From the Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York, New York 10029-6574
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ABSTRACT |
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WW domains can be divided into three groups based
on their binding specificity. By random mutagenesis, we switched the
specificity of the Yes-associated protein (YAP) WW1 domain, a Group I
WW domain, to that of the FE65 WW domain, which belongs to Group II. We
showed that a single mutation, leucine 190 ( Since the isolation of Src homology 2 (SH2)1 domain (1), the
protein-protein interaction field has seen the emergence of several
other protein modules, such as SH3, PTB, PDZ, and WW domains (reviewed
in Refs. 2 and 3). Most of these domains have their own binding
specificity; for example, SH3 domain ligands contain a PXXP
motif, whereas SH2 domain ligands possess a phosphotyrosine residue
followed by a hydrophobic amino acid at position +3 (4). Usually, the
core consensus of the ligands is short, allowing different domains
sometimes to recognize the same binding motif. This was notably
described for the WW and SH3 domains of formin-binding proteins (FBPs)
that interact with the same 10-mer motif found in formin isoforms,
which are implicated in limb and kidney development (5, 6). In addition
to the ligand core sequences, other "surrounding" amino acids are
also necessary for a stable and specific interaction with protein
modules. For example, the
phosphotyrosine-hydrophilic-hydrophilic-hydrophobic motif binds to the
Src SH2 domain, whereas the phosphotyrosine- hydrophobic-x-hydrophobic
motif preferentially interacts with the phospholipase C- The understanding of the mechanisms that dictate the specificity of
domain-ligand interactions is important because several diseases were
shown to be related to domain dysfunction. We and others have described
that the loss of interaction between selected WW domains and their
ligands could underlie such human diseases as Liddle's syndrome,
Duchenne and/or Becker muscular dystrophies, and Alzheimer's disease
(10-15).
WW domains are composed of 38 amino acids that place them among the
smallest globular domains known to mediate protein-protein interactions. Their major features are (i) the conservation of two
tryptophans separated by 20-22 amino acids, (ii) the presence of
aromatic amino acids in the middle of the two tryptophans, (iii) and
the presence of a proline at position +2 in relation to the second
tryptophan (reviewed in Refs. 10 and 11). WW domains interact with
polyproline-rich motifs through a small hydrophobic pocket formed by
three antiparallel YAP, which contains the first described WW domain, exists in two
isoforms: the shorter possesses one WW domain, and the longer contains
two WW domains. Both of these YAP isoforms also possess an SH3 binding
motif that allows the connection to c-Yes kinase SH3 domain (20). Even
though the precise function of YAP remains unclear, its overall
organization suggests that it may function as an adaptor protein. YAP
may mediate signaling between tyrosine and serine kinases (20). YAP WW1
domain, which is common for the two YAP isoforms, has a strong affinity
for the PPPPY motif (17, 21). The YAP WW1 domain/cognate peptide NMR
structure showed that leucine 190 ( FE65 is an adaptor protein that in addition to its WW domain possesses
two PTB domains. The carboxyl-terminal PTB domain of FE65 binds to the
Group III is the less characterized, because its description is very
recent. Only the two WW domains of FBP21, which is a protein that can
bind to a proline-rich region of formin, belong thus far to this group
(18). The FBP21 WW1 and WW2 domains bind a proline-rich motif, where
methionines and glycines are present.
To address the question of ligand selectivity of WW domains, we
screened mutants of the YAP WW1 domain for those that would acquire
ligand predilection of the FE65 WW domain. Our results show that the
YAP Leu-190 substitution by Trp is necessary and sufficient for the
switch. Moreover, this substitution is enough to precipitate the two
isoforms of Mena. However, an additional substitution, His-192 to Gly,
enhanced the ability of the mutated YAP WW1 domain to act like the FE65
WW domain. Our data also suggest that a block of three aromatic amino
acids located in the second Library and Constructions of Mutants--
All inserts were
cloned into pGEX-2TK vector (Amersham Pharmacia Biotech) at
BamHI and EcoRI sites, after polymerase chain reaction amplification using the Deep Vent DNA polymerase from New
England Biolabs. All constructs were verified by Sanger sequencing using either T7 Sequenase v2.0 kit (Amersham Pharmacia Biotech) or an
automated DNA sequencer (ABI model 373). Primer nomenclature is as
follows: underlined nucleotides indicate introduced mutation(s), whereas boldface nucleotides show the generated restriction site(s). For annealed primers, only the coding strands are shown.
