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Originally published In Press as doi:10.1074/jbc.M702678200 on July 25, 2007

J. Biol. Chem., Vol. 282, Issue 39, 28893-28903, September 28, 2007
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A Novel Interaction between Atrophin-interacting Protein 4 and beta-p21-activated Kinase-interactive Exchange Factor Is Mediated by an SH3 Domain*

Jay M. Janz{ddagger}1, Thomas P. Sakmar{ddagger}2, and K. Christopher Min§3

From the {ddagger}Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, New York, New York 10021 and the §Department of Neurology, Columbia University, New York, New York 10032

Received for publication, March 28, 2007 , and in revised form, June 29, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cross-talk between G protein-coupled receptors and receptor tyrosine kinase signaling pathways is crucial to the efficient relay and integration of cellular information. Here we identify and define the novel binding interaction of the E3 ubiquitin ligase atrophin-interacting protein 4 (AIP4) with the GTP exchange factor beta-p21-activated kinase-interactive exchange factor (betaPIX). We demonstrate that this interaction is mediated in part by the betaPIX-SH3 domain binding to a proline-rich stretch of AIP4. Analysis of the interaction by isothermal calorimetry is consistent with a heterotrimeric complex with one AIP4-derived peptide binding to two betaPIX-SH3 domains. We determined the crystal structure of the betaPIX-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 betaPIX-SH3 domain as a canonical Class I ligand with an additional type II polyproline helix that makes extensive contacts with another face of betaPIX. Taken together, the novel interaction between AIP4 and betaPIX represents a new regulatory node for G protein-coupled receptor and receptor tyrosine kinase signal integration. Our structure of the betaPIX-SH3·AIP4 complex provides important insight into the mechanistic basis for betaPIX scaffolding of signaling components, especially those involved in cross-talk.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Two prominent transmembrane receptor families facilitate cellular communication with the extracellular environment: G protein-coupled receptors (GPCRs)4 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 (3-5). Representative E3 ligases include atrophin-interacting protein 4 (AIP4), 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).

beta-PAK-interactive exchange factor (betaPIX also referred to as Cool-1) is composed 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, betaPIX acts as a 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 betaPIX in breast cancer pathogenesis as well as aspects of CXCR4-induced cellular chemotaxis (24-27).

Like other SH3 domains, betaPIX-SH3 binds to proline-rich sequences with a polyproline II helix (PPII) conformation. Although most SH3 domain ligands contain a PXXP motif, betaPIX-SH3 binds ligands with a non-canonical PXXXPR sequence and serves as a scaffolding point for a number of protein-protein interactions as demonstrated by the direct coupling of betaPIX to p21-activated kinase (PAK) family members (20). The betaPIX-SH3 domain also binds to the PXXXPR motif of Cbl family members to facilitate the clustering of proteins involved in the down-regulation of EGF receptor signaling (26, 28). Interestingly, the betaPIX-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 and the functional ramifications of competition among multiple ligands for overlapping binding sites on betaPIX remain to be elucidated.

Here we identify the E3 ubiquitin ligase AIP4 as a binding partner for the betaPIX scaffolding protein. We demonstrate an endogenous betaPIX·AIP4 complex in breast cancer cell lines and show that complex formation is mediated in part by betaPIX-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 betaPIX-SH3 domains. Fluorescence assays suggest that the mode of interaction for the betaPIX-SH3·AIP4 complex is unique compared with known betaPIX-SH3 interactions. We determined the crystal structure of the betaPIX-SH3·AIP4 complex to 2.0-Å resolution. AIP4 binds the betaPIX-SH3 domain as a canonical Class I ligand with an additional PPII helix that makes extensive contacts with another face of betaPIX. Our structure of the betaPIX-SH3·AIP4 complex provides important insight into the mechanistic basis for betaPIX scaffolding of signal components. The novel interaction between betaPIX and AIP4 represents a new regulatory node for GPCR and RTK signal integration.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and Antibodies—Except where noted, all buffers and chemicals were purchased from either Fisher or Sigma. Monoclonal anti-Myc and monoclonal anti-AIP4 were purchased from Santa Cruz Biotechnology. Polyclonal anti-betaPIX antibody was from Chemicon International. Polyclonal anti-HA antibody was purchased from Covance. Monoclonal anti-FLAG M2 antibody was purchased from Sigma. Monoclonal anti-Itch antibody was purchased from BD Biosciences. Goat anti-mouse IgG and goat anti-rabbit IgG, horseradish peroxidase-conjugated secondary antibodies were from Upstate%20Biotechnology">Upstate Biotechnology. SDF1{alpha} was purchased from R&D Systems, and EGF was purchased from Calbiochem.

Cell Culture, Plasmids, and Transfection—HEK293T, MCF7, MDA-MB-231, and NIH 3T3 cells were all cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The pcDNA3-FLAG-CIN85 mammalian expression construct was a kind gift from Dr. Sachiko Kajigaya (National Institutes of Health, Bethesda, MD). The pEBB-betaPIX-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, Ontario, Canada). The pGEX-6P1-betaPIX-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 (Invitrogen), and cells were assayed or harvested ~48 h after transfection. Harvested cells were either used immediately or snap frozen in liquid nitrogen and kept at -80 °C.

