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Mechanisms of Signal Transduction:
A Novel interaction between AIP4 and β
PIX is mediated by an SH3 domain
Jay M. Janz, Thomas P. Sakmar and K.
Christopher Min
J. Biol. Chem. published online July 25, 2007
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A NOVEL INTERACTION BETWEEN AIP4 AND βPIX IS MEDIATED BY AN SH3 DOMAIN
Jay M. Janz1,*, Thomas P. Sakmar1, and K. Christopher Min2
1
Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, 1230 York Avenue,
New York, NY, 10021. 2Department of Neurology, Columbia University, 710 West 168th Street, New
York, NY 10032.
RUNNING TITLE: Structural insight into the βPIX/AIP4 complex
*To whom correspondence should be addressed: The Rockefeller University, 1230 York Avenue, Box
187, New York, NY, 10021. Tel: 212-327-8283 Fax: 212-327-7904 Email: [email protected]
Two prominent transmembrane receptor
families facilitate cellular communication with the
extracellular environment – G protein-coupled
receptors (GPCRs) and receptor tyrosine kinases
(RTKs). A wide array of ligands bind to these
receptors to orchestrate signaling networks
integral to many cellular functions. Internalization
and degradation of receptor molecules from both
families
regulate
signal
duration
and
termination(1,2). E3 ubiquitin ligases, which
catalyze the ubiquitination of receptors, control
intracellular sorting, recycling and degradation (3-
1
Copyright 2007 by The American Society for Biochemistry and Molecular Biology, Inc.
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5).
Representative E3 ligases include AIP4
(Atrophin-Interacting
Protein
4),
which
ubiquitinates the GPCR chemokine receptor
CXCR4 (6), and Cbl (Casitas B-Lymphoma
protein), which ubiquitinates the RTK epidermal
growth factor (EGF) receptor (7-9).
The
importance of signal termination control is
exemplified by recent work implicating prolonged
signaling as a cause of cellular transformation
(10,11).
In addition to mediating the ubiquitination
and sorting of CXCR4, a number of recent studies
detail the regulatory role AIP4 plays in
developmental, immunological and oncogenic
signaling pathways (12-18).
The AIP4/Itch
protein is composed of an N-terminal C2 domain,
followed by a proline-rich region, four WW
domains and a C-terminal catalytic HECT domain
(3,4,12,13). AIP4 is abundantly expressed in most
human tissues and displays tissue specificity
similar to that of Cbl (19).
βPIX (β-PAK-interactive exchange factor,
also referred to as Cool-1) is comprised of
modular domains including an N-terminal SH3
domain followed by DH (Dbl, diffuse B-cell
lymphoma homology) domain, PH (pleckstrin
homology) domain, and a leucine zipper (20,21).
Functionally, βPIX acts as a GEF (guanine
nucleotide exchange factor) for Rac/Cdc42 and
was first identified as an exchange factor for PAK
(20,22,23). Recent studies illustrate a role for
βPIX in breast cancer pathogenesis as well as
aspects of CXCR4 induced cellular chemotaxis
(24-27).
Like other SH3 domains, βPIX–SH3 binds
to proline-rich sequences with a polyproline II
helix (PPII) conformation. Although most SH3
domain ligands contain a PxxP motif, βPIX–SH3
binds ligands with a noncanonical PxxxPR
sequence and serves as a scaffolding point for a
number of protein-protein interactions as
Cross-talk between GPCR and RTK signaling
pathways are crucial to the efficient relay and
integration of cellular information. Here we
identify and define the novel binding
interaction of the E3 ubiquitin ligase AIP4 with
the GTP exchange factor βPIX.
We
demonstrate that this interaction is mediated in
part by the βPIX–SH3 domain binding to a
proline-rich stretch of AIP4. Analysis of the
interaction by isothermal calorimtery is
consistent with a heterotrimeric complex with
one AIP4 derived peptide binding to two βPIX–
SH3 domains. We determined the crystal
structure of the βPIX–SH3/AIP4 complex to 2.0
Å resolution. In contrast to the calorimetry
results, the crystal structure shows a
monomeric complex in which AIP4 peptide
binds the βPIX–SH3 domain as a canonical
Class I ligand with an additional type II
polyproline helix that makes extensive contacts
with another face of βPIX. Taken together, the
novel interaction between AIP4 and βPIX
represents a new regulatory node for GPCR
and RTK signal integration. Our structure of
the
βPIX–SH3/AIP4
complex
provides
important insight into the mechanistic basis for
βPIX scaffolding of signaling components,
especially those involved in cross-talk.
demonstrated by the direct coupling of βPIX to
p21-activated kinase (PAK) family members (20).
The βPIX–SH3 domain also binds to the PxxxPR
motif of Cbl family members to facilitate the
clustering of proteins involved in the downregulation
of
EGFR
signaling
(26,28).
Interestingly, the βPIX–SH3 domain also binds
proteins including Rac1 and SAP (Signaling
Lymphocyte Activation Molecule Associated
Protein) (29,30) that possess neither the PxxxPR,
nor the canonical PxxP motifs. The structural
basis for these interactions as well as the
functional ramifications of competition among
multiple ligands for overlapping binding sites on
βPIX remains to be elucidated.
Here we identify the E3 ubiquitin ligase
AIP4 as a binding partner for the βPIX scaffolding
protein.
We demonstrate an endogenous
βPIX/AIP4 complex in breast cancer cell lines and
show that complex formation is mediated in part
by βPIX–SH3 domain binding to a proline-rich
stretch of AIP4. We show using isothermal
titration calorimetry (ITC) that the interaction is
heterotrimeric in solution, with one AIP4 derived
peptide binding to two βPIX–SH3 domains.
Fluorescence assays suggest that the mode of
interaction for the βPIX–SH3/AIP4 complex is
unique compared to known βPIX–SH3
interactions. We determined the crystal structure
of the βPIX–SH3/AIP4 complex to 2.0 Å
resolution. AIP4 binds the βPIX–SH3 domain as
a canonical Class I ligand with an additional PPII
helix that makes extensive contacts with another
face of βPIX. Our structure of the βPIX–
SH3/AIP4 complex provides important insight
into the mechanistic basis for βPIX scaffolding of
signal components. The novel interaction between
βPIX and AIP4 represents a new regulatory node
for GPCR and RTK signal integration.
was purchased from Sigma. Monoclonal anti-Itch
antibody was purchased from BD Biosciences.
Goat anti-Mouse IgG and goat anti-Rabbit IgG,
HRP-conjugated secondary antibodies were from
Upstate Biotechnology. SDF1α was purchased
from R&D Systems and EGF was purchased from
Calbiochem.
Immunoblotting, Co-immunoprecipitation and
Pull-down Assays – Harvested cells were lysed in
lysis buffer (50 mM Tris-HCL, pH 8, 150 mM
NaCl, 5 mM EDTA, 0.5% sodium deoxycholate
(w/v), 1% Nonident P-40 (v/v), 0.1% SDS (w/v)),
plus protease inhibitor tablets (Roche). Cell
lysates
were
subsequently
cleared
by
centrifugation and the supernatant incubated with
the appropriate antibody at 4 °C for 1h to
overnight followed by incubation with protein A
or G argarose for 2 h at 4 °C. Immunoprecipitates
were then washed 3 - 5 times with cold lysis
buffer, eluted by boiling in SDS-buffer and
separated by SDS-PAGE.
