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Originally published In Press as doi:10.1074/jbc.M600307200 on March 22, 2006

J. Biol. Chem., Vol. 281, Issue 20, 14376-14382, May 19, 2006
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Comparative Peptide Binding Studies of the PABC Domains from the Ubiquitin-protein Isopeptide Ligase HYD and Poly(A)-binding Protein

IMPLICATIONS FOR HYD FUNCTION*Formula

Nadia S. Lim{ddagger}1, Guennadi Kozlov{ddagger}1, Tsung-Cheng Chang§, Olivia Groover{ddagger}, Nadeem Siddiqui{ddagger}, Laurent Volpon{ddagger}, Gregory De Crescenzo, Ann-Bin Shyu§, and Kalle Gehring, A Chercheur National of the Fonds de la Recherche en Santé de Québec{ddagger}2

From the {ddagger}Department of Biochemistry, McGill University, 3655 Promenade Sir William Osler, Montréal, Québec H3G 1Y6, Canada, the §Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas 77030, and the Département de Génie Chimique, École Polytechnique de Montréal, 2500 chemin de Polytechnique, Montréal, Québec H3T 1J4, Canada

Received for publication, January 11, 2006 , and in revised form, March 7, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The PABC domain is a peptide-binding domain that is specifically found in poly(A)-binding protein (PABP) and a HECT ubiquitin-protein isopeptide ligase (E3) known as HYD (hyperplastic discs), EDD (E3 isolated by differential display), or Rat100. The PABC domain of PABP recruits various regulatory proteins and translation factors to poly(A) mRNAs through binding of a conserved 12-amino acid peptide motif, PAM2 (PABP-interacting motif 2). In contrast, little is known about the specificity or function of the domain from HYD. Here, we used isothermal calorimetry and surface plasmon resonance titrations to show that the PABC domain of HYD binds PAM2 peptides with micromolar affinity. NMR chemical shift perturbations were used to map the peptide-binding site in the PABC domain of HYD. The structural features of binding are very similar to those of the interactions with the domain of PABP, which explains the overlapping peptide specificity and binding affinity. We identified the anti-proliferative Tob proteins as potential binding partners of HYD. This was confirmed by glutathione S-transferase pulldown and immunoprecipitation experiments demonstrating the interaction with full-length Tob2. Altogether, our results point to a role of the PABC domain as a protein-protein interaction domain that brings together the processes of translation, ubiquitin-mediated protein degradation, and cell cycle control.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The tumor suppressor protein HYD (hyperplastic discs), also known as EDD (E3 3 isolated by differential display) or Rat100, is a member of the family of HECT (homologous to E6-associated protein carboxyl terminus) E3 ligases, which target specific proteins for ubiquitin-mediated proteolysis. The highly conserved ubiquitin/proteasome pathway controls the degradation of many critical regulatory proteins. Targeted proteins are post-translationally conjugated to a 76-residue ubiquitin moiety through a combined set of reactions involving activating (ubiquitin-activating enzyme), conjugating (ubiquitin carrier protein), and ligating (E3) enzymes. E3 enzymes physically interact with their substrates and are thus critical determinants of the specificity of ubiquitination. Two main groups of E3 ligases are the ring finger and HECT domain ligases (1, 2).