GST-YAP corresponds to the human cDNA region (nucleotides 758-926;
GenBankTM accession no. P46937) coding for the YAP WW1
domain (amino acids 162-217; see Fig. 1A) (17). This
construct includes some flanking sequences of the WW domain to ensure
better folding. To generate YAFE libraries, we first introduced, by
polymerase chain reaction, three unique restriction sites,
NaeI, SpeI, and StyI, into GST-YAP
without changing the amino acid sequence. Primers used for polymerase
chain reaction to introduce the Nae I and StyI sites
were as follows: 5' NaeI,
5'-AACGGATCCCAGTCTTCTTTTGAGATACCTGATGATGTACCTCTGCCGGCAGGTTG-3'; 3' StyI,
5'-TCTGAATTCGACTGGTGGGGGCTGTGACGTTCATCTGGGACAGCATGGCCTTCCTAGGGTCC3-'. Then, the Spe I site was introduced by ligating, into NaeI
and ClaI sites, the following annealed primers:
5'-GGCAGGTTGGGAGATGGCAAAGACTAGTTCTGGTCAGAGATACTTCTTAAATCACAT-3'. The YAFE-LH library was obtained by ligating annealed primers XE-LH
(5'-CTAGCTCTGGTCAGAGATACTTCNNNAATNNNAT-3') into
Spe I and ClaI sites of GST-YAP. The YAFE-Q library was made
by ligating annealed primers XE-Q
(5'-CGACNNNACAACAACATGGCAGGACC-3') into the ClaI
and StyI sites of GST-YAP. The YAFE-WH, YAFE-LY, and YAFE-RY
constructs were also generated with annealed primers XE-WH (5'-CTAGCTCTGGTCAGAGATACTTCTGG - - -CACAT-3'), XE-LY
(5'-CTAGCTCTGGTCAGAGATACTTCTACAATCACAT-3'), and XE-RY
(5'-CTAGCTCTGGTCAGTACTACTTCTTAAATCACAT-3'), respectively.
GST-FE65 contains the rat FE65 cDNA
(GenBankTM accession no. P46933) that corresponds to the WW
domain (nucleotides 458-568; amino acids 43-79; see Fig.
1A) (12).
7-PPLP comes from clone 7, which was isolated from a mouse expression
library screened with GST-FE65 (12). This clone codes for PPPPPPLPPPPPP
peptide in GST fusion.
PY5 was obtained by polymerase chain reaction using
XE-BamHI (5'-ACTTAGGGATCCAATCCAGAGGCTCCACATGTGC-3')
and EX-EcoRI
(5'-ACATCGAATTCGCGGGCGCATGCTCACCGAGTC-3') primers on human
p53BP-2 cDNA (GenBankTM accession no.
U58334). The amplified fragment codes for the PY motif (amino acids
729-768) that interacts with the YAP WW1 domain.2
Preparation and Labeling of GST Fusion Proteins--
The GST
fusion proteins expressed from the pGEX-2TK vector possess a protein
kinase A site at the end of the GST part, allowing for radiolabeling
under proper conditions. GST fusion proteins were induced by adding 1 mM IPTG to exponentially growing bacteria for 2 h at
30 °C. Bacterial protein extracts were obtained by sonication in
phosphate-buffered saline + 1% Triton X-100. The extracts were then
incubated, at 4 °C under vigorous shaking, for at least 2 h
with glutathione-Sepharose beads (Amersham Pharmacia Biotech). The GST
fusion proteins were purified by rapid centrifugations and
phosphate-buffered saline washes.
About 50 µg of the GST fusion proteins bound to the beads were
labeled with 150 units of protein kinase A from bovine heart (Sigma) in
40 µl of kinase buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 12 mM MgCl2) with 1 mM dithiothreitol in the presence of 30 µCi of
[ Pull-down Experiments and Western Blots--
Rat brains were
lysed in radioimmune precipitation buffer (10 mM Tris, pH
7.4, 300 mM NaCl, 0.1% SDS, 1% Triton X-100, 5 mM EDTA, 1% sodium deoxycholate) in the presence of a
protease inhibitor mixture (CompleteTM, Boehringer Mannheim). Protein
extracts were clarified by two or three centrifugations (12,000 rpm for
10 min at 4 °C). Lysates were diluted 10 times in Tween buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 1 mM
EDTA, 0.1% Tween-20, 1% bovine serum albumin) and were incubated, at
4 °C for 14 h under agitation, with GST fusion proteins (50 µg) bound onto glutathione beads. Then GST fusion proteins/beads were
washed three times with Tween buffer without bovine serum albumin.