Proteins were
subsequently
transferred
to
nitrocellulose
membranes and probed with the appropriate
antibody of interest. To probe for endogenous
AIP4 levels a monoclonal antibody to mouse Itch
EXPERIMENTAL PROCEDURES
Reagents and Antibodies – Except where noted, all
buffers and chemicals were purchased from either
Fisher or Sigma.
Monoclonal Anti-myc,
monoclonal Anti-AIP4 were purchased from Santa
Cruz Biotechnology.
Polyclonal anti-βPIX
antibody was from Chemicon International.
Polyclonal anti-HA antibody was purchased from
Covance. Monoclonal anti-FLAG M2 antibody
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Cell Culture, Plasmids and Transfection –
HEK293T, MCF7, MDA-MB-231 and NIH 3T3
cells were all cultured in DMEM supplemented
with 10% fetal bovine serum. The pcDNA3FLAG-CIN85 mammalian expression construct
was a kind gift from Dr. Sachiko Kajigaya (NIH;
Bethesda, MD). The pEBB-βPIX-HA mammalian
expression construct was a kind gift from Dr.
Bruce Mayer (University of Connecticut,
Farmington, CT).
The pRK5-myc-AIP4
mammalian expression construct was a kind gift
from Dr. Tony Pawson (University of Toronto,
Toronto, ON, Canada). The pGEX-6P1-βPIX–
SH3 domain expression construct was a kind gift
from Dr. André Hoelz (Rockefeller University,
New York, NY). All truncation and mutant
constructs were generated by site-directed
mutagenesis using high fidelity thermostable DNA
polymerase Pfu (Stratagene). All transfection
studies were performed using DNA-Lipofectamine
(Gibco/Invitrogen) and cells were assayed or
harvested approximately 48 h after transfection.
Harvested cells were either used immediately or
snap frozen in liquid nitrogen and kept at -80 °C.
Fluorescence Spectroscopy – Fluorescence
measurements were performed essentially as
described previously (31). All experiments were
carried out in 50 mM Hepes, 150 mM NaCl, pH
7.3, at 20°C in a 4 mm × 4 mm quartz cuvette.
Intrinsic Trp fluorescence was measured by
exciting samples at 295 nm (1 nm bandpass) and
monitoring emission from 315 to 450 nm (10 – 15
nm bandpass). The EC50 values were determined
by titrating βPIX–SH3 domain samples with
increasing amounts of WT AIP4(206 – 229) peptide
and monitoring both the shift in Trp emission
wavelength and overall fluorescence intensity.
Competition experiments were performed in a
similar manner using the PAK2(176 – 199) and Cblb(899-914) peptides. The PAK2(176 – 199) peptide
sequence contains a tyrosine residue, and it is
possible that some energy transfer to this residue
from the Trps in the SH3 domain takes place upon
binding, thereby reducing the fluorescence
emission intensity. However, a PAK2 peptide in
which this Tyr residue is converted to a Phe does
not significantly increase the fluorescence
emission intensity (data not shown).
Data was
analyzed in SigmaPlot using nonlinear regression
and fitted to a sigmoidal dose-response (variable
slope) equation four-parameter logistic equation;
Y = minimum + (maximum – minimum)/1 +
10(logEC50-x)*Hillslope.
Protein Expression and Purification – The SH3
domain βPIX was expressed and purified
essentially as described previously (31).
Peptide Synthesis and Preparation – All peptides
were synthesized by the Proteomics Resource
Center at the Rockefeller University (New York,
NY), purified by HPLC and verified by mass
spectrometry. Peptides that correspond to regions
of AIP4 are as follows:
AIP4(206–229),
Y206GFKPSRPPRPSRPPPPTPRRPASV229;
AIP4(211 – 226), 211RPPRPSRPPPPTPRRP226;
AIP4(201 – 216), 201SLSNGGFKPSRPPRPS216;
AIP4(216 – 232), 216SRPPPPTPRRPASVNGS232;
AIP4(216 – 226), 216SRPPPPTPRRP226; AIP4-R217A,
Y206GFKPSRPPRPSAPPPPTPRRPASV229. The
following peptide corresponds to the βPIX–SH3
binding domain of human PAK2; PAK2(176 – 199),
176EETAPPVIAPRPDHTKSIYTRSVI199 and that
of
human
Cbl-b;
Cbl-b(899-914),
Note that a
899SQAPARPPKPRPRRTA914Y.
tyrosine residue was added on to the N-terminus of
peptides AIP4(206-229) and AIP4-R217A or the Cterminus of Cbl-b(899-914) to aid in concentration
determination.
All protein and peptide
concentrations were determined by UV
spectroscopy and/or amino acid analysis.
Isothermal titration calorimetry (ITC) –
All ITC measurements were performed at 20 °C
using a MicroCal (Northampton, MA) VP-ITC
calorimeter essentially as described previously
(31). Typically, the sample cell contained 50 µM
solution of the βPIX–SH3 domain and was titrated
at equal intervals with 10-µl aliquots 500 µM of
AIP4 peptide solution for a total of approximately
290 µL. The heat generated due to dilution of the
titrants (peptide) was subtracted for baseline
correction. Baseline corrected data were analyzed
with MicroCal ORIGIN Ver 6.0 software. All
experiments were performed at least twice
Crystallography, Data Collection and Structure
Determination – The SH3 domain (3 mM in 20
mM Tris, pH 8.0, 100 mM NaCl, 5 mM DTT) was
mixed at a 1:2 molar ratio with AIP4(206-229)
peptide dissolved in the same buffer and
crystallized at 20ºC by the vapor diffusion method.
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was used. This antibody readily detects AIP4, as
Itch and AIP4 are highly homologous. In overexpression studies, all AIP4 constructs encode an
N-terminal myc-tag, βPIX constructs encode a Cterminal HA-tag and CIN85 constructs encode a
N-terminal FLAG-tag to aid in immunoblot
detection.
For GST pull-down assays, lysates of
transformed E. coli BL21(DE3) cells expressing
either GST-βPIX–SH3 or GST alone were bound
to glutathione-Sepharose beads (GE Amersham
Biosciences) in a Tris buffer (50 mM Tris, 150
mM NaCl, 2 mM DTT, pH 7.3) for one hour at 4
ºC. The beads were subsequently washed in the
same buffer and incubated with HEK 293T
cellular lysate expressing either myc-tagged AIP4
or an empty vector. The complexes were pulleddown, washed extensively and resolved via SDSPAGE. Proteins were subsequently transferred to
nitrocellulose and probed for AIP4 binding using
an anti-myc antibody.
hypothesized that AIP4 interacts directly with
βPIX through binding of the proline-rich region of
AIP4 to the SH3 domain of βPIX. First we found
that βPIX–SH3 alone was sufficient to pull down
AIP4 in a GST pull-down assay with HEK293T
lysate expressing myc-tagged AIP4 (Figure 1B).