Immunoblotting, Co-immunoprecipitation, and Pulldown 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% Nonidet P-40 (v/v), 0.1% SDS (w/v)), plus protease inhibitor tablets (Roche Applied Science). Cell lysates were subsequently cleared by centrifugation, and the supernatant was incubated with the appropriate antibody at 4 °C for 1 h to overnight followed by incubation with protein A- or G-agarose for 2 h at 4 °C. Immunoprecipitates were then washed three to five 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 was used. This antibody readily detects AIP4, because Itch and AIP4 are highly homologous. In overexpression studies, all AIP4 constructs encode an N-terminal Myc tag, betaPIX constructs encode a C-terminal HA tag, and CIN85 constructs encode an N-terminal FLAG tag to aid in immunoblot detection.

For GST pulldown assays, lysates of transformed Escherichia coli BL21(DE3) cells expressing either GST-betaPIX-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 dithiothreitol, pH 7.3) for 1 h at 4 °C.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 pulled down, washed extensively, and resolved via SDS-PAGE. Proteins were subsequently transferred to nitrocellulose and probed for AIP4 binding using an anti-Myc antibody.

Protein Expression and Purification—The SH3 domain betaPIX 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 high-performance liquid chromatography, and verified by mass spectrometry. Peptides that correspond to regions of AIP4 were as follows: AIP4206-229, Y206GFKPSRPPRPSRPPPPTPRRPASV229; AIP4211-226, 211RPPRPSRPPPPTPRRP226; AIP4201-216, 201SLSNGGFKPSRPPRPS216; AIP4216-232, 216SRPPPPTPRRPAS-VNGS232; AIP4216-226, 216SRPPPPTPRRP226; and AIP4-R217A, 205YGFKPSRPPRPSAPPPPTPRRPASV229. The following peptide corresponds to the betaPIX-SH3 binding domain of human PAK2 (PAK2176-199,176EETAPPVIAPRPDHTKSIYTRSVI199) and that of human Cbl-b (Cbl-b899-914, 899SQAPARPPKPRPRRTA914Y). Note that a tyrosine residue was added on to the N terminus of peptides AIP4206-229 and AIP4-R217A or the C terminus of Cbl-b899-914 to aid in concentration determination. All protein and peptide concentrations were determined by UV spectroscopy and/or amino acid analysis.

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-x 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 betaPIX-SH3 domain samples with increasing amounts of WT AIP4206-229 peptide and monitoring both the shift in Trp emission wavelength and overall fluorescence intensity. Competition experiments were performed in a similar manner using the PAK2176-199 and Cbl-b899-914 peptides. The PAK2176-199 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 did not significantly increase the fluorescence emission intensity (data not shown). Data were analyzed in Sigma-Plot using nonlinear regression and fitted to a sigmoidal dose-response (variable slope) equation four-parameter logistic equation: Y = minimum + (maximum - minimum)/1 + 10(log EC50 - x)*Hill slope.

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 betaPIX-SH3 domain and was titrated at equal intervals with 10-µl aliquots of 500 µM AIP4 peptide solution for a total of ~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 version 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 dithiothreitol) was mixed at a 1:2 molar ratio with AIP4206-229 peptide dissolved in the same buffer and crystallized at 20 °C by the vapor diffusion method. 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) polyethylene glycol-MME 5000). The crystals were allowed to grow at 20 °C 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 were collected using a Raxis IV (Rigaku) with 1° oscillations. Data were 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). Super-position of the C{alpha} positions was calculated using Lsqman (35). Electrostatic surfaces were calculated using Grasp (36). All molecular graphics in the figures were made using the program PyMOL (DeLano Scientific, San Carlos, CA).

Accession Codes—Structural coordinates and structure factors have been deposited in the Protein Data Bank and have been assigned the ID code 2P4R.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The Proline-rich Region of AIP4 Binds to the SH3 Domain of betaPIX—AIP4 regulates the stability and degradation of p73 and p63 oncogenic transcription factors, and both AIP4 and betaPIX 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 betaPIX and AIP4 in two breast cancer cell lines as well as a fibroblast line also known to express both proteins (11, 13). Our co-immunoprecipitation experiments reveal that endogenous betaPIX forms a stable complex with endogenous AIP4 in MDA-MB-231 and NIH3T3 cell lines (Fig. 1A). Interestingly, we could not detect an endogenous interaction between these proteins in the MCF7 line (Fig. 1A).

To test whether AIP4 and betaPIX interact directly to form a complex, we conducted a series of pulldown and co-immunoprecipitation experiments using affinity-tagged constructs. We hypothesized that AIP4 interacts directly with betaPIX through binding of the proline-rich region of AIP4 to the SH3 domain of betaPIX. First we found that betaPIX-SH3 alone was sufficient to pull down AIP4 in a GST pulldown assay with HEK293T lysate-expressing Myc-tagged AIP4 (Fig. 1B). We further confirmed the importance of the proline-rich region of AIP4 using co-immunoprecipitation assays in HEK293T cells. HA-tagged betaPIX was co-expressed with full-length or truncated forms of Myc-tagged AIP4; immunoprecipitation was performed with anti-Myc antibody, and immunoblots were performed to test for the association of betaPIX. We found that deletion of the proline-rich region of AIP4 abolished the interaction with betaPIX in this assay (Fig. 1C).