Pellets were resuspended volume to volume in loading buffer (50%
glycerol, 125 mM Tris, pH 6.8, 10 µg/ml bromphenol blue,
5%
Samples were run on SDS-polyacrylamide gels (10.5%) and then
electrotransferred onto nitrocellulose membranes. Membranes were blocked for 1 h at room temperature in TBS-T buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween-20)
in presence of 5% low fat dried milk. The rabbit polyclonal antibody
against Mena, N-Mena (23), was diluted 1/1000 into TBS-T buffer + 0.5%
low fat dried milk. To reveal Mena, we used the enhanced
chemiluminescence kit (NEN Life Science Products) with the anti-rabbit
antibody conjugated with horseradish peroxidase (diluted 1/8000;
Amersham Pharmacia Biotech).
Far Western Blots--
Immediately after electrotransfer,
nitrocellulose membranes were blocked, for 4-14 h at 4 °C, in
Western wash solution (10 mM Tris, pH 7.4, 0.1% Triton
X-100, and 150 mM NaCl) with 5% low fat dried milk and
incubated with the radiolabeled probe for 14 h at 4 °C under
constant shaking. Then blots were washed four times with the same
Western wash solution.
Library Screenings and Membrane Binding Assays--
Bacteria
were plated onto LB plates containing 50 µg/ml ampicillin and 0.5 mM IPTG. 14 h later, nitrocellulose membranes were applied onto colonies for 10 min. Membranes were lifted and placed inside a chloroform chamber for 20 min. Then, membranes were incubated for 2-12 h in lysis buffer (50 mM Tris, pH 7.4, 400 µg/ml lysozyme, 5% low fat dried milk, 1 unit/ml DNase I, 5 mM MgCl2, 150 mM NaCl). Then, blots
were transferred into Western wash solution (see above) with 5% low
fat dried milk and incubated with the probe under constant shaking for
14 h at 4 °C. Washing conditions were described previously.
"SPOTs" Techniques--
Peptides were synthesized on a
derivatized cellulose membrane provided by Genosys Biotechnologies,
Inc. as described by Frank and co-workers (26, 27). SPOTs membranes
were blocked, probed, and washed with Western wash solution + blocking
buffer (1×; Genosys). 50 µg of radiolabeled GST fusion proteins were
incubated with blots for 14 h at 4 °C with constant agitation.
The Single Substitution Leu-190 to Trp Is Sufficient to Switch the
Specificity of the YAP WW1 Domain from Group I to That of Group
II--
Because the NMR structure of the YAP WW1 domain shows that
Leu-190, His-192, and Gln-195 seem to directly interact with the Tyr of
the ligand, we decided to randomly mutate these three residues to
determine whether one of these mutants can acquire the specificity of
the FE65 WW domain. To achieve this goal, we constructed two libraries
in pGEX-2TK vector containing the YAP WW1 domain with random sequences
at the position corresponding either to Leu-190 and His-192 together
(YAFE-LH) or to Gln-195 (YAFE-Q) alone. Approximately 20,000 colonies/library were plated onto LB plates supplemented with IPTG to
allow the expression of GST fusion proteins. The membranes were lifted
from the plates and probed with the radiolabeled GST-7-PPLP (7-PPLP), a
ligand of FE65 WW domain. This technique was sensitive and specific,
because a single colony expressing the GST-FE65 WW domain (GST-FE65)
could be readily detected with the radiolabeled 7-PPLP probe, whereas
bacterial colonies expressing the GST-YAP WW1 domain (wild type;
GST-YAP) were not detectable (Fig.
1A). The converse was also
true, because GST-YAP was the only construct recognized by the GST-PY5
probe (PY5), which corresponds to the region of the p53BP-2 protein
that interacts with YAP through its WW1 domain.2 In both
cases, GST alone could not interact with these two probes (Fig.
1A).
From the YAFE-LH library, we obtained more than 200 positive clones,
whereas no positive clones were isolated in the YAFE-Q library. Six
YAFE-LH clones were fully analyzed, using two complementary techniques:
a membrane binding assay and far Western blot. The membrane binding
assay maintains the native conformation of GST fusion proteins but is
not quantitative, whereas far Western blot is quantitative but requires
one step of denaturation due to SDS treatment followed by one
renaturation step. Renaturation in some cases can be difficult. For
example, GST-FE65 does not fold well under far Western blot conditions,
whereas GST-YAP seems to fold well, as judged by the intensity of their
respective signals when probed with 7-PPLP or PY5 (Fig. 1D).