We further confirmed the importance of the
proline-rich region of AIP4 using coimmunoprecipitation assays in HEK293T cells.
HA-tagged βPIX was co-expressed with fulllength or truncated forms of myc-tagged AIP4;
immunoprecipitation was performed with anti-myc
antibody and immunoblots were performed to test
for the association of βPIX. We found that
deletion of the proline-rich region of AIP4
abolished the interaction with βPIX in this assay
(Figure 1C).
AIP4 binds to the CIN85 scaffolding
protein – The βPIX–SH3 domain is
phylogenetically similar to the three SH3 domains
of CIN85, an adaptor protein involved in Cblmediated down-regulation of RTKs (9,26). Like
βPIX, the SH3 domains of CIN85 recognize and
bind the non-canonical PxxxPR motifs in Cbl and
PAK proteins (26,28,39). When both FLAGtagged CIN85 and myc-tagged AIP4 are coexpressed in HEK293T cells we find that CIN85
pulls down AIP4, albeit to a lesser extent than
βPIX (Figure 1D). Notably, this interaction
dissipates as cells co-express increasing amounts
of βPIX (Figure 1D).
AIP4 forms a heterotrimeric complex with
βPIX–SH3 domains in solution – Guided by our
cellular binding assays we designed a series of
peptides corresponding to the proline-rich region
of AIP4 shown to bind the βPIX–SH3 domain.
We synthesized two WT peptides AIP4(206-229) and
AIP4(211-226) that contain the proline-rich stretch,
but vary in the number of residues flanking these
regions, and three peptides AIP4(201-216), AIP4(216232) and AIP4(216-226) that contain only the N- or Cterminal PxxxPR motif (Figure 2B). Our ITC
studies reveal that both peptides AIP4(206-229) and
AIP4(211-226) bind to the SH3 domain of βPIX with
high affinity, 7.38 ± 0.9 µM and 10.1 ± 0.6 µM,
respectively (Figure 2C). The C-terminal portion
of the sequence is required in the binding
interaction since peptide AIP4(216-232) bound to the
SH3 domain with a Kd of 72.2 ± 5.0 µM, while
peptide AIP4(201-216) did not show any detectable
Accession codes – Structural coordinates and
structure factors have been deposited in the PDB
and have been assigned the ID code 2P4R.
RESULTS
The proline-rich region of AIP4 binds to the SH3
domain of βPIX – AIP4 regulates the stability and
degradation of p73 and p63 oncogenic
transcription factors and both AIP4 and βPIX have
been shown independently to be involved in breast
cancer metastasis (15-18,26,37,38). With this in
mind we first assayed for the endogenous
association of βPIX and AIP4 in two breast cancer
cell lines as well as a fibroblast line also known to
express both proteins (11,13).
Our coimmunoprecipitation experiments reveal that
endogenous βPIX forms a stable complex with
endogenous AIP4 in MDA-MB-231 and NIH3T3
cell lines (Figure 1A). Interestingly, we could not
detect an endogenous interaction between these
proteins in the MCF7 line (Figure 1A).
In order to test whether AIP4 and βPIX
interact directly to form a complex, we conducted
a series of pull-down and co-immunoprecipitation
experiments using affinity-tagged constructs. We
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Drops containing 1 µl of the complex were mixed
with 1 µl of the well solution (0.1 M Tris-HCl, pH
7.1-7.9, 0.2 M ammonium sulfate, 32-38% (w/v)
PEG-MME 5000). The crystals were allowed to
grow at 20ºC and appeared as long rectangular
rods, with typical dimensions of 40 µm x 30 µm x
600 µm. The crystals were snap-frozen in a stream
of liquid nitrogen in the mother liquor
supplemented by 10% glycerol. Data was
collected using an Raxis IV (Rigaku) with 1o
oscillations. Data was integrated and scaled using
the program HKL2000 (32). The phases were
determined by molecular replacement, using the
model from 2AK5 from the Protein Data Bank and
the program Phaser (33).
The model was
manually modified with the correct peptide
residues with iterative building and refinement
using the programs COOT, REFMAC5 and
ARP/warp (34). Superposition of the Cα positions
were calculated using Lsqman (35). Electrostatic
surfaces were calculated using Grasp (36). All
molecular graphics in the figures were made using
the program PyMOL (http://www.pymol.org).
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fluorescence emission λmax generates an EC50 of
6.3 µM, which is similar to the binding affinity
observed for the same AIP4(206-229) peptide in our
ITC studies (Figure 2E). Fitting the increase in
fluorescence emission intensity also yields similar
values, (data not shown). We used this assay to
test the preferential binding specificity for the
βPIX–SH3 domain and find that the binding of the
AIP4(206-229) peptide can be displaced by a PAK
derived peptide (Figure 2G). Taken together, the
observations that the emission spectra for the
βPIX–SH3 domain differ significantly for the
binding of AIP4(206-229) than either the PAK or Cblb derived peptides suggests that these peptides
may be interacting differently with the SH3
domain, possibly exhibiting alternate modes of
binding.
We decided to pursue the crystal
structure of AIP4/βPIX–SH3 to better understand
the molecular basis for these differences.
Structure determination – Purified
recombinant βPIX–SH3 was mixed with AIP4(206229) and crystallized using PEGMME 5000 by the
vapor diffusion method. The phases were solved
by molecular replacement. The crystals were of
space group P6122, with one SH3 domain per
asymmetric unit. The final model contained all of
the residues of βPIX–SH3; there was an additional
residue at the N-terminus arising from the remnant
of the GST fusion after cleavage by PreScission
protease.
In addition, the model contained
residues 209-224 of the AIP4 synthetic peptide;
the C-terminal residue was modeled as Ala where
density supporting the side chain was missing.
There was one sulfate molecule and one glycerol
molecule, as well as 69 solvent molecules in the
model after multiple rounds of refinement to a
final resolution of 2.0 Å (Rfree = 23.5%).
Refinement statistics are presented in Table 2.
Overall structure of βPIX–SH3/AIP4
peptide complex – The structure of βPIX–SH3
with the AIP4 peptide showed a 1:1 complex of
peptide ligand to protein. The structure of the SH3
domain itself is mainly composed of a 5-stranded
antiparallel β-sheet consistent with previous
reports. The root mean squared deviation of the
Cα positions compared with previous βPIX–SH3
structures is ~0.55 Å. The model clearly shows the
core residues that constitute the binding site in
AIP4 are residues 217-223 (Figure 3A), which are
in a left-handed PPII helix configuration in the
binding (Figure 2C). The significant decrease in
affinity observed between peptide AIP4(216-232)
relative to AIP4(206-229) and AIP4(211-226) further
indicates that residues adjacent to the C-terminal
proline-rich region are necessary to impart high
affinity binding.
Importantly, a binding
stoichiometry for AIP4 peptide to βPIX–SH3
domain of 1:2 is consistently observed for all of
these peptides (Table 1). This suggests that
binding the proline-rich region of AIP4 by βPIX
may induce the formation of heterotrimeric
complexes in solution, similar to what has been
previously reported for CIN85–SH3/Cbl-b and
βPIX–SH3/Cbl-b (28).