In Drosophila, HYD is required for the regulation of cell proliferation during development (3). It has been inferred that HYD participates in signal transduction downstream of the signaling receptors to initiate and/or maintain proliferation as well as to terminate proliferation (3). Indeed, it has been shown that cells with mutations in HYD fail to properly terminate proliferation, leading to tumors. Furthermore, these mutations also result in developmental abnormalities such as adult sterility due to germ cell defects (4). HYD is frequently overexpressed in breast and ovarian cancers, supporting a role in cancer development (5, 6). HYD is also involved in DNA damage signaling, in which TopB1, a target for ubiquitinylation by HYD (7), co-localizes with BRAC1 at stalled replication forks (7, 8). HYD also interacts with the calcium- and integrin-binding protein CIB in a DNA damage-dependent manner (9). Finally, HYD is an in vivo substrate for ERK1 and ERK2 (10). The ERK pathway is an evolutionarily conserved signaling pathway that regulates a variety of cellular processes, including proliferation, differentiation, transcription, and migration (11). In this context, it has been proposed that HYD phosphorylation by ERK2 modulates the activity of HYD (10). Conversely, HYD may modulate the ubiquitination of ERK2, as this kinase has been shown to be ubiquitinated and degraded by the proteasome (12). However, the precise relationship between HYD and ERK2 remains unknown (12). Despite the accumulating knowledge about HYD, its exact biochemical roles have yet to be determined.

Structurally, HYD ligases contain a ubiquitin-associated domain at their N termini, two nuclear localization signals, a zinc finger-like UBR domain involved in recognition of type 1 N-terminal degrons (13), a domain highly homologous to the PABC (poly(A)-binding protein C-terminal) domain, and a HECT domain at their extreme C termini (see Fig. 1). The solution and crystal structures of the ~70-residue long PABC domains from human HYD and various poly(A)-binding proteins (PABPs) have shown that these domains all consist of a bundle of four or five {alpha}-helices (14-17). Previous NMR studies showed that the PABC domain is a peptide-binding domain that specifically recognizes a conserved PAM2 (PABP-interacting motif 2) sequence (14). This PAM2 site was initially identified in the PABP-interacting proteins Paip1 and Paip2 and in eukaryotic release factor 3 (eRF3). These proteins modulate translational activity by either stabilizing (Paip1 and eRF3) or destabilizing (Paip2) the closed loop structure of mRNA, the formation of which involves the simultaneous interactions of PABP with the poly(A) tail of mRNA and the 5'-cap binding complex (18-20). The structures of the PABC domain from human PABP in complex with peptides from Paip1 and Paip2 revealed that PAM2 binds to the most conserved {alpha}2-, {alpha}3-, and {alpha}5-helices of the PABC domain as a series of two beta-turns (21). Recently, a bioinformatics survey highlighted the existence of multiple proteins containing PAM2 sites, including ataxin-2, Tob1 (transducer of Erb1), Tob2, USP10, dNF-x1, TPRD/TTC3, and dMAP205 (22).

The PABC domain of human HYD was shown previously to bind to truncated Paip1 containing PAM2 by glutathione S-transferase (GST) pulldown assays. This finding suggests that HYD may be able to interact with other PABP-interacting proteins through a similar mechanism (15). In common with yeast PABP, which has unusual peptide specificity, the PABC domain of HYD is composed of only four {alpha}-helices (16). The position of the PABC domain directly adjacent to the catalytic HECT domain suggests HYD involvement in substrate recognition. A better understanding of the PAM2 binding properties of HYD should help elucidate its function through identification of novel physiological partners and provide useful insight into the potential competition between HYD and PABP for shared binding partners. This is especially important in the light of recent results demonstrating a role for HYD in the proteasome-mediated turnover of Paip2 (23). Here, we report a comprehensive characterization of the peptide binding properties of the PABC domain of HYD and identify the anti-proliferative Tob proteins as potential in vivo partners of HYD.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
PABC Domain Expression, Purification, and Peptide Synthesis—The PABC domain of rat HYD was cloned into the BamHI and XhoI restriction sites of the pGEX-6P-1 vector (Amersham Biosciences), and the construct was transformed into the Escherichia coli BL21-Gold(DE3) expression host (Stratagene). The protein was expressed and purified by affinity chromatography to yield the 60 residues of the PABC domain plus a five-residue (Gly-Pro-Leu-Gly-Ser) N-terminal extension. The amino acid composition was confirmed by mass spectrometry. For NMR analysis, the protein was exchanged into NMR buffer (50 mM Tris, 100 mM NaCl, and 1 mM NaN3, pH 6.8). The final yield of purified protein was ~7 mg/liter of LB culture medium. PAM2 peptides were synthesized by Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid-phase peptide synthesis and purified by reverse-phase chromatography on a Vydac C18 column. The composition and purity of the peptides were verified by ion spray quadrupole mass spectroscopy.