As depicted in Fig. 1, C and D, these six clones
can bind to the 7-PPLP probe but not to the PY5 probe in either the
membrane binding assay or far Western blot. In fact, overexposure shows
that the clone YAFE-LH3 can still interact weakly with PY5 motif.
The sequence analysis of these six mutants shows that Leu-190 is
substituted each time by Trp, which is the amino acid in the FE65 WW
domain (Trp-61) corresponding to Leu-190 of YAP. In addition, His-192
can be maintained or substituted by at least five different amino
acids: Lys, Arg, Ser, Asp, or Gly (Table I), suggesting that position 192 is not
critical for this switch.
The Amino Acid in Position 192 Also Interacts with Ligands--
In
order to determine whether position 192 is neutral, as the previous
experiment suggested, we screened a PPPPXP peptide repertoire synthesized onto cellulose membrane (SPOTs technique; X means any amino acid) with the YAFE-LH mutants. We used
the PPPPXP repertoire because our previous studies have
shown that the YAP and FE65 WW domains bind with high affinity to
PPPPYP and PPPPPP peptides, respectively. Because peptides synthesized by the SPOTs technique are anchored by their carboxyl termini, we added
four Ala residues at the carboxyl terminus of each peptide to provide a
flexible spacer between the proline stretch and the membrane. Eight
repertoires were generated and probed with the six radiolabeled mutants
and with YAP and FE65 WW domains used as controls (Fig.
2). Although the six mutants showed a
strong affinity for the PPPPPP peptide as the FE65 WW domain, only the YAFE-LH10 mutant displayed exactly the same pattern as that of the FE65
WW domain. This pattern showed, in addition to the strong binding to
the PPPPPP peptide, a moderate binding to PPPPRP and a weak binding to
PPPPKP peptide. The YAFE-LH6 mutant can also bind to those two basic
peptides but with an affinity the same as or stronger than that of
the PPPPPP motif. Interestingly, the YAFE-LH3 mutant can interact
with both PPPPPP and PPPPYP peptides. This result is consistent with
the weak interaction observed in Fig. 1, C and D,
between YAFE-LH3 and the PY5 motif. Taken together, these data suggest
that position 192 plays a role in the interaction with ligands.
The Single Point Mutation Leu-190 to Trp Is Sufficient to
Precipitate FE65 WW Domain Ligands--
To determine whether the
YAFE-LH mutants can also interact with the other FE65 WW domain
ligands, we performed pull-down experiments from rat brain lysates with
YAFE-LH mutants. As negative controls, we used GST alone and a FE65 WW
domain mutant (FE65 mut), where Trp-69 and Pro-72 were substituted by
Phe and Ala, respectively. These two mutations render the FE65 WW
domain inactive in terms of ligand binding (Fig.
3B) (12). As shown in Fig.
3A, almost all of the ligands precipitated by FE65 WW domain
were also precipitated by YAFE-LH mutants, whereas none of the major
ligands of YAP WW1 domain were precipitated by the mutants.
Interestingly, as shown in the SPOT technique data (Fig. 2), the
YAFE-LH10 mutant is most similar to the FE65 WW domain, because the
precipitation patterns of YAFE-LH10 and FE65 are identical except for
the faint 90-kDa protein band that can only be pulled down by the FE65
WW domain. It is worth noticing that the YAFE-LH3 mutant, although
displaying a hybrid specificity on SPOT technique (PPPPPP and PPPPYP;
Fig. 2), cannot precipitate any YAP ligands, confirming that its
affinity for PPXY is poor.
To show that the co-migrating protein bands observed in the mutants and
FE65 pull-down experiments correspond to the same proteins, we probed
them with an antibody against Mena, which interacts in vivo
with FE65 through the WW domain (12). As shown in Fig. 3C,
all of the mutants, like the FE65 WW domain, precipitated the two
isoforms of Mena (80 and 140 kDa), whereas YAP can only precipitate the
neuronal form of Mena (140 kDa) due to the presence of a PPSY motif
(12). As expected, a third band (60 kDa), visualized only with FE65 and
the mutants, can also be detected with this antibody, suggesting that
another form of Mena or a related protein interacts with the FE65 WW
domain. It is worth noticing that the Mena antibody also recognizes a
band migrating above the Mena-related protein. This band is apparently
none specific, because the signal is present in every lane, including
the GST lane, when the film is exposed for a longer period (data not
shown). These results confirm that all the co-migrating bands observed
with the mutants and with FE65 correspond to the same proteins.