Alternate mode of binding for the
AIP4/βPIX–SH3 complex in solution – We next
investigated the role that residue R217 of AIP4
plays in formation of βPIX–SH3/AIP4
heterotrimeric complexes. The analogous Arg in
Cbl-b (R904) is necessary for the formation of
heterotrimeric complexes of βPIX–SH3/Cbl-b
(28). We find that the R217A mutant of AIP4
associates with βPIX when each are co-expressed
in HEK 293T cells and subjected to
immunoprecipitation (Figure 2A). As expected,
our ITC tests on an AIP4-R217A peptide indicate
that this mutation reduces the affinity of the βPIX–
SH3/AIP4 interaction with an apparent Kd of 43.4
± 3.5 µM (Figure 2C). To our surprise, this
mutation did not alter the binding stoichiometry
for the βPIXSH3/AIP4 complex, n = 0.452 ± 0.04,
in contrast with the effect of an analogous Arg
mutation on the stoichiometry observed for both
the CIN85/Cbl-b and βPIX/Cbl-b complexes (28).
The observation that the AIP4-R217A
mutation does not alter the binding stoichiometry
to a 1:1 complex suggests that AIP4 may bind
differently to the βPIX–SH3 domain than either
PAK or Cbl proteins. To test this hypothesis we
employed a fluorescence assay that monitors
βPIX–SH3 domain intrinsic Trp fluorescence
emission during ligand binding. Similar to our
previously reported results for PAK peptides (31),
titration of βPIX–SH3 with increasing amounts of
peptide AIP4(206-229) results in a blue-shift in the
fluorescence emission λmax (Figure 2D and 2E).
However, unlike the binding of PAK or Cbl-b
peptides, binding of AIP4(206-229) also results in a
large increase in fluorescence emission intensity
(Figure 2F). This effect is titratable and fitting the
packing site of this PPII helix and would be
uniquely favored for binding (41). Furthermore,
two potential hydrogen bonds, between the
backbone carbonyl atom of P212 of AIP4 and
W54 of βPIX–SH3, and between the guanido
group of R211 of AIP4 and the carboxylate group
of E45 of βPIX-SH3, may help stabilize the ligand
interactions. The total buried surface area of 1042
Å2 is ~30% larger than is typical for other
ligand/SH3 complexes (28). The extent of the
interactions between the N-terminal PPII helix of
AIP4 and βPIX–SH3 may contribute to the higher
affinity observed for peptides AIP4(206-229) and
AIP4(211-226), in the ITC experiments (Figure 2B
and 2C).
Comparison to βPIX-SH3/Cbl-b and
βPIX-SH3/PAK2 complex structures – The
structure of βPIX-SH3 has previously been
determined in a complex with two other peptide
ligands; however, unlike AIP4, neither ligand
contained a canonical PxxP motif, resulting in a
kink in the PPII helix for these ligands. The
structure of βPIX–SH3 with a peptide derived
from Cbl-b is a heterotrimeric complex formed
like a sandwich with the peptide in-between two
βPIX–SH3 domains in opposite orientations (28).
We shall limit our comparison to the Class I
configuration. The Cbl-b peptide does not contain
a PxxP motif, and one of the external packing
sites, P0, has a Lys residue instead of Pro (Figure
4A and 4C). Consequently, the PPII secondary
structure, which ideally contains φ and ψ angles of
-78º and 149º respectively, is perturbed at this
position resulting in the Cα and Cβ atoms of this
Lys residue occupying positions similar to the
backbone nitrogen and Cα of the proline found in
the AIP4 complex (Figure 4C) (42). A further
difference is found at the specificity pocket P-3
site, which for Cbl-b is occupied by R904 on
another face of a local surface protrusion, partly to
accommodate the positive charge of the lysine at
P0 (Figure 4A).
In comparing the present structure to that
of the βPIX-SH3/PAK2 complex, the PAK2
peptide was in the opposite (Class II) orientation
in the binding groove (Figure 4B and 4C) (31). In
this orientation, the external packing sites are at P-1
and P2 (41). Like the Cbl-b b motif, PAK2 lacks
the expected PxxP motif, with an Ala in the P-1
position instead of Pro. In spite of this, PAK2 was
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binding groove of βPIX–SH3 in a Class I
orientation.
The binding surface of SH3 domains is
characterized by several shallow pockets, which
have been referred to as P-3, P-1, P0, P+2 and P+3
(40). SH3 ligands adopt the secondary structure of
a PPII helix, which has a triangular cross-section
arising from the perfect helical repeat every three
residues: residues along two of the edges interact
with the surface of SH3 domains. Non-proline side
chains on the first interacting edge point away
from the surface, resulting in a poorer fit to the
SH3 domain surface and are referred to as external
packing sites: the unique substitution of the
backbone nitrogen with the delta carbon favors
Pro at these positions (41). Residues on the
second edge point toward the surface so many
residues can fit into the shallow pockets of the
SH3 domain, and positions on the second edge are
referred to as internal packing sites. The third
edge points away from the SH3 domain and thus
can normally accommodate any residue, although
Pro residues are often found at these positions,
likely stabilizing the PPII secondary structure.
Thus the characteristic PxxP motif of SH3 ligands
arises from the presence of Pro residues at two
subsequent external packing sites along the PPII
helix. The P0 and P+3 positions of Class I ligands
are external packing sites and strongly favor Pro
residues (41) as shown in our model of AIP4
bound to βPIX–SH3 (Figure 3A and 4C). In
addition there are three hydrogen bonds
characteristic of SH3–ligand interactions observed
between βPIX–SH3 side chains and three
backbone carbonyl atoms from AIP4: at positions
P1 with Y59, at P-3 with W43, and a watermediated bond at P0 with N58 (Figure 3A and D).
The B factor of that water molecule is 11.4,
comparable to the main chain atoms of βPIX–
SH3. An Arg residue (R217) interacts with a
negatively charged “specificity pocket” at P-3
(Figure 3B and 3D).
The N-terminal region of the AIP4 peptide
contains a second PPII helix encompassing
residues 209-215 immediately adjacent to core
SH3 ligand (Figure 3A). The two helices are
joined by a turn of 107º through S216. One
anchor of this second PPII helix is P215 of AIP4,
which binds a hydrophobic cleft between W43 and
W54 of βPIX–SH3. P215 is on an external
found to have an affinity to βPIX–SH3 similar to
that of AIP4 (31). Once again there is a
perturbation of the PPII helix at P0 that appears to
allow the isoleucine residue at that position to
interact more fully with the surface of βPIX–SH3
(Figure 4B and 4C). In this case the high affinity
may also be attributed to interactions of an
additional 11 residues of PAK2 that appear to be
loosely coiled on the adjacent surface of βPIX–
SH3 (31).
DISCUSSION
A growing body of evidence indicates that
signaling cascades initiated by either GPCRs or
RTKs are not mutually exclusive of one another.