NMR Spectroscopy—NMR resonance assignments of the PABC domain of the HYD protein were determined by HNCACB and CBCA-(CO)NH experiments (24) performed on a Bruker Avance 600 NMR spectrometer using a 13C,15N-labeled protein sample. Additional 1H-15N edited nuclear Overhauser effect and total correlation spectra were used to assign the resonances in the PABC domain-peptide complex. All NMR experiments were recorded at 293 K. Peptide NMR titrations were carried by adding peptides to 1.0-1.5 mM samples of the 15N-labeled PABC domain from the HYD protein and monitored by 1H-15N heteronuclear single quantum correlation spectra.

Isothermal Titration Calorimetry (ITC) Measurements—Experiments were carried out on a MicroCal VP-ITC titration calorimeter using VPViewer software for instrument control and data acquisition. The buffer used for ITC experiments contained 50 mM Tris and 100 mM NaCl, pH 6.8. During a titration experiment, samples of the PABC domain from the HYD protein were thermostatted at 288 K in a stirred (310 rpm) reaction cell of 1.4 ml. Fifty-nine injections (5-µl volume and 10-s duration each, with 5-min intervals between injections) were carried out using a 296-µl syringe filled with the peptide solution. Titration experiments were performed with 30-60 µM PABC domain solution in the cell and 250-550 µM peptide solution in the syringe to ensure a final peptide/PABC domain molar ratio of at least 2:1 in the reaction cell. The binding constants and thermodynamic parameters were determined as described previously (21).

Surface Plasmon Resonance-based Biosensor Analysis—The interaction of the PABC domain from PABP with the synthetic PAM2 peptides was analyzed by surface plasmon resonance (25) using both Biacore 1000 and Biacore 3000 optical biosensors (Biacore AB, Uppsala, Sweden). The C2 fragment of PABP (residues 498-636) was immobilized on a CM5 biosensor chip using amine coupling chemistry as described previously (25). For each peptide interaction, the biosensor experiment was repeated three times over different PABC domain surfaces (between 2000 and 4000 resonance units coupled) using the two biosensors. A separate control flow cell was activated and blocked to correct for refractive index changes. The experiments were performed at 25 °C using a flow rate of 5 µl/min. For each peptide, at least five different concentrations were injected over the PABC domain and control flow cells. The ranges of concentrations used were 6.25-100 µM for USP10, dNF-x1, TPRD/TTC3, and dMAP205; 412 nM to 100 µM for ataxin-2; 620 nM to 500 µM for Tob2-(131-147); and 3.7-300 µM for Tob2-(251-267). All peptide dilutions were performed in running buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, and 0.005% Tween 20). After each injection, regeneration was performed using two pulses of HCl (120 mM, 15 ml), followed by an EXTRACLEAN and a RINSE procedure as recommended by the manufacturer.

Thermodynamic dissociation constants (Kd) were determined by Scatchard plot analysis, Req/C = f(Req), using the control-corrected plateau values (Req) corresponding to the injection of the peptide at concentration C. The Kd was derived as the reciprocal of the slope of the linear regression of the plot.