The Extra Length of YAFE Mutants Is Not Responsible for Their
Failure to Precipitate the 90-kDa Band--
A close observation shows
that the YAP WW1 domain possesses 21 amino acids between the two
conserved tryptophans, whereas in the FE65 WW domain there are only 20 (Fig. 1A). In order to address the importance of this extra
amino acid for the switch and to examine whether the length is the
factor that prevents the 90-kDa band precipitation, we deleted the
Asn-191 in the YAFE-LH3 construct (YAFE-WH). The YAFE-WH mutant
possesses the same second Three Aromatic Amino Acids in the Middle of YAP WW1 Domain Are
Necessary but Not Always Sufficient to Allow the Switch--
A rapid
comparison between Group I and II WW domains showed that Group I WW
domains possess only two consecutive aromatic amino acids in the
middle, whereas Group II WW domains have three aromatics (Table
II). Because the YAFE-LH mutants are
consistent with this observation, we investigated whether three
aromatics in the YAP WW1 domain are sufficient, when we changed the
composition and the position of the aromatic block.
To address the issue of the composition, we replaced Leu-190 with a
different aromatic amino acid, such as Tyr (YAFE-LY), in case we had
missed it in our screening of the YAFE-LH library. With this YAFE-LY
mutant, we observed a weak and probably insignificant signal with
either the 7-PPLP or PY5 probes (compare Fig.
5C and Fig. 1D).
This conclusion is supported by the observations that the YAFE-LY
mutant cannot pull down Mena from rat brain lysate and that SPOT
analysis revealed a faint interaction only with the PPPPYP peptide
(data not shown).
To address the importance of the position of the aromatic amino acids,
we substituted Arg-187 with Tyr (YAFE-RY). As shown in Fig. 5, the
YAFE-RY mutant acts as the YAP wild type. We confirmed this result
using the SPOT technique. In this experiment, both mutant and wild type
presented the same pattern (data not shown).
Taken together, these data show that the position and the composition
of the aromatic amino acids within the second Based on their binding specificity, WW domains can currently be
divided into three groups. In this report, we focused our study on the
specificity that drives Group I and Group II WW domains to interact
with PPXY core and with a stretch of prolines, respectively. To achieve this aim, we switched the specificity of the YAP WW1 domain
of Group I to that of the FE65 WW domain of Group II.
Using two biased YAP WW1 domain libraries, in which Leu-190 and His-192
together or Glu-195 alone has been randomly replaced, we have isolated
and characterized six mutants that can bind to 7-PPLP probe, as does
the FE65 WW domain. None of these mutants came from the YAFE-Q library,
but all were isolated from the YAFE-LH library, suggesting that the
Gln-195 is not crucial for this switch. Sequence analysis showed that
the six mutants possess, like FE65, a Trp instead of Leu-190, whereas
His-192 can be maintained or replaced by, at least, five other amino
acids: Lys, Arg, Ser, Asp, and Gly. These substitutions of His-192
represent four different amino acid categories: basic, acidic, polar,
and nonpolar. This wide spectrum suggests that the switch is mainly due
to Trp-190 and that position 192 may not play an important role in the
interaction with the 7-PPLP ligand. Although all the YAFE-LH mutants
can precipitate the two Mena isoforms with the same strength as FE65,
position 192 also imparts domain specificity, because analysis by SPOT and pull-down experiments revealed that these mutants can mimic the
behavior of the FE65 WW domain to different degrees (Figs. 2 and 3).
This observation is confirmed by the fact that a single mutation in YAP
that changes this His-192 to a Phe switches the specificity from PPPPYP
to PPPPFP, again suggesting that position 192 is also involved in
imparting binding specificity.3
According to SPOT and pull-down data, only two point mutations in the
YAP WW1 domain, L190W and H192G (YAFE-LH10), generate a WW domain that
acts similar to FE65. The only difference is a faint 90-kDa protein
band that is precipitated by FE65 and not by YAFE-LH10 (Figs. 2 and 3).