AIP4 Binds to the CIN85 Scaffolding Protein—The betaPIX-SH3 domain is phylogenetically similar to the three SH3 domains of CIN85, an adaptor protein involved in Cbl-mediated down-regulation of RTKs (9, 26). Like betaPIX, the SH3 domains of CIN85 recognize and bind the non-canonical PXXXPR motifs in Cbl and PAK proteins (26, 28, 39). When both FLAG-tagged CIN85 and Myc-tagged AIP4 were co-expressed in HEK293T cells we found that CIN85 pulled down AIP4, albeit to a lesser extent than betaPIX (Fig. 1D). Notably, this interaction dissipated as cells co-expressed increasing amounts of betaPIX (Fig. 1D).

AIP4 Forms a Heterotrimeric Complex with betaPIX-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 betaPIX-SH3 domain. We synthesized two WT peptides AIP4206-229 and AIP4211-226 that contain the proline-rich stretch, but vary in the number of residues flanking these regions, and three peptides AIP4201-216, AIP4216-232, and AIP4216-226 that contain only the N- or C-terminal PXXXPR motif (Fig. 2B). Our ITC studies reveal that both peptides AIP4206-229 and AIP4211-226 bind to the SH3 domain of betaPIX with high affinity, 7.38 ± 0.9 µM and 10.1 ± 0.6 µM, respectively (Fig. 2C). The C-terminal portion of the sequence is required in the binding interaction, because peptide AIP4216-232 bound to the SH3 domain with a Kd of 72.2 ± 5.0 µM, whereas peptide AIP4201-216 did not show any detectable binding (Fig. 2C). The significant decrease in affinity observed between peptide AIP4216-232 relative to AIP4206-229 and AIP4211-226 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 betaPIX-SH3 domain of 1:2 was consistently observed for all of these peptides (Table 1). This suggests that binding the proline-rich region of AIP4 by betaPIX may induce the formation of heterotrimeric complexes in solution, similar to what has been previously reported for CIN85-SH3·Cbl-b and betaPIX-SH3·Cbl-b (28).


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  		TABLE 1
Thermodynamic parameters for betaPIX-SH3 domain binding AIP4 peptides

Each peptide was titrated at least three times, and the average and standard error are reported.

 


Figure 1
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  		FIGURE 1.
The proline-rich region of AIP4 binds preferentially to the SH3 domain of betaPIX. A, interaction of endogenous AIP4 and betaPIX. betaPIX was immunoprecipitated (IP) from the indicated cell lines with antibody specific for betaPIX and subjected to SDS-PAGE followed by immunoblot (IB) analysis using antibody specific for Itch/AIP4 or betaPIX (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 pulldown with the SH3 domain of betaPIX 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 Coomassie 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 betaPIX in HEK293T cells. Top, schematic domain representations of the C-terminally HA-tagged betaPIX, betaPIX-SH3 GST fusion, and N-terminally Myc-tagged AIP4 constructs used in the pulldown and co-immunoprecipitation assays. For betaPIX, the SH3 domain, Dbl-homology domain (DH), pleckstrin-homology domain (PH), G protein-coupled 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-{Delta}PolyPro-AIP4 lacks the proline-rich stretch of residues from 206 to 229. Bottom, HEK 293T cells were transfected with HA-tagged betaPIX and Myc-tagged AIP4 or mutants as indicated, and the cell lysates were immunoprecipitated with anti-Myc antibody and subjected to SDS-PAGE followed by IB analysis. The filter was probed with anti-HA or anti-Myc antibodies to detect betaPIX 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 betaPIX. HEK 293T cells were transfected with HA-tagged betaPIX, 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 co-expressing AIP4 and CIN85, increasing amounts of betaPIX expression abrogates the ability of CIN85 to co-immunoprecipitate AIP4.