GST Pulldown Assay—GST pulldown assays were performed using the MagneGST pulldown system (Promega Corp.) according to the manufacturer's instruction. Briefly, GST fusion proteins were expressed in E. coli BL21 cells (Amersham Biosciences) and then induced with 0.5 mM isopropyl beta-D-thiogalactopyranoside at 30 °C for 3 h. The cells were harvested and lysed in cell lysis reagent containing protease inhibitor mixture (Roche Applied Science) and DNase (Promega Corp.). The lysate was clarified by centrifugation at 14,000 x g for 10 min. The expressed GST fusion protein in the clear lysate was immobilized on MagneGST particles and then incubated with 5 µl of the in vitro translated protein in 250 µl of binding buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.5% Nonidet P-40, and protease inhibitor mixture). After incubation with rotation for 1 h at 4°C, the MagneGST particles were washed four times with 400 µl of radioimmune precipitation assay buffer (10 mM sodium phosphate, pH 7.2, 150 mM NaCl, 1% Nonidet P-40, and 0.5% sodium deoxycholate). The bound fraction was eluted in 1x SDS loading buffer and analyzed by SDS-PAGE. The gels were stained with Coomassie Blue to show equal loading of the GST fusion protein and then dried for autoradiography to detect the pull-down protein. For in vitro translation, [35S]methionine-labeled proteins were produced in the rabbit reticulocyte lysate using an in vitro coupled transcription/translation system (Promega Corp.) in the presence of [35S]methionine (Amersham Biosciences). The in vitro translated products were treated with DNase I (0.5 units/µl) and RNase A (0.1 mg/ml) for 15 min at 30 °C.


Figure 1
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  		FIGURE 1.
HYD, an E3 ligase, contains a fragment that is highly homologous to the PABC domain. A, HYD domain organization. UBA, ubiquitin-associated domain; NLS, nuclear localization signal; UBR, the UBR-type zinc finger domain. B, sequence alignment of the PABC domains from the human (hHYD), rat (rHYD), and Drosophila (dHYD) HYD proteins and human PABP1. The consensus sequence is shown below. The secondary structure is shown above; helices are labeled {alpha}2-{alpha}5 according to the PABC domain of PABP (14).

 


Figure 2
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  		FIGURE 2.
1H-15N heteronuclear single quantum correlation spectrum of the rat HYD fragment containing the PABC domain (residues 2393-2452). Signals are labeled with residue numbers and one-letter amino acid code. Residues originating from the cloning vector are labeled with asterisks. The signals from side chains of asparagines and glutamines are connected with horizontal lines. The middle regions of the spectrum are enlarged for clarity and shown in the insets.

 
Immunoprecipitation—COS-7 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin at 37 °C and 5% CO2. The cells were split to a density of 4.5 x 106/100-mm dish 24 h before transfection. For transient transfection, cells were transfected with pcDNA6-V5 or pcDNA6-Tob2-V5 DNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Cells were harvested 40 h after transfection and suspended in lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20, 1 mM sodium orthovanadate, 1 mM sodium pyrophosphate, 1 mM NaF, 100 nM okadaic acid, and protease inhibitor mixture). Cell suspensions were subjected to freezing/thawing for three cycles, and lysates were clarified by centrifugation at 14,000 x g for 10 min. For immunoprecipitation, lysates were incubated with anti-V5 antibody-agarose (Sigma) in the presence of RNase A (0.1 mg/ml) with rotation for 2 h at 4 °C. Following precipitation, the resin was washed five times with 0.5 ml of lysis buffer. The precipitates were resolved by SDS-PAGE. Western blotting was performed with horseradish peroxidase-conjugated anti-V5 antibody (Invitrogen), goat anti-HYD polyclonal antibody (Santa Cruz Biotechnology, Inc.), and anti-glyceraldehyde-3-phosphate dehydrogenase monoclonal antibody (Research Diagnostics, Inc.).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The PAM2-binding Surface of the PABC Domain from HYD Is Similar to That from PABP—To study the PAM2 binding properties of the PABP-homologous domain of HYD, the corresponding fragment of the rat HYD protein (residues 515-574) was cloned and expressed in medium supplemented with 15NH4Cl. This fragment corresponds to residues 2393-2452 of human HYD, with only two amino acids differing in their corresponding sequences (E2443D and V2450I) (Fig. 1B). To be consistent within the family of HYD ligases, the residue numbering for human HYD is used in this study, as the sequence of the full-length 300-kDa rat HYD protein (26) is currently not available in protein data bases. The 1H-15N correlation spectrum of the unliganded domain showed a good dispersion of signals characteristic of a folded protein (Fig. 2). Standard heteronuclear experiments were performed using triple-labeled protein for the sequence-specific assignments of the signals. As a result, all detectable signals were assigned; the amides of His2393, Arg2404, and Ser2414 were missing from the spectrum.