To ask whether the 90-kDa band precipitation is due to the size of FE65
WW domain (one amino acid less compared with the YAP WW1 domain), we
generated the YAFE-WH mutant, which possesses the substitution Leu-190
to Trp and also a Asn-191 deletion. We chose the Asn-191 because its
deletion results in a sequence similar to that of FE65. Interestingly
this YAFE-WH mutant can still fold properly because it can interact
with the 7-PPLP probe and precipitate Mena. However, this mutant, like
the other, cannot pull down the 90-kDa band, suggesting that the
interaction with this band is not due to the length of FE65 WW domain.
The 90-kDa band might correspond to a protein that, in addition to its
interaction with the binding pocket of the FE65 WW domain, requires
additional contacts with surrounding amino acids of the WW domain to
stabilize its binding. This protein may also bind to FE65 via a
nonlinear motif, as it has been previously described with Mbh I, an
in vitro FE65 ligand that does not contain any stretches of
prolines (12). This latter option is possible because Mbh I, expressed
as a GST fusion protein, cannot interact with the YAFE-LH mutants or
with YAFE-WH (data not shown).
A rapid comparison between Group I and Group II WW domains shows that
Group I possesses two consecutive aromatic amino acids on their second
In order to confirm our hypothesis concerning the importance of the
number of aromatic amino acids in the second In this study, we have shown that a double or even a single mutation
can switch the YAP WW1 domain specificity to another specificity. Now
it would be interesting to generate modified WW domains that can
restore the disrupted binding due to a point mutation in the
proline-rich sequence. Such mutations have been described in the
PPXY motif of a sodium channel subunit, B5) to tryptophan, is
required to switch from Group I to Group II. Although this single
substitution in YAP WW1 domain is sufficient to precipitate the two
protein isoforms of Mena, an in vivo ligand of FE65, we
showed that an additional substitution, histidine 192 (
B7) to
glycine, significantly increased the ability of YAP to mimic FE65. This
double mutant (L190W/H192G) precipitates eight of the nine protein
bands that FE65 pulls down from rat brain protein lysates. Based on
both our data and a sequence comparison between Group I and Group II WW
domains, we propose that a block of three consecutive aromatic amino
acids within the second
-sheet of the domain is required, but not
always sufficient, for a WW domain to belong to Group II. These data
deepen our understanding of WW domain binding specificity and provide a
basis for the rational design of modified WW domains with potential
therapeutic applications.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
carboxyl-terminal SH2 domain (4). Based on this observation,
subconsensus sequences have emerged that lead to the subdivision of the
domains into different classes according to their binding preferences
(reviewed in Refs. 3 and 4). It seems that only a few amino acids
within domains are responsible for these binding preferences, because a
single substitution in SH2 or SH3 domains can switch their binding
specificity (7-9).
-sheets (16). Similar to other protein modules,
WW domains can also be subdivided thus far into three groups. Group I
WW domains, like Yes-associated protein (YAP) WW1 and WW2 domains,
interact with the core sequence PPXY (17). Group II WW
domains, such as the FE65 WW domain, bind to a long stretch of prolines
often interrupted by a leucine (PPLP motif) (5, 6, 12). Group III WW
domains, represented by the FBP21 WW1 and WW2 domains, interact with a
repeated polyproline-rich region containing glycines and methionines,
called the PGM motif (18). Recently, a
phosphoserine/phosphothreonine-containing peptide has been shown to
interact with some WW domains, such as the mitotic rotamase Pin 1, this
finding might generate a fourth group (19).
B5) and histidine 192 (
B7)
interact with the tyrosine of the ligand and that tryptophan 199 (
C5) binds to the two "central" prolines of the ligand
(PPXY) through Van der Waals contacts. Glutamine 195 (
C1)
may also establish a hydrogen bond with the tyrosine of the ligand
(16).
-amyloid precursor protein, which is a transmembrane protein
implicated in Alzheimer's disease. It is proposed that this
interaction regulates
-amyloid precursor protein processing (reviewed in Ref. 22). FE65 also interacts through its WW domain with
Mena (mammalian homologue of Drosophila enabled), which is implicated in the regulation of the cytoskeleton dynamics (12, 23). We
have shown that the PPPPPPLPPPPPP motif, which is present several times
in Mena, is required for the efficient binding of the FE65 WW domain
(12). In fact, a stretch of six or more prolines in a row can also bind
to the FE65 WW domain in vitro. The three-dimensional structure of the FE65 WW domain is not yet known. However, the crystal
structure of the Pin 1 WW domain displays the same overall structure as
the YAP WW1 domain, including the three protruding amino acids in the
pocket (Phe-25, His-27, and Trp-34) (24). Based on this evidence and on
modeling programs, such as Modeller (25), which also predicts three
-sheets for the FE65 WW domain, one can suppose that the overall
structure of the FE65 WW domain is similar to those of the YAP and Pin
1 WW domains.
-sheet of WW domains is required, but is
not always sufficient, for a WW domain to belong to Group II.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP, for 30 min on ice. After several
phosphate-buffered saline washes, GST fusion proteins were eluted from
beads on a flow-through column, by 10 mM free glutathione
in 50 mM Tris, pH 8.