 
Alternate Mode of Binding for the AIP4·betaPIX-SH3 Complex in Solution—We next investigated the role that residue Arg-217 of AIP4 plays in formation of betaPIX-SH3·AIP4 heterotrimeric complexes. The analogous Arg in Cbl-b (Arg-904) is necessary for the formation of heterotrimeric complexes of betaPIX-SH3·Cbl-b (28). We find that the R217A mutant of AIP4 associated with betaPIX when each were co-expressed in HEK 293T cells and subjected to immunoprecipitation (Fig. 2A). As expected, our ITC tests on an AIP4-R217A peptide indicated that this mutation reduced the affinity of the betaPIX-SH3·AIP4 interaction with an apparent Kd of 43.4 ± 3.5 µM (Fig. 2C). To our surprise, this mutation did not alter the binding stoichiometry for the betaPIX-SH3·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 betaPIX·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 betaPIX-SH3 domain than either PAK or Cbl proteins. To test this hypothesis we employed a fluorescence assay that monitors betaPIX-SH3 domain intrinsic Trp fluorescence emission during ligand binding. Similar to our previously reported results for PAK peptides (31), titration of betaPIX-SH3 with increasing amounts of peptide AIP4206-229 results in a blue shift in the fluorescence emission {lambda}max (Fig. 2, D and E). However, unlike the binding of PAK or Cbl-b peptides, binding of AIP4206-229 also resulted in a large increase in fluorescence emission intensity (Fig. 2F). This effect was titratable, and fitting the fluorescence emission {lambda}max generated an EC50 of 6.3 µM, which was similar to the binding affinity observed for the same AIP4206-229 peptide in our ITC studies (Fig. 2E). Fitting the increase in fluorescence emission intensity also yielded similar values (data not shown). We used this assay to test the preferential binding specificity for the betaPIX-SH3 domain and found that the binding of the AIP4206-229 peptide could be displaced by a PAK-derived peptide (Fig. 2G). Taken together, the observations that the emission spectra for the betaPIX-SH3 domain differ significantly for the binding of AIP4206-229 than either the PAK- or Cbl-b-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·betaPIX-SH3 to better understand the molecular basis for these differences.

Structure Determination—Purified recombinant betaPIX-SH3 was mixed with AIP4206-229 and crystallized using polyethylene glycol-MME 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 betaPIX-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.


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  		TABLE 2
Crystallographic data

 
Overall Structure of betaPIX-SH3·AIP4 Peptide Complex—The structure of betaPIX-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 five-stranded antiparallel beta-sheet consistent with previous reports. The root mean squared deviation of the C{alpha} positions compared with previous betaPIX-SH3 structures was ~0.55 Å. The model clearly showed the core residues that constitute the binding site in AIP4 are residues 217-223 (Fig. 3A), which are in a left-handed PPII helix configuration in the binding groove of betaPIX-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 betaPIX-SH3 (Figs. 3A and 4C). In addition there are three hydrogen bonds characteristic of SH3-ligand interactions observed between betaPIX-SH3 side chains and three backbone carbonyl atoms from AIP4: at positions P1 with Tyr-59, at P-3 with Trp-43, and a water-mediated bond at P0 with Asn-58 (Fig. 3, A and D). The B factor of that water molecule is 11.4, comparable to the main-chain atoms of betaPIX-SH3. An Arg residue (Arg-217) interacts with a negatively charged "specificity pocket" at P-3 (Fig. 3, B and D).


Figure 2
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  		FIGURE 2.
The betaPIX-SH3·AIP4 complex is trimeric in solution. A, the AIP4-R217A mutant immunoprecipitates betaPIX in HEK293T cells. HEK 293T cells were transfected with HA-tagged betaPIX, 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 betaPIX 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 proline-rich region of AIP4 binding to the betaPIX-SH3 domain. Upper left panel, buffer subtracted ITC data for binding of the WT AIP4206-229 peptide to the betaPIX-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 betaPIX-SH3 domain. Lower right panel, representative fitted binding isotherms for the AIP4-R217A peptide (blue) in comparison with the peptide AIP4206-229 (gray), illustrating that the R217A mutation decreases the binding affinity for betaPIX but does not alter the binding stoichiometry. All thermodynamic binding parameters are compiled in Table 1. D, the titration of 1 µM betaPIX-SH3 domain (black) with increasing amounts of peptide AIP4206-229 results in an increase in fluorescence emission intensity and a blue-shift in emission {lambda}max. 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 AIP4206-229 (red), but not that of PAK (blue)- or Cbl-b (green)-derived peptides results in a large increase in betaPIX-SH3 domain intrinsic tryptophan fluorescence. G, the binding of peptide AIP4206-229 to the betaPIX-SH3 domain can be competed off by a PAK-derived peptide, PAK2176-199. Steady-state emission spectra of 1 µM betaPIX-SH3 domain alone (black) and in the presence of either 10 µM AIP4206-229 (red) or 10 µM of PAK2176-199 (blue), and in the presence of both 10 µM AIP4206-229 and 100 µM PAK2176-199 (orange).

 
The N-terminal region of the AIP4 peptide contains a second PPII helix encompassing residues 209-215 immediately adjacent to core SH3 ligand (Fig. 3A). The two helices are joined by a turn of 107° through Ser-216. One anchor of this second PPII helix is Pro-215 of AIP4, which binds a hydrophobic cleft between Trp-43 and Trp-54 of betaPIX-SH3. Pro-215 is on an external 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 Pro-212 of AIP4 and Trp-54 of betaPIX-SH3, and between the guanido group of Arg-211 of AIP4 and the carboxylate group of Glu-45 of betaPIX-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 betaPIX-SH3 may contribute to the higher affinity observed for peptides AIP4206-229 and AIP4211-226, in the ITC experiments (Fig. 2, B and C).