The PABC domain of HYD was shown previously to bind to Paip1 (residues 113-480) by GST affinity assays (15). To better characterize this interaction, the 15N-labeled HYD fragment was titrated with a shorter fragment of Paip1 (residues 123-144) containing PAM2. Addition of this peptide caused the appearance in slow exchange of new signals for HYD residues involved in peptide binding (Fig. 3A). The residues displaying the largest chemical shift changes upon Paip1 binding were Ala2396 (0.49), Tyr2402 (0.38), Ala2445 (0.38), and Met2419 (0.35), identifying the peptide-binding site (Fig. 3B). Mapping of chemical shift changes on the available crystal structure of the human HYD fragment (Fig. 3C) clearly indicated that the peptide-binding surface of the PABC domain from HYD is analogous to that from its PABP ortholog (14). The PAM2 peptides bound to the PABC domain of PABP along the large surface involving the same helices that were affected in the PABC domain of HYD (Fig. 3D). Similar results were obtained with the PAM2 peptides from ataxin-2 (residues 912-928) and dMAP205 (residues 234-250) (supplemental Fig. 1), confirming the existence of a single and well defined PAM2-binding site on the HYD PABC domain.


Figure 3
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  		FIGURE 3.
NMR mapping of the PAM2-binding site in the PABC domain of HYD. A, overlay of the 1H-15N heteronuclear single quantum correlation spectra of the unliganded (blue) and Paip1-(123-144)-bound (red) PABC domains from rat HYD. Signal shifts are shown with black lines. B, plot of amide chemical (chem.) shift changes in the PABC domain of HYD upon addition of the Paip1 peptide. The chemical shift changes were calculated as ({Delta}HN2 + (0.15*{Delta}N)2)1/2. The most affected amides are labeled. C, mapping of chemical shift changes upon Paip1 peptide binding in the crystal structure of the PABC domain from human HYD (Protein Data Bank code 1I2T [PDB] ). The width of the sausage is proportional to the magnitude of chemical shift changes. Blue-to-red color transition reflects an increase in chemical shift changes. The helices are labeled according to the PABC domain of PABP. The most affected residues are labeled. The figure was generated using MolMol (34). D, ribbon and mesh surface representation of the complex structure between the PABC domain of PABP (blue) and the PAM2 peptide from Paip2 (red) (21). The helices are labeled as described for C, and a similar orientation of the PABC domain is used.

 
Ionic Interactions Have Greater Importance for HYD-PAM2 Binding—To further characterize the binding, we applied ITC to determine the binding constants and thermodynamic parameters of interactions between the PABC domain of HYD and a series of PAM2 peptides (Fig. 4). The binding of PAM2 peptides to the PABC domain of HYD led to favorable enthalpy effects, which gave rise to heat release. This primarily reflects the contributions of van der Waals, hydrogen bond, and electrostatic interactions to the binding. Alternatively, changes in entropy mainly reflect counteracting contributions from solvation entropy (water release upon complex formation) and conformational entropy. Negative entropy values were measured for the Paip1, eRF3, ataxin-2 and dMAP205 peptides (Fig. 4C), indicating that the heat release was accompanied by a loss of disorder due mostly to the immobilization of the peptide backbone. In contrast, we observed an entropy increase in the binding of the Tob2-(251-267), dNF-x1, USP10, and TPRD/TTC3 peptides to the PABC domain of HYD. Here, desolvation effects more likely dominated the conformational restrictions resulting from complex formation.