-mercaptoethanol, 2% SDS).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
At least six YAFE-LH mutants can bind to
7-PPLP probe. A, full amino acid sequences of the YAP
WW1 and FE65 WW domains, and also the YAFE-LH and YAFE-Q libraries. All
these constructs were expressed as GST fusion proteins. Open
arrows indicate the position of the three -sheets of the YAP
WW1 domain. X indicates where random mutagenesis was
introduced. Numbers indicate amino acid position in FE65 and
the short isoform of YAP proteins. B, upper
panels, two LB agar plates containing 0.5 mM IPTG were
streaked with GST, GST-YAP, and GST-FE65. Lower panels,
membranes lifted from these two plates were probed with either the
radiolabeled 7-PPLP probe (left) or the radiolabeled PY5
probe (right). C, membrane binding assay.
Upper panels, GST, YAP, FE65, and six clones isolated from
the YAFE-LH library screening were plated onto two LB plates plus IPTG.
Lower panels, filters obtained from the previous plates were
probed with the radiolabeled 7-PPLP probe (left) or with the
radiolabeled PY5 probe (right). D, far Western
blots. The same quantity of GST fusion proteins (~2 µg) was loaded
in parallel on three 10.5% SDS-polyacrylamide gels. Two gels were
electrotransferred and probed with either the 7-PPLP (left)
or the PY5 (right) probes. The third gel was stained with
Coomassie Blue to show the calibration. Molecular mass markers are
indicated in kDa.
Partial peptide sequences of YAFE-LH mutants
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Fig. 2.
Spot analyses of YAFE-LH mutants. Eight
membranes with a repertoire of 20 peptides of the type
PPPPXPAAAA (where X indicates any amino acid),
were probed with eight different radiolabeled probes, indicated on the
left. Spot intensity cannot be compared from one membrane to
another because labeling was not constant from one probe to
another.
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Fig. 3.
Like FE65 WW domain, YAFE mutants can pull
down the non-neuronal isoform of Mena. A, pull-down
experiments. 800 µg of rat brain lysates were incubated with nine
different GST fusion proteins. GST fusion proteins were precipitated
with glutathione beads, extensively washed, and then loaded on 10.5%
SDS-polyacrylamide gels. Proteins were electrotransferred onto a
membrane that was cut into nine strips according to the nine loaded
samples. Each of the strips was probed back with the radiolabeled GST
fusion protein used for the pull-down. The two isoforms of Mena are
indicated by solid arrows, whereas the Mena-related protein
is indicated with an open arrow. The open circle
indicates the 90-kDa band. B, pull-down controls. We also
performed a pull-down with a mutant of FE65 (FE65 mut), in
which its WW domain was inactivated in terms of binding by two point
mutations, W69F and P72A. We used either FE65 mut or FE65 as a probe
onto FE65 mut and FE65 pull-down experiments to see whether any of the
nine bands precipitated by FE65 are nonspecific. Lower panels
correspond to the calibration of the loaded GST proteins. These two
same blots were stripped and probed back with a rabbit polyclonal
antibody against GST. C, Mena can be precipitated by the
YAFE-LH mutants. Using the same pull-down experiments as in
A, we ran and electrotransferred them onto a membrane that
we probed them with a rabbit polyclonal antibody against Mena, N-Mena
(upper panel). Two solid arrows indicate the two
Mena isoforms, and the open arrow indicates the Mena-related
protein. The lower panel is the calibration of the previous
blot with a GST antibody.
-sheet end
(YFW-HID) as FE65 WW domain
(YYWHIP). Fig. 4 shows that
YAFE-WH can still fold properly because it can bind to the 7-PPLP
probe. YAFE-WH can also precipitate Mena (Fig. 4C, right panel), but not the 90-kDa band. In addition, its precipitation pattern is not as close to FE65 as that of YAFE-LH10 (Fig. 4C, left panel). Interestingly SPOT analysis shows that YAFE-WH
exclusively binds to the PPPPPP peptide and not to the PPPPYP peptide
as YAFE-LH3 does.