Comparison with betaPIX-SH3·Cbl-b and betaPIX-SH3·PAK2 Complex Structures—The structure of betaPIX-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 betaPIX-SH3 with a peptide derived from Cbl-b is a heterotrimeric complex formed like a sandwich with the peptide in between two betaPIX-SH3 domains in opposite orientations (28). We shall limit our comparison to the Class I configuration. The Cbl-b peptide did not contain a PXXP motif, and one of the external packing sites, P0, had a Lys residue instead of Pro (Fig. 4, A and C). Consequently, the PPII secondary structure, which ideally contains {varphi} and {psi} angles of -78° and 149°, respectively, was perturbed at this position resulting in the C{alpha} and Cbeta atoms of this Lys residue occupying positions similar to the backbone nitrogen and C{alpha} of the proline found in the AIP4 complex (Fig. 4C) (42). A further difference was found at the specificity pocket P-3 site, which for Cbl-b is occupied by Arg-904 on another face of a local surface protrusion, partly to accommodate the positive charge of the lysine at P0 (Fig. 4A).

In comparing the present structure to that of the betaPIX-SH3·PAK2 complex, the PAK2 peptide was in the opposite (Class II) orientation in the binding groove (Fig. 4, B and C) (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. Despite this, PAK2 is found to have an affinity to betaPIX-SH3 similar to that of AIP4 (31). Once again there was a perturbation of the PPII helix at P0 that appeared to allow the isoleucine residue at that position to interact more fully with the surface of betaPIX-SH3 (Fig. 4, B and C). 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 betaPIX-SH3 (31).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
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 down-stream 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 betaPIX-SH3 domain exemplifies a protein module at the nexus of many cellular processes. These include the down-regulation of EGF receptor through binding and sequestering Cbl as well as regulation of GPCRs through formation of betaPIX-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 betaPIX. Because AIP4 regulates CXCR4 signaling and betaPIX is involved in EGF receptor down-regulation, their association represents a potential new regulatory node for GPCR and RTK signal integration.


Figure 3
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  		FIGURE 3.
AIP4 binds to betaPIX-SH3 as a Class I ligand. A, AIP4 peptide binds to a largely hydrophobic surface of betaPIX 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 209-224, although an Ala was placed for the C-terminal Arg, because 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 Arg-217 occupying the P-3 specificity pocket, and Pro-220 and Pro-223 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, Arg-214, lies near a negatively charged surface, Arg-211 appears to form a salt bridge with betaPIX-SH3, and again two Pro residues, Pro-212 and Pro-215, are buried in shallow pockets on this surface of betaPIX-SH3. In the asymmetric unit the N terminus of the peptide appears to point into empty space, but 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 schematic representation of the SH3 backbone. B, a representative section of the calculated electron 2fo - fc density map is shown at 1.2 {sigma} in a stereo view. AIP4 peptide residues are represented with yellow carbons, whereas those from betaPIX-SH3 are shown in green. Arg-217 of AIP4 is shown in this view in close proximity to Glu-24 of betaPIX-SH3. C, the N-terminal region of the AIP4 peptide is shown in gray in this view with the symmetry mates as packed in the crystal. The corresponding betaPIX-SH3 is shown as molecular surface also in gray 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 between betaPIX-SH3 domains appear to be mediated by electrostatic interactions, whereas this region of AIP4 peptide appears to lie across a hydrophobic surface of a betaPIX-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 217-223). In addition, the second PPII helix also has one hydrogen bond with a backbone carbonyl, and Arg-211 forms a charge-stabilized hydrogen bond with betaPIX-SH3. The peptide is shown with bonds in purple. betaPIX-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.

 
Our cellular binding assays demonstrate that betaPIX can indeed interact with AIP4 and further show that this interaction is mediated by binding of the betaPIX-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 betaPIX 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 betaPIX. Although we attribute this 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 betaPIX-SH3 serves as a scaffold for numerous signal transduction proteins, including PAK, Cbl, Rac, and 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 betaPIX with micromolar affinity and furthermore form heterotrimeric complexes, with one AIP4-derived peptide binding to two betaPIX-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 is wrapped around another face of betaPIX-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 betaPIX-SH3·Cbl-b complexes showed that mutation of an Arg residue (analogous to Arg-217 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 betaPIX-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·betaPIX-SH3 complex (28). In the case of Cbl-b, the peptide is the center of pseudosymmetry with the matching betaPIX-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, Arg-224 hypothetically would occupy the P-3 specificity pocket of another betaPIX-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 Thr-222 (Fig. 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 Pro-favoring pockets. In examining the AIP4 and Cbl-b peptide sequence, we would propose a new heterotrimeric motif for betaPIX-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 RXP(P/Z)X(P/Y)PR.

To prove this model, future experiments will be needed to further characterize the complex of AIP4 peptide and betaPIX-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 betaPIX-SH3 domain of the heterotrimer would be limited to interactions with the core ligand, whereas the betaPIX-SH3 domain observed in the crystal structure has additional contacts with the N-terminal 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 (Fig. 3C), it 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 betaPIX-SH3·AIP4 peptide, as well as crystallization trials under different conditions and with different AIP4-derived peptides. The full molecular understanding of how betaPIX andAIP4 interact to influence their respective pathways will require eventual structural studies of the full-length proteins.

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 multiprotein 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 EGF receptors (19). In addition, it is also possible that AIP4 may, like Cbl, directly ubiquitinate betaPIX or exert a cooperative effect on betaPIX 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).


Figure 4
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  		FIGURE 4.
The canonical binding of AIP4 tobetaPIX compared with PAK2 and Cbl-b. A, the binding of Cbl-b peptide (in magenta) in the Class I orientation is shown in comparison with AIP4 peptide (in yellow) after a least squares fit of betaPIX-SH3 domains from the two models. Residues from betaPIX 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 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 Lys-907 of Cbl-b, shown in the center of the figure, resulting in a displacement of the backbone so that the C{alpha} and Cbeta atoms of that residue occupy similar positions to the backbone N and C{delta} positions of P220 of AIP4. B, PAK2 peptide (in cyan) binds to betaPIX 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 Ile-183 and the surface of betaPIX. 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, 904-910, and 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 {varphi}/{psi} 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 gray squares drawn around the residues.

 


Figure 5
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  		FIGURE 5.
Proposed schematic arrangement of dimer interface of betaPIX 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 report for the AIP4·betaPIX complex and for the Class I orientation for the Cbl-b·betaPIX complex. Above the peptide sequences are the same positions in the Class II orientation as described in the Cbl-b·betaPIX complex, and the proposed Class II orientation of an additional betaPIX-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 Thr-222 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 {varphi}/{psi} angles, which show how the PPII helix in the case of Cbl-b and PAK2 are distorted. The pockets that favor the presence of Pro residues are indicated by gray squares drawn around the residues.

 
The association of AIP4 with betaPIX may also regulate GPCR signaling. Following activation of C5a receptors, released Gbeta{gamma} proteins bind to PAK, which in turn forms a complex with {alpha}PIX 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 betaPIX by Gbeta{gamma}·PAK complexes. Liberated AIP4 could in turn regulate CXCR4 ubiquitination and endocytotic sorting. Our fluorescence experiments demonstrate competition between PAK- and AIP4-derived peptides for the betaPIX-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 GPCR-kinase interacting proteins (GITs). 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 multiprotein 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 ubiquitin ligase AIP4 and the signaling scaffold protein betaPIX. Given the extensive involvement of these proteins in GPCR and RTK signaling, their interaction represents a new node in GPCR/RTK signal integration. Future studies will aim to elucidate the role this interaction plays in receptor down-regulation and cancer tumor progression and metastasis.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 2P4R) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported in part by the Allene Reuss Memorial Trust and the Howard Hughes Medical Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

2 Formerly an Ellison Medical Foundation Senior Scholar. Back

3 Supported by NIH Grant K08-EY015540. Back

1 Supported in part by National Institutes of Health (NIH) Grant T3ZEY007138. To whom correspondence should be addressed: The Rockefeller University, 1230 York Ave., Box 187, New York, NY 10021. Tel.: 212-327-8283; Fax: 212-327-7904; E-mail: jjanz{at}rockefeller.edu.

4 The abbreviations used are: GPCR, G protein-coupled receptor; RTK, receptor tyrosine kinase; E3, ubiquitin-protein isopeptide ligase; Cbl, Casitas B-lymphoma protein; EGF, epidermal growth factor; AIP4, atrophin-interacting protein 4; PAK, p21-activated kinase; betaPIX, beta-PAK-interactive exchange factor; PPII, polyproline II helix; ITC, isothermal titration calorimetry; HA, hemagglutinin; GST, glutathione S-transferase; GIT, GPCR-kinase interacting protein; IB, immunoblotting. Back