Figure 4
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  		FIGURE 4.
Thermodynamics of peptide binding to the PABC domain of HYD. A, calorimetric titration of the PABC fragment from HYD with Paip1-(123-144). The upper panel shows the heat released following injections of Paip1 into a solution of the PABC domain. The lower panel shows the integrated heat after correction for the heat of dilution and normalization of the amount of Paip1 injected (boxes). The curve represents the best fit to a model involving a single site. B, temperature dependence of the enthalpy change upon binding of the Paip1 (diamonds), ataxin-2 (triangles), eRF3 (boxes), and Tob2-(251-267) (circles) peptides to the PABC domain. C, thermodynamic parameters of PAM2-containing peptides measured at 25 °C as {Delta}H (kcal/mol), {Delta}S (cal mol-1 K-1), {Delta}G (kcal/mol), and {Delta}Cp (cal mol-1 K-1). Peptide sequences are aligned, and the most conserved residues are shown in boldface. The residue numbering corresponds to the following NCBI entries: Paip1 (GI:17061092), eRF3 (GI:46094014), ataxin-2 (GI:51479160), Tob2 (GI:7706739), dNF-x1 (GI:11359837), dMAP205 (dMAP 205kda; GI:126746), USP10 (GI:1136438), and TPRD/TTC3 (GI:49640011).

 
The change in heat capacity upon binding ({Delta}Cp) is determined by changes in the degree of surface hydration in the unliganded versus complexed molecules and can be used to estimate the ratio of polar to non-polar surface buried upon complex formation. The negative {Delta}Cp values for the Paip1 and eRF3 peptides (Fig. 4C) point to an increase in hydrophobic interactions upon binding. Interestingly, {Delta}Cp for eRF3 (-81 cal mol-1 K-1) was significantly higher for HYD binding than for PABP binding (-294 cal mol-1 K-1) (21). This noticeable difference in {Delta}Cp highlights the contribution of ionic interactions in the eRF3-HYD complex. Taken together, the entropy and heat capacity changes hint at small but distinct differences in peptide binding by HYD and PABP that modulate their binding affinity for different PAM2 sequences.

Specificity of PAM2 Recognition by HYD and PABP—To address these differences in specificity, the binding constants for the PABC domains of HYD and PABP were measured for 10 PAM2 peptides derived from confirmed and potential binding partners (Table 1). We previously measured the binding constants of the interactions between the Paip1, Paip2, and eRF3 peptides and the PABC domain of PABP using the surface plasmon resonance-based Biacore sensor (21). Here, we complemented these data with affinity measurements for other PAM2 peptides by ITC and surface plasmon resonance. The Kd values measured by surface plasmon resonance (Table 1, in parentheses) and ITC are in a very good agreement, underlining the reliability of both techniques for affinity measurements.


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  		TABLE 1
Comparison of HYD and PABP binding affinities for PAM2 peptides

 
Most of the peptides tested bound to the HYD PABC domain with low micromolar affinity, which is typical for PABP-PAM2 interactions (21). This suggests that all of the proteins are potential physiological partners of HYD and PABP. Indeed, ataxin-2 and Tob2 were shown recently to bind PABP in vivo (27, 28), and HYD was shown to interact with Paip2 (23). The affinity of PAM2 peptides for HYD is generally weaker than for PABP, with notable exceptions for Tob2-(251-267) and USP10, which bind both domains with equal affinity. The largest differences (>10-fold higher affinity for PABP) were observed for the Paip2, ataxin-2, and Tob2-(131-147) peptides. The Tob2-(131-147) sequence was the only one that did not bind HYD. Although differences exist, the data demonstrate a great deal of overlap in PAM2 binding by HYD and PABP and potential cross-talk in their in vivo interactions.