Cross-talk between receptor classes via
downstream adaptors and effectors allows the cell
to fine-tune and integrate signals (43). Modular
protein domains mediate transient protein-protein
interactions that are crucial to relaying and
regulating signaling cascades (44). The βPIX–
SH3 domain exemplifies a protein module at the
nexus of many cellular processes. These include
the downregulation of EGFR through binding and
sequestering Cbl as well as regulation of GPCRs
through formation of βPIX–SH3/PAK complexes
during leukocyte chemotaxis in response to
CXCR4 activation (27). In the present study, we
have identified the E3 ubiquitin ligase AIP4 as a
binding partner of βPIX. As AIP4 regulates
CXCR4 signaling and βPIX is involved in EGFR
down-regulation, their association represents a
potential new regulatory node for GPCR and RTK
signal integration.
Our cellular binding assays demonstrate
that βPIX can indeed interact with AIP4 and
further show that this interaction is mediated by
binding of the βPIX–SH3 domain to a proline-rich
stretch of AIP4.
Interestingly, while this
interaction is readily detectable in MDA-MB-231
and NIH3T3 cells, it was undetectable in MCF7
cells under our assayed conditions. This raises the
possibility that the localization and regulation of
AIP4 and βPIX might be cell-type specific as has
been shown for PAK and PIX in different breast
cancer cell lines (38). We found that AIP4 could
also interact with CIN85 and that this interaction is
abrogated
upon
increasing
the
cellular
concentration of βPIX. While we attribute this
7
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interaction to CIN85 SH3 domains, we cannot rule
out the possibility that other parts of the proteins
are involved in recognition and binding. Thus the
βPIX–SH3 serves as a scaffold for numerous
signal transduction proteins including PAK, Cbl,
Rac, SAP and as the present report now shows,
AIP4.
How ligands may compete among
themselves for the same SH3 domain as well as
how all of these processes are regulated in the
context of the full-length proteins merits further
investigation.
Our ITC experiments demonstrate that
AIP4 residues 206–229, which encompass the
proline-rich region, bind to βPIX with micromolar
affinity and furthermore form heterotrimeric
complexes, with one AIP4 derived peptide binding
to two βPIX–SH3 domains. These experiments
also reveal that residues outside the core PxxP
motif are important for high affinity, and the
crystal structure of the complex shows that an
additional PPII helix outside the core wrapped
around another face of βPIX–SH3.
This
additional PPII helix is a novel feature for an SH3
ligand and to our knowledge has never been
described by any of the SH3/ligand complex
structures in the Protein Data Bank. Previous
studies on βPIX–SH3/Cbl-b complexes showed
that mutation of an Arg residue, (analogous to
R217 on AIP4), reduced the complex to a
monomeric interaction with a dramatically lower
affinity. In contrast, our ITC results indicate that
the AIP4-R217A mutant peptide does not alter
complex stoichiometry.
We speculate that
additional interactions provided by the secondary
PPII helix (including specific hydrophobic as well
as salt-bridge interactions) with βPIX-SH3 are
sufficient to overcome this substitution.
Although we have no direct structural
evidence for the configuration of a heterotrimeric
complex, we would propose that it might be
similar to that observed for the Cbl-b/βPIX–SH3
complex (28). In the case of Cbl-b, the peptide is
the center of pseudosymmetry with the matching
βPIX–SH3 laying on top of the Class I complex in
the opposite orientation. Thus the same peptide
becomes a Class II oriented ligand with respect to
the pseudosymmetry mate.
The interactions
between the two SH3 domains in that case were
minimal. In our case, R224 hypothetically would
occupy the P-3 specificity pocket of another βPIX–
8
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CXCR4 signaling is attenuated in part by
AIP4, which mediates the ubiquitination and
lysosomal sorting of the activated receptor (6).
Notably, AIP4 itself contains four modular WW
domains, and these domains often serve as
platforms for assembly of multi protein networks
(45). AIP4 is capable of binding Cbl family
members via these WW domains, and importantly,
this interaction facilitates AIP4 catalyzed
ubiquitination and proteasomal degradation of Cbl
(19,46). In doing so, AIP4 can impede Cbl
mediated ubiquitination of activated EGFRs (19).
In addition, it is also possible that AIP4 may, like
Cbl, directly ubiquitinate βPIX or exert a
cooperative effect on βPIX ubiquitination by
regulating Cbl ligase activity. Indeed, others have
shown that E3 ligases of the RING and HECT
families may interact cooperatively with a shared
common substrate to regulate signaling cascades
(47,48). This process is exemplified by regulation
of the NOTCH protein that can be ubiquitinated
and regulated by both AIP4 and Cbl (49,50).
The association of AIP4 with βPIX may
also regulate GPCR signaling.
Following
activation of C5a receptors, released Gβγ proteins
bind to PAK which in turn forms a complex with
αPIX that further initiates cascades resulting in
cellular chemotaxis (51). We speculate that a
similar process may occur following activation of
CXCR4, with AIP4 being displaced from βPIX by
Gβγ/PAK complexes. Liberated AIP4 could in
turn regulate CXCR4 ubiquitination and
endocytotic sorting. Our fluorescence experiments
demonstrate competition between PAK and AIP4derived peptides for the βPIX–SH3 domain and
would support such a mechanism; however,
further work is needed to examine this potential
regulatory loop. Finally, we note that PIX is a
binding partner for GITs (GPCR-kinase
interacting proteins).
GIT proteins serve as
signaling scaffolds and regulate numerous cellular
processes including cytoskeletal dynamics,
membrane trafficking and clathrin-dependent
endocytosis of GPCRs (52). GIT and PIX form
large multi-protein complexes within the cell, and
this raises the possibility that a GIT-PIX-AIP4
complex may transiently exist in the cell perhaps
regulating CXCR4 endocytosis and trafficking.
In conclusion, we have identified and
defined the binding interaction between the E3
SH3 molecule similarly arranged to SH3B of the
Cbl-b complex (28). As in the case the Cbl-b, one
of the pockets normally occupied by Pro would
instead accommodate another residue: for our
hypothetical heterotrimer, the P1 pocket would be
occupied by T222 (Figure 5) whereas P2 would be
occupied by Pro. In such an arrangement, Arg
residues would occupy both specificity pockets,
and Pro residues would occupy three of four Profavoring pockets. In examining the AIP4 and Cblb peptide sequence, we would propose a new
heterotrimeric motif for βPIX–SH3 and other
closely related SH3 domains such as those from
CIN85. Given the inherent symmetry of PPII
helices that allow for the Class I and II
orientations, what appears to be distinct about the
two peptides are the arginine residues anchoring
the ends: the motif could thus be considered R—
X—P—P/Z—X—P/Y—P—R.