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Fig. 4.
The short size of the FE65 WW domain is not
responsible for the 90-kDa band precipitation.
A, membrane binding assay. Upper panels,
bacterial colonies containing GST, YAP, YAFE-WH, or FE65 were induced
by IPTG on LB plates. Lower panels, membranes issued from
these two plates and probed with either 7-PPLP or with PY5.
B, far Western blots. Upper panels, filters, with
GST fusion proteins electrotransferred onto them, were probed with the
radiolabeled 7-PPLP or PY5 proteins. Lower panel, Coomassie
Blue calibration of the GST fusion proteins analyzed in far Western
blots. Molecular mass markers are in kDa. C, pull-down
experiments with YAFE-WH. Conditions were the same as in Fig.
3A. D, pull-down experiments probed with an
anti-Mena antibody, N-Mena. Conditions were the same as in Fig.
3C. E, spot analysis. Spot membrane, with the
PPPPXPAAAA peptide repertoire, was probed with the
radiolabeled YAFE-WH probe.
Partial listing of Group I and Group II WW domains for which ligands
have been characterized
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Fig. 5.
Three aromatics are not enough to allow the
switch to the 7-PPLP ligand. A, membrane binding assay.
Upper panels, bacterial colonies expressing GST, YAP,
YAFE-RY, YAFE-LY, or FE65. Lower panels, filters from the
upper single colonies were hybridized with either the 7-PPLP or PY5
probes. B, far Western blots. Conditions were the same as in
Fig. 1D.
-sheet of the YAP WW1
domain are important for the switch of the specificity.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheet, whereas Group II has three (Table II). Although YAFE-LH
mutants strengthen this observation, data from YAFE-RY and YAFE-LY
mutants show that the position and the composition of the central
aromatic amino acids are also important for imparting binding
specificity. To summarize, three aromatic amino acids in the second
-sheet seem to be required for a WW domain to belong to Group II,
but probably not all WW domains with three aromatics belong to Group
II. Interestingly, the human and mouse FBP21-WW1 and -WW2 (Group III)
have four aromatics, Y/FXYYY, except
the mouse FBP21-WW1, which has three aromatics plus a His, a basic amino acid with an imidazole ring, HCYYY. More
studies are needed to conclude that group III specificity is dictated
by its four central aromatics or "rings."
-sheet, we are trying
to generate the reverse switch, by creating a modified FE65 WW domain
that binds to the PY5 probe. Thus far, we have changed Trp-61 to a Leu
(FEYA) with or without the addition of the extra amino acid, Asn. As
expected, these FEYA mutants can no longer bind to 7-PPLP probe, but
they also cannot bind to the PY5 probe (data not shown). Because these
mutants can neither bind to any PPPPXP peptides nor
precipitate ligands from rat brain lysates (data not shown), we think
that they cannot fold properly. This result suggests that to switch the
FE65 WW domain binding affinity to the PY5 probe, we probably need to
introduce, in addition to Leu, other changes to stabilize the structure
and/or increase the affinity to the PY5 probe.
EnaC. These
mutations prevent sodium channel degradation by Nedd4, a ubiquitin
ligase possessing WW domains, and lead to Liddle's syndrome, a form of
severe hypertension (31-33).
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ACKNOWLEDGEMENTS |
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We thank Hillary Linn, Kira Ermekova, and Frank Gertler (Massachusetts Institute of Technology, Boston, MA) for the SPOT membrane synthesis, the FE65 construct, and the antibody against Mena, respectively. We are grateful to Anthony Korosi, Alex Chang, and Stacey Rentschler for their valuable comments on the manuscript and to Paul Klotman for support.
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FOOTNOTES |
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* This work was supported by United States Public Health Service grants DK50795, CA45757, and CAO1605 and by grant from the Muscular Dystrophy Association.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.
Recipient of fellowships from the Association pour la Recherche
contre le Cancer and from the Phillipe Foundation.
§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, One Gustave Levy Place, New York, NY 10029-6574. Tel.: 212-241-9431; Fax: 212-426-1483; E-mail: M_Sudol{at}smtplink.mssm.edu.
2 X. Espanel and M. Sudol, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
YAP, Yes-associated
protein;
FBP, formin-binding protein;
GST, glutathione
S-transferase;
SH, Src homology;
IPTG, isopropyl-1-thio--D-galactopyranoside.
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REFERENCES |
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