Interaction between Tob2 and the PABC Domain of HYD—The anti-proliferative protein Tob2 contains two distinct PAM2 sequences, one at its N terminus (residues 131-147) and the other in its central region (residues 251-267), but only the second sequence binds to HYD. We applied pulldown and immunoprecipitation assays to complement our in vitro binding assays with Tob2. The GST pulldown experiment showed that the PABC domains of both HYD and PABP bound to full-length Tob2 (Fig. 5A). The binding of Tob2 to HYD was somewhat weaker than to PABP, which might reflect the inability of the N-terminal PAM2 site to interact with HYD. The reverse immunoprecipitation experiment showed that V5-tagged Tob2 specifically coprecipitated a small amount of HYD, but not the control abundant glyceraldehyde-3-phosphate dehydrogenase protein (Fig. 5B). No band was detected when the cells were transfected with vector alone. NMR titrations confirmed that the Tob2-(251-267) peptide bound to the same surface on the PABC domain of HYD as the other studied PAM2 sequences (Fig. 5C). These experiments demonstrate a specific, albeit weak, interaction between HYD and Tob2 in crude cell extracts.


Figure 5
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  		FIGURE 5.
Interaction of Tob2 with HYD. A, GST pulldown assay. In vitro translated and [35S]methionine-labeled Tob2 and luciferase proteins were incubated with MagneGST particle-immobilized GST, GST-PABP PABC domain (amino acids 498-636), and GST-HYD PABC domain (amino acids 2393-2452), and the resulting complexes were resolved by SDS-PAGE followed by Coomassie Blue staining (lower panel) for equal bait loading and then autoradiography (upper panel). Luciferase and GST were used as negative controls. B, immunoprecipitation (IP) of Tob2 with HYD. COS-7 cells were transfected with vector alone or the vector expressing V5-tagged Tob2. Lysates were prepared and incubated with anti-V5 antibody-agarose in the presence of RNase A. Following precipitation, bound proteins were analyzed by SDS-PAGE. Western blotting (WB) was performed with the indicated antibodies. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an abundant cytoplasmic protein, was used as a negative control. C, plot of amide chemical (chem.) shift changes in the PABC domain of HYD upon NMR titration of the 15N-labeled PABC domain of HYD with the Tob2-(251-267) peptide. The most affected signals are labeled.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have presented the first comprehensive study of the PAM2 binding properties of the PABC domain from HYD, an E3 ligase. We have demonstrated that HYD and PABP employ the same surface features to interact with PAM2-containing peptides, bind the peptides in the same way, and have a large degree of overlap in their binding specificity. Careful affinity measurements hint at subtle but distinct differences in peptide specificity for HYD compared with PABP. Two factors are likely to contribute to these differences. The first is the PABC domain primary sequence, which may affect/modify peptide-protein intermolecular contacts, thus altering the weight of charged versus hydrophobic interactions. The second is the absence of the first N-terminal helix in the HYD PABC domain. In PABPs, this helix does not directly contact the bound peptide, but may indirectly affect binding through its effect on the {alpha}2-helix. The solution structures of peptides from Paip1 and Paip2 bound to the PABP PABC domain show the greatest differences in the peptide C termini, which bind to the {alpha}2-helix (21).

The interactions of Paip1, Paip2, and eRF3 with PABP have been well characterized (18-20). These proteins bind to the PABC domain of PABP through PAM2 sequences located at either the N or C termini in unfolded regions of the proteins. Paip1 and Paip2 also bind to a second site in the RNA-binding portion of PABP via a less well characterized motif termed PAM1. One important conclusion of these studies is that PAM2-containing proteins are recruited to the mRNA through binding to PABP and that they compete for binding to PABP. Sequence analysis identified a number of proteins containing PAM2 as potential PABP-binding proteins. Recent in vivo studies have confirmed two of these predictions: ataxin-2 and Tob2 (27, 28).