In order to prove this model, future
experiments will be needed to further characterize
the complex of AIP4 peptide and βPIX-SH3 and
to resolve the discrepancy in the stoichiometry of
the complex between the crystal structure and that
which is consistent with the ITC results. One
possible reason for this discrepancy might be due
to the inherent asymmetry of the proposed
complex. The proposed partner βPIX-SH3 domain
of the heterotrimer would be limited to
interactions with the core ligand whereas the
βPIX-SH3 domain observed in the crystal
structure has additional contacts with the Nterminal PPII helix resulting in higher affinity to
the peptide. Under our crystallization conditions
with excess AIP4 peptide, it may be that the
observed monomeric complex formed to a
significant degree, and that due to favorable
crystal packing interactions (Figure 3C) led to the
formation of a lattice of the monomeric complex
to the exclusion of the heterotrimeric complex.
We aim to carry out further studies, including
other biophysical methods to further characterize
the complex of βPIX-SH3/AIP4 peptide, as well
as crystallization trials under different conditions
and with different AIP4 derived peptides. The full
molecular understanding of how βPIX andAIP4
interact to influence their respective pathways will
require eventual structural studies of the fulllength proteins.
signal integration. Future studies will aim to
elucidate the role this interaction plays in receptor
downregulation and cancer tumor progression and
metastasis.
ubiquitin ligase AIP4 and the signaling scaffold
protein βPIX. Given the extensive involvement of
these proteins in GPCR and RTK signaling, their
interaction represents a new node in GPCR/RTK
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FOOTNOTES
FIGURE LEGENDS
Figure 1. The proline-rich region of AIP4 binds preferentially to the SH3 domain of βPIX. (A)
Interaction of endogenous AIP4 and βPIX. βPIX was immunoprecipitated (IP) from the indicated cell
lines with antibody specific for βPIX and subjected to SDS-PAGE followed by immunoblot (IB) analysis
using antibody specific for Itch/AIP4 or βPIX (upper panels). Total cell lysate (TCL) was subjected to
direct IB analysis to check for expression levels and control for gel loading (lower panels). (B) GST pulldown assay. Lysates from HEK293T cells expressing either myc-AIP4 or a control vector were subjected
to GST pull-down with the SH3 domain of βPIX or GST alone and the filter was blotted with anti-myc to
probe for AIP4 binding. The relative levels of GST and GST-fusion protein are revealed following
Coomasie staining of the same filter. The expression levels of AIP4 in the TCL are shown in the lower
panel. (C) The proline-rich region of AIP4 is required to co-immunoprecipitate βPIX in HEK293T cells.
(Top) Schematic domain representations of the C-terminally HA-tagged βPIX, βPIX–SH3 GST fusion
and N-terminally myc-tagged AIP4 constructs used in the pull-down and co-immunoprecipitation assays.
For βPIX, the SH3 domain, Dbl-homology domain (DH), pleckstrin-homology domain (PH), G proteincoupled receptor kinase interactor binding domain (GBD) and leucine zipper (LZ) region are shown. For
AIP4, the calcium binding C2 domain, the four WW domains and the ubiquitin ligase HECT domain are
shown. The deletion mutant myc-∆PolyPro-AIP4 lacks the proline-rich stretch of residues from 206 to
229. (Bottom) HEK 293T cells were transfected with HA-tagged βPIX and myc-tagged AIP4 or mutants
as indicated, and the cell lysates were immunoprecipitated with anti-myc antibody and subjected to SDSPAGE followed by IB analysis. The filter was probed with anti-HA or anti-myc antibodies to detect
βPIX and AIP4, respectively (upper panels). TCL was subjected to direct IB analysis to check for
expression levels and control for gel loading (lower panels). (D) AIP4 binds to CIN85 possibly in
competition with βPIX. HEK 293T cells were transfected with HA-tagged βPIX, myc-tagged AIP4 and
FLAG-tagged CIN85 as indicated and the cell lysates were immunoprecipitated with anti-FLAG antibody
and subjected to SDS-PAGE followed by IB analysis. The filter was probed with anti-myc or anti-FLAG
antibodies to detect AIP4 and CIN85, respectively (upper panels). TCL was subjected to direct IB
analysis to check for expression levels and control for gel loading (lower panels). In cells transiently coexpressing AIP4 and CIN85, increasing amounts of βPIX expression abrogates the ability of CIN85 to coimmunoprecipitate AIP4.
11
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The authors wish to thank Wayne A. Hendrickson for his generous support and valuable discussions, and
for use of the X-ray facility at Columbia University. We thank Joseph P. Lidestri for help in the
maintenance of the X-ray facility and with data collection. We also thank Christoph Seibert, Pallavi
Sachdev, Paul Lee, and members of the Sakmar and Hendrickson labs for insightful discussions. We
gratefully acknowledge Dr. Sachiko Kajigaya, Dr. Bruce Mayer, Dr. Tony Pawson and Dr. André Hoelz
for providing plamids. J.M.J. was supported in part by NIH grant T3ZEY007138, K.C.M. was supported
in part by NIH grant K08-EY015540, T.P.S. was formerly an Ellison Medical Foundation Senior Scholar.
Additional funding was provided by the Allene Reuss Memorial Trust and the Howard Hughes Medical
Institute.
Figure 3. AIP4 binds to βPIX–SH3 as a Class I ligand. (A) AIP4 peptide binds to a largely
hydrophobic surface of βPIX between the RT (depicted in magenta) and n-src (depicted in cyan) loops.
In the left-hand view the SH3 ligand-binding surface is seen from above with the peptide lying on the
surface. In the right-hand view, the model has been rotated at on oblique angle to give a better view of
the second PPII helix. Electron density for the peptide was seen for residues 209 to 224, although an Ala
was placed for the C-terminal Arg, as the side chain density was not observed. There are two left-handed
PPII helices, from residues 209-215 and from 217-223. The second helix represents the core SH3 ligand
with R217 occupying the P-3 specificity pocket, and P220 and P223 occupying the obligate Pro-preferring
P0 and P3 pockets. A better view of the first PPII helix is seen in the right-hand view. One Arg residue,
R214, lies near a negatively charged surface, R211 appears to form a salt bridge with βPIX–SH3, and
again two Pro residues, P212 and P215, are buried in shallow pockets on this surface of βPIX–SH3. In
the asymmetric unit the N-terminus of the peptide appears to point into empty space, but panel D shows
that this end of the peptide interacts closely with a symmetry mate. The surface is colored to show the
electrostatic potential as calculated in GRASP, with positive charge represented in blue and negative
charge shaded in red. Underlying the surface is a cartoon representation of the SH3 backbone. (B) A
representative section of the calculated electron 2fo-fc density map is shown at 1.2 σ in a stereo view.
AIP4 peptide residues are represented with yellow carbons while those from βPIX–SH3 are shown in
green. R217 of AIP4 is shown in this view in close proximity to E24 of βPIX–SH3. (C) The N-terminal
region of the AIP4 peptide is shown in grey in this view with the symmetry mates as packed in the
crystal. The corresponding βPIX–SH3 is shown as molecular surface also in grey with the protein atoms
shown also in stick representation. The various symmetry mates are colored so that the corresponding
peptide carbon atoms correspond to the color of the molecular surface. Several of the crystal contacts
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Figure 2. The βPIX–SH3/AIP4 complex is trimeric in solution. (A) The AIP4-R217A mutant
immunoprecipitates βPIX in HEK293T cells. HEK 293T cells were transfected with HA-tagged βPIX,
and myc-tagged AIP4 or AIP4-R217A as indicated and the cell lysates were immunoprecipitated with
anti-myc antibody and subjected to SDS-PAGE followed by IB analysis with anti-HA to probe for βPIX
binding as well as anti-myc (upper panels). TCL was subjected to direct IB analysis to check for
expression levels and control for gel loading (lower panels). (B) AIP4 derived peptides used in ITC
binding studies. Note that a Tyr residue has been added to the N-terminus of the WT and R217A peptides
to aid in concentration determination. The symbols preceding each peptide name are the same as those
used on the ITC graph and amino acid numbers are given as subscripts. (C) ITC analysis of the prolinerich region of AIP4 binding to the βPIX–SH3 domain. (Upper left panel) Buffer subtracted ITC data for
binding of the WT AIP4(206-229) peptide to the βPIX–SH3 domain. (Lower left panel) Representative fitted
binding isotherms of AIP4 derived peptides, colors and symbols correspond to peptides depicted in (B).
(Upper right panel) Buffer subtracted raw ITC data for the binding of the AIP4-R217A peptide to the
βPIX–SH3 domain. (Lower right panel) Representative fitted binding isotherms for the AIP4-R217A
peptide (blue) in comparison with the peptide AIP4(206-229) (grey), illustrating that the R217A mutation
decreases the binding affinity for βPIX but does not alter the binding stoichiometry. All thermodynamic
binding parameters are compiled in Table I. (D) The titration of 1 µM βPIX–SH3 domain (black) with
increasing amounts of peptide AIP4(206-229) results in an increase in fluorescence emission intensity and a
blue-shift in emission λmax. (E) Sigmoidal dose-response fit for the emission data from (A), EC50 = 6.3
µM. The color of the individual data points corresponds to the spectra depicted in (A). (F) The binding
of AIP4(206-229) (red), but not that of PAK (blue) or Cbl-b (green) derived peptides results in a large
increase in βPIX–SH3 domain intrinsic tryptophan fluorescence. (G) The binding of peptide AIP4(206-229)
to the βPIX–SH3 domain can be competed off by a PAK derived peptide, PAK2(176-199). Steady-state
emission spectra of 1 µM βPIX–SH3 domain alone (black) and in the presence of either 10 µM AIP4(206229), (red) or 10 µM of PAK2(176-199), (blue) and in the presence of both 10 µM AIP4(206-229) and 100 µM
PAK2(176-199), (orange).
between βPIX–SH3 domains appear to be mediated by electrostatic interactions, whereas this region of
AIP4 peptide appears to lie across a hydrophobic surface of a βPIX–SH3 symmetry mate. (D) A
schematic representation of ligand binding as generated by LIGPLOT. Hydrogen bonds with three AIP4
backbone carbonyl atoms, one of them water-mediated, are shown in the core SH3 ligand (residues 217223). In addition, the second PPII helix also has one hydrogen bond with a backbone carbonyl and R211
forms a charge-stabilized hydrogen bond with βPIX–SH3. The peptide is shown with bonds in purple.
βPIX–SH3 residues which form hydrogen bonds with the peptide are shown with yellow bonds, whereas
Van der Waals interactions are depicted by half circles. There is one water-mediated hydrogen bond
shown with a cyan sphere representing the water molecule.
Figure 5. Proposed schematic arrangement of dimer interface of βPIX with AIP4 peptide. An
alignment of the core binding regions of the AIP4 and Cbl-b peptides is shown relative to the shallow
hydrophobic binding pockets as sites P-3 to P+3 as defined previously in two orientations. Below the
peptide sequences are the positions of the residues with respect to the structure reported in this paper for
the AIP4/βPIX complex and for the Class I orientation for the Cbl-b/βPIX complex. Above the peptide
sequences are the same positions in the Class II orientation as described in the Cbl-b/βPIX complex, and
the proposed Class II orientation of an additional βPIX–SH3 domain based on the ITC data and an
analysis of the sequence for AIP4. The proposed interaction of AIP4 in a class II orientation would result
in T222 occupying the P-1 site, which normally favors the presence of a Pro residue. The core sequences
are residues 217-224 for AIP4 and 904-911 for Cbl-b, respectively. Underneath each residue are the
respective φ/ψ angles which show how the PPII helix in the case of Cbl-b and PAK2 are distorted. The
pockets which favor the presence of Pro residues are indicated by grey squares drawn around the residues.
13
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Figure 4. The canonical binding of AIP4 to βPIX compared to PAK2 and Cbl-b. (A) The binding of
Cbl-b peptide (in magenta) in the Class I orientation is shown in comparison to AIP4 peptide (in yellow)
after a least squares fit of βPIX–SH3 domains from the two models. Residues from βPIX that appear to
form hydrogen bonds are shown under the molecular surface. The alignment of the respective core
binding sequences from the peptides is shown in panel C. A Pro residue usually occupies the P0 and P3
positions in canonical Class I SH3 ligands. There is a distortion in the PPII helix at the K907 of Cbl-b,
shown in the center of the figure, resulting in a displacement of the backbone so that the Cα and Cβ
atoms of that residue occupy similar positions to the backbone N and Cδ positions of P220 of AIP4. (B)
PAK2 peptide (in cyan) binds to βPIX in the opposite Class II orientation, but again contains a
perturbation from a PPII helix in a similar position to the Cbl-b peptide. Unlike typical Class II ligands,
the PAK2 peptide contains an Ala residue in the P-1 position. In contrast to the Cbl-b peptide, this
perturbation does not result in a residue making up for the lack of a Pro, but rather appears to allow
extensive contacts between I183 and the surface of βPIX. The high affinity observed for PAK2 by ITC
may be due to the residues in the peptide lying outside of the SH3 ligand-binding surface. (C) An
alignment of the core binding regions of the three peptides is shown relative to the shallow hydrophobic
binding pockets as sites P-3 to P+3 as defined previously. The core sequences are residues 217-223, 904910, 180-186 for AIP4, Cbl-b, and PAK2, respectively. The sequences are color coded to match panels A
and B, with AIP4 in yellow, Cbl-b in magenta, and PAK2 in cyan. Underneath each residue are the
respective φ/ψ angles, with numbers in red where the PPII helix in the case of Cbl-b and PAK2 are
distorted. The pockets, which favor the presence of Pro residues, are indicated by grey squares drawn
around the residues.
Table 1. Thermodynamic parameters for βPIX–SH3 domain binding AIP4 peptidesa
∆Ha (kcal/mol)
∆Sa(cal/mol/deg)
Stoichiometry
AIP4(206-229)
7.38 ± 0.9
-21.2 ± 0.2
-48.9 ± 0.3
0.442 ± 0.07
AIP4(211-226)
10.1 ± 0.6
-14.2 ± 0.6
-25.6 ± 1.1
0.601 ± 0.08
AIP4(201-216)
No Bindingb
-
-
-
AIP4(216-232)
72.2 ± 5.0
-18.9 ± 0.4
-45.5 ± 0.5
0.536 ± 0.05
AIP4(216-226)
64.5 ± 4.2
-16.4 ± 0.4
-34.1 ± 0.4
0.508 ± 0.05
AIP4-R217A(206-229)
43.4 ± 3.5
-16.5 ± 2.0
-36.1 ± 2.2
0.452 ± 0.04
a
Each peptide was titrated at least three times and the average and standard error are reported.
b
Peptide AIP4(201-216) did not display a significant change in heat capacity when titrated, and did not
appear to bind using fluorescence emission shift assays (data not shown).
14
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Kd (µM)
Peptide
Table 2. Crystallographic data
Data Collection
Space group
P61 2 2
Unit Cell Parameters
a=b=41.75 Å, c=149.3 Å
α=β=90º, γ=120º
36.15-2.01 (2.05-2.01)
Total observations
78,603
Unique observations
5612
Completenessa,%
96.3 (94.2)
<I/σ(I)>a
36.7 (17.2)
Rsyma,%
8.3 (18.7)
Refinement Data Statistics
(20.87-2.001Å)
Work/Test
5,349
Completenessa
97.3 (94.4)
Atoms, total
670
Protein
470
Ligand
120
Sulfate Ion
5
Glycerol
6
Water
69
R/Rfree,%
21.7
Mean B Value
15.5
r.m.s.d. bond length
0.006
r.m.s.d. bonds angles
1.026
a
254
24.6
Numbers in parentheses refer to the values for the highest resolution shell.
15
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Bragg Spacing (Å)
Figure 1
B
GSTβPIX-SH3
GST
Ve
ct
or
AI
P4
Ve
ct
or
AI
P4
MD
AM
MC B-2
31
F7
NI
H3
T3
A
115
115
IB: anti-myc
IB: anti-Itch/AIP4
GST-βPIX-SH3
IB: anti-βPIX
82
Coomasie
GST
IP: βPIX
115
IB: anti-βPIX
82
TCL
βPIX-HA:
HA
GST-βPIX-SH3:
myc-AIP4:
myc
myc-∆PolyPro-AIP4:
myc
TCL
D
Vector
myc-AIP4
CIN85-FLAG
βPIX-HA
+
-
+
-
+
+
-
+
+
+
+
-
+
+
+
+
115
∆GGFKPSRPPRPSRPPRPSRPPPPTPRRPASV
myc-AIP4-Nterm:
IB: anti-myc
+
+
IB: anti-myc
IB: anti-FLAG
82
myc
IP: FLAG (CIN85)
Vector
βPIX-HA
myc-AIP4
myc-AIP4-Nterm
myc-∆PolyPro-AIP4
+ - - - - - - + + + +
- + - + - - - - - + - - - - - +
IB: anti-HA
82
115
IB: anti-myc
IP: myc (AIP4)
82
IB: anti-HA
60
IB: anti-Tubulin
TCL
IB: anti-myc
115
82
IB: anti-FLAG
82
IB: anti-HA
60
IB: anti-Tubulin
TCL
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C
GST Pull-down
IB: anti-Itch/AIP4
115
Figure 2
A
Vector
myc-AIP4
myc-R217A
βPIX-HA
+
-
+
+
-
+
+
B
+
+
AIP4 Peptides:
115
Y206 GFKPSRPPRPSRPPPPTPRRPASV
AIP4 (206-229)
IB: anti-HA
82
RPPRPSRPPPPTPRRP
AIP4 (211-226)
AIP4 (201-216) SLSNGGFKPSRPPRPS
IB: anti-myc
SRPPPPTPRRPASVNGS
AIP4 (216-232)
IP: myc (AIP4)
AIP4 (216-226)
IB: anti-HA
82
115
SRPPPPTPRRP
Y206 GFKPSRPPRPSAPPPPTPRRPASV
AIP4-R217A
IB: anti-myc
60
IB: anti-Tubulin
TCL
C
0
50
Time (min)
100
150
200
0
50
Time (min)
100
150
200
0.0
-0.8
-1.2
-1.2
0
0
R217A
-4
-4
-8
WT
WT:
K d = 7.38 µM
n = 0.44
-12
-8
R217A:
K d = 43.4 µM
n = 0.45
-12
-16
-16
+ 10 µM AIP4
4
+ 10 µM Cbl-b
+ 10 µM PAK
2
βPIX SH3
44
0
42
0
40
0
38
0
36
0
32
0
34
0
0
Wavelength (nm)
G
335
9
7
8
6
5
4
Log [Peptide] (M)
6
3
+ 10 µM AIP4
+ 10 µM AIP4,
100 µM PAK
4
+ 10 µM PAK
2
βPIX SH3
0
44
0
6
340
0
Wavelength (nm)
345
42
0
44
0
42
0
38
0
40
0
36
0
32
0
34
0
0
2.0
350
0
2
1.5
40
4
1.0
Molar Ratio
38
6
E
0.5
36
0
8
0.0
2.0
34
0
1.5
32
0
1.0
Molar Ratio
Emission λ (nm)
0.5
Fluorescence
-5
Intensity (c.p.s.) X10
kcal/mole of injectant
Fluorescence
Intensity (c.p.s.) X10 -5
Fluorescence
Intensity (c.p.s.) X10-5
F
-0.4
-0.8
0.0
D
R217A
WT
-0.4
Wavelength (nm)
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µcal/sec
0.0
Figure 3
A
n-src Loop
P219
R214
R214
T222
P221
P218
n-src Loop
R211
P209
R224
S210
P213
R217 P220
P215
S216
S216 P219
P213
P212
P223
P218
P215
P221
R217
P220
R224
P223
P209
RT Loop
RT Loop
C
B
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D
Glu 45
Pro 56
Phe 15
Asn 58
Ala 224
Trp 54
Gly 42
2.67
Glu 24
Arg 211
Pro 223
3.00
3.04
Arg 214
Pro 209
Lys 208
Ser 210
2.74
2.45
Tyr 59
Thr 222
Ser 216
Arg 217
2.74
2.60
HOH
Pro 215
3.05
Pro 218
Pro 219
Pro 212
Ala 207
Ala 206
Pro 220
Pro 221
Asp 23
Pro 213
Trp 43
Figure 4
A
P220
K907
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B
P220
I183
C P P P P P P P
-3
-2
-1
0
1
2
3
N
R P P P P T P
C
R P P K P R P
C
R P A
N
-72/152 -68/152 -64/142 -72/151 -69/155 -66/145 -63/139
N
--/93.9 -63/150 -59/156 -92/125 -68/147 -76/157 -61/155
C
I
V P P
-57/122 -65/148 -65/151 -95/117 -73/130 -60/160 --/163
Figure 5
AIP4
N
Cbl-b
N
Class I
R P P P P T P R
C
R P P K P R P R
C
-72/152 -68/152 -64/142 -72/151 -69/155 -66/145 -63/139 -94/-
--/93.9 -63/150 -59/156 -92/125 -68/147 -76/157 -61/155 -87/-
P-3 P-2 P-1 P0 P1 P2 P3
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P3 P2 P1 P0 P-1 P-2 P-3
Class II