   ACKNOWLEDGMENTS
 
We 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 laboratories for insightful discussions. We gratefully acknowledge Dr. Sachiko Kajigaya, Dr. Bruce Mayer, Dr. Tony Pawson, and Dr. André Hoelz for providing plasmids.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Katzmann, D. J., Odorizzi, G., and Emr, S. D. (2002) Nat. Rev. Mol. Cell. Biol. 3, 893-905[CrossRef][Medline] [Order article via Infotrieve]
  2. Sorkin, A., and Von Zastrow, M. (2002) Nat. Rev. Mol. Cell. Biol. 3, 600-614[CrossRef][Medline] [Order article via Infotrieve]
  3. Liu, Y. C. (2004) Annu. Rev. Immunol 22, 81-127[CrossRef][Medline] [Order article via Infotrieve]
  4. Ingham, R. J., Gish, G., and Pawson, T. (2004) Oncogene 23, 1972-1984[CrossRef][Medline] [Order article via Infotrieve]
  5. Haglund, K., and Dikic, I. (2005) EMBO J. 24, 3353-3359[CrossRef][Medline] [Order article via Infotrieve]
  6. Marchese, A., Raiborg, C., Santini, F., Keen, J. H., Stenmark, H., and Benovic, J. L. (2003) Dev. Cell 5, 709-722[CrossRef][Medline] [Order article via Infotrieve]
  7. Joazeiro, C. A., Wing, S. S., Huang, H., Leverson, J. D., Hunter, T., and Liu, Y. C. (1999) Science 286, 309-312[Abstract/Free Full Text]
  8. Levkowitz, G., Waterman, H., Ettenberg, S. A., Katz, M., Tsygankov, A. Y., Alroy, I., Lavi, S., Iwai, K., Reiss, Y., Ciechanover, A., Lipkowitz, S., and Yarden, Y. (1999) Mol. Cell 4, 1029-1040[CrossRef][Medline] [Order article via Infotrieve]
  9. Soubeyran, P., Kowanetz, K., Szymkiewicz, I., Langdon, W. Y., and Dikic, I. (2002) Nature 416, 183-187[CrossRef][Medline] [Order article via Infotrieve]
  10. Wu, W. J., Tu, S., and Cerione, R. A. (2003) Cell 114, 715-725[CrossRef][Medline] [Order article via Infotrieve]
  11. Feng, Q., Baird, D., Peng, X., Wang, J., Ly, T., Guan, J. L., and Cerione, R. A. (2006) Nat. Cell Biol. 8, 945-956[CrossRef][Medline] [Order article via Infotrieve]
  12. Perry, W. L., Hustad, C. M., Swing, D. A., O'Sullivan, T. N., Jenkins, N. A., and Copeland, N. G. (1998) Nat. Genet. 18, 143-146[CrossRef][Medline] [Order article via Infotrieve]
  13. Wood, J. D., Yuan, J., Margolis, R. L., Colomer, V., Duan, K., Kushi, J., Kaminsky, Z., Kleiderlein, J. J., Sharp, A. H., and Ross, C. A. (1998) Mol. Cell Neurosci. 11, 149-160[CrossRef][Medline] [Order article via Infotrieve]
  14. Gao, M., Labuda, T., Xia, Y., Gallagher, E., Fang, D., Liu, Y. C., and Karin, M. (2004) Science 306, 271-275[Abstract/Free Full Text]
  15. Rossi, M., De Laurenzi, V., Munarriz, E., Green, D. R., Liu, Y. C., Vousden, K. H., Cesareni, G., and Melino, G. (2005) EMBO J. 24, 836-848[CrossRef][Medline] [Order article via Infotrieve]
  16. Rossi, M., Aqeilan, R. I., Neale, M., Candi, E., Salomoni, P., Knight, R. A., Croce, C. M., and Melino, G. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 12753-12758[Abstract/Free Full Text]
  17. Kitching, R., Wong, M. J., Koehler, D., Burger, A. M., Landberg, G., Gish, G., and Seth, A. (2003) Biochim. Biophys. Acta 1639, 104-112[Medline] [Order article via Infotrieve]
  18. Li, H., and Seth, A. (2004) Oncogene 23, 1801-1808[CrossRef][Medline] [Order article via Infotrieve]
  19. Courbard, J. R., Fiore, F., Adelaide, J., Borg, J. P., Birnbaum, D., and Ollendorff, V. (2002) J. Biol. Chem. 277, 45267-45275[Abstract/Free Full Text]
  20. Manser, E., Loo, T. H., Koh, C. G., Zhao, Z. S., Chen, X. Q., Tan, L., Tan, I., Leung, T., and Lim, L. (1998) Mol. Cell 1, 183-192[CrossRef][Medline] [Order article via Infotrieve]
  21. Bagrodia, S., Taylor, S. J., Jordon, K. A., Van Aelst, L., and Cerione, R. A. (1998) J. Biol. Chem. 273, 23633-23636[Abstract/Free Full Text]
  22. Bagrodia, S., Bailey, D., Lenard, Z., Hart, M., Guan, J. L., Premont, R. T., Taylor, S. J., and Cerione, R. A. (1999) J. Biol. Chem. 274, 22393-22400[Abstract/Free Full Text]
  23. Zhao, Z. S., Lim, J. P., Ng, Y. W., Lim, L., and Manser, E. (2005) Mol. Cell 20, 237-249[CrossRef][Medline] [Order article via Infotrieve]
  24. Ahn, S. J., Chung, K. W., Lee, R. A., Park, I. A., Lee, S. H., Park, D. E., and Noh, D. Y. (2003) Cancer Lett. 193, 99-107[CrossRef][Medline] [Order article via Infotrieve]
  25. Bae, J. Y., Ahn, S. J., Lee, J. E., Kim, J. E., Han, M. R., Han, W., Kim, S. W., Shin, H. J., Lee, S. J., Park, D., and Noh, D. Y. (2005) J. Cell. Biochem. 94, 1010-1016[CrossRef][Medline] [Order article via Infotrieve]