The BTG/Tob proteins are a large family of anti-proliferative proteins involved in various functions, including cell cycle control (29) and differentiation (30), transcription control (28), and potentially mRNA metabolism. The family is defined by a highly conserved 110-amino acid N-terminal region designated the BTG/Tob homology domain. Of the BTG/Tob family of proteins, only the Tob proteins contain PAM2 sites. Tob2 contains two distinctive PAM2 sites, whereas Tob1 contains a single PAM2 site, which corresponds to Tob2-(251-267). The Tob2-(131-147) sequence was shown to be important for PABP interactions (28), whereas we have shown that HYD specifically binds Tob2-(251-267). In theory, PABP and HYD could be brought together by simultaneously binding Tob2; however, the biological significance of this is untested. Another intriguing observation is that both HYD and Tob2 are targets for the MAPK ERK2 (10, 31). The ERK pathway is an evolutionarily conserved signaling pathway that regulates many cellular processes, including proliferation, differentiation, transcription, and migration (11). Tob proteins have already been implicated in the negative regulation of the cell cycle via their C-terminal regions; Tob inhibits cell growth by suppressing cyclin D1 expression, whereas ERK1- and ERK2-mediated Tob phosphorylation negatively regulates Tob anti-proliferation (31-33). HYD and Tob2 could be part of a related signaling pathway that is involved in the control of cell cycle progression.


Figure 6
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  		FIGURE 6.
Protein interaction network including HYD, PABP, and PAM2-containing proteins. HYD and PABP contain PABC domains that bind to the PAM2 site of Paip2, a protein involved in translation regulation. PABP also binds to the PAM2 site of the anti-proliferative protein Tob2; PABP and Tob2 bind to different subunits of the principal deadenylating complex, consisting of CCR4, NOT1, and CAF1 (35, 36). Tob2 possesses two independent PAM2 sites, one of which binds to the PABC domain of HYD. Tob2 is the substrate of the MAPK ERK2, which was also shown to bind HYD. All three proteins are involved in development and cell cycle regulation.

 
The BTG/Tob proteins are known to be multiubiquitinated and degraded by the ubiquitin/proteasome pathway (32). Our finding that HYD interacts with Tob2 proteins in cell extracts suggests a mechanism that is analogous to the regulation of Paip2 levels by competition between PABP and HYD. When PABP levels are depressed, Paip2, which is bound to the higher affinity site of PABP, is released and ubiquitinated by HYD, leading to degradation by the proteasome (23). It is plausible that HYD similarly competes with PABP for the PAM2 site on Tob2. More generally, our study suggests that many PAM2-containing proteins may be targets for ubiquitination by HYD through binding to its PABC domain.

The intriguing occurrence of the PABC domain in just two proteins, PABPs and HYD E3 ligases, is not coincidental. Recent studies have uncovered several connections between these proteins through shared binding partners (Fig. 6), and more links will undoubtedly surface in the near future. It appears that PABP and HYD are part of a shared network that brings together the fundamental processes of translation, ubiquitination, and cell cycle control.


   FOOTNOTES
 
* This work was supported in part by Canadian Institutes of Health Research Grant MA14219 (to K. G.) and National Institutes of Health Grant GM46454 (to A.-B. S.). 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

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1. Back

1 Both authors contributed equally to this work. Back

2 To whom correspondence should be addressed. Tel.: 514-398-7287; Fax: 514-398-7384; E-mail: kalle.gehring{at}mcgill.ca.

3 The abbreviations used are: E3, ubiquitin-protein isopeptide ligase; ERK, extracellular signal-regulated kinase; PABPs, poly(A)-binding proteins; eRF3, eukaryotic release factor 3; GST, glutathione S-transferase; ITC, isothermal titration calorimetry; MAPK, mitogen-activated protein kinase. Back


   ACKNOWLEDGMENTS
 
We thank Simon Wing for the gift of rat HYD cDNA, Demetra Elias for cloning the PABC domain, and Tara Sprules for assistance with NMