain; PVDF, polyvinylidene difluoride; CMV, cytomegalovirus; PBS, phosphate-buffered saline; PST, proline, serine, and threonine residues.
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