  26. Schmidt, M. H., Husnjak, K., Szymkiewicz, I., Haglund, K., and Dikic, I. (2006) Oncogene 25, 3071-3078[CrossRef][Medline] [Order article via Infotrieve]
  27. Volinsky, N., Gantman, A., and Yablonski, D. (2006) Biochem. J. 397, 213-222[CrossRef][Medline] [Order article via Infotrieve]
  28. Jozic, D., Cardenes, N., Deribe, Y. L., Moncalian, G., Hoeller, D., Groemping, Y., Dikic, I., Rittinger, K., and Bravo, J. (2005) Nat. Struct. Mol. Biol. 12, 972-979[Medline] [Order article via Infotrieve]
  29. Ten Klooster, J. P., Jaffer, Z. M., Chernoff, J., and Hordijk, P. L. (2006) J. Cell Biol. 172, 759-769[Abstract/Free Full Text]
  30. Gu, C., Tangye, S. G., Sun, X., Luo, Y., Lin, Z., and Wu, J. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 14447-14452[Abstract/Free Full Text]
  31. Hoelz, A., Janz, J. M., Lawrie, S. D., Corwin, B., Lee, A., and Sakmar, T. P. (2006) J. Mol. Biol. 358, 509-522[CrossRef][Medline] [Order article via Infotrieve]
  32. Otwinowski, Z., and Minor, W. (1997) Methods Enzymol. 276, 307-326
  33. McCoy, A. J., Grosse-Kunstleve, R. W., Storoni, L. C., and Read, R. J. (2005) Acta Crystallogr. D Biol. Crystallogr. 61, 458-464[CrossRef][Medline] [Order article via Infotrieve]
  34. Collaborative Computational Project (1994) Acta Crystallogr. D Biol. Crystallogr. 50, 760-763[CrossRef][Medline] [Order article via Infotrieve]
  35. Kleywegt, G. J. (1999) Acta Crystallogr. D. Biol. Crystallogr. 55, 1878-1884[CrossRef][Medline] [Order article via Infotrieve]
  36. Nicholls, A., Sharp, K. A., and Honig, B. (1991) Proteins 11, 281-296[CrossRef][Medline] [Order article via Infotrieve]
  37. Flanders, J. A., Feng, Q., Bagrodia, S., Laux, M. T., Singavarapu, A., and Cerione, R. A. (2003) FEBS Lett. 550, 119-123[CrossRef][Medline] [Order article via Infotrieve]
  38. Stofega, M. R., Sanders, L. C., Gardiner, E. M., and Bokoch, G. M. (2004) Mol. Biol. Cell 15, 2965-2977[Abstract/Free Full Text]
  39. Kowanetz, K., Szymkiewicz, I., Haglund, K., Kowanetz, M., Husnjak, K., Taylor, J. D., Soubeyran, P., Engstrom, U., Ladbury, J. E., and Dikic, I. (2003) J. Biol. Chem. 278, 39735-39746[Abstract/Free Full Text]
  40. Yu, H., Chen, J. K., Feng, S., Dalgarno, D. C., Brauer, A. W., and Schreiber, S. L. (1994) Cell 76, 933-945[CrossRef][Medline] [Order article via Infotrieve]
  41. Lim, W. A., Richards, F. M., and Fox, R. O. (1994) Nature 372, 375-379[CrossRef][Medline] [Order article via Infotrieve]
  42. Ramachandran, G. N., and Sasisekharan, V. (1968) Adv. Protein Chem. 23, 283-438[Medline] [Order article via Infotrieve]
  43. Natarajan, K., and Berk, B. C. (2006) Methods Mol. Biol. 332, 51-77[Medline] [Order article via Infotrieve]
  44. Pawson, T., and Nash, P. (2003) Science 300, 445-452[Abstract/Free Full Text]
  45. Ingham, R. J., Colwill, K., Howard, C., Dettwiler, S., Lim, C. S., Yu, J., Hersi, K., Raaijmakers, J., Gish, G., Mbamalu, G., Taylor, L., Yeung, B., Vassilovski, G., Amin, M., Chen, F., Matskova, L., Winberg, G., Ernberg, I., Linding, R., O'Donnell, P., Starostine, A., Keller, W., Metalnikov, P., Stark, C., and Pawson, T. (2005) Mol. Cell. Biol. 25, 7092-7106[Abstract/Free Full Text]
  46. Magnifico, A., Ettenberg, S., Yang, C., Mariano, J., Tiwari, S., Fang, S., Lipkowitz, S., and Weissman, A. M. (2003) J. Biol. Chem. 278, 43169-43177[Abstract/Free Full Text]
  47. Lai, E. C. (2004) Development 131, 965-973[Abstract/Free Full Text]
  48. Lai, E. C. (2002) Curr. Biol. 12, R74-R78[CrossRef][Medline] [Order article via Infotrieve]
  49. Jehn, B. M., Dittert, I., Beyer, S., von der Mark, K., and Bielke, W. (2002) J. Biol. Chem. 277, 8033-8040[Abstract/Free Full Text]
  50. Qiu, L., Joazeiro, C., Fang, N., Wang, H. Y., Elly, C., Altman, Y., Fang, D., Hunter, T., and Liu, Y. C. (2000) J. Biol. Chem. 275, 35734-35737[Abstract/Free Full Text]
  51. Li, Z., Hannigan, M., Mo, Z., Liu, B., Lu, W., Wu, Y., Smrcka, A. V., Wu, G., Li, L., Liu, M., Huang, C. K., and Wu, D. (2003) Cell 114, 215-227[CrossRef][Medline] [Order article via Infotrieve]
  52. Hoefen, R. J., and Berk, B. C. (2006) J. Cell Sci. 119, 1469-1475[Abstract/Free Full Text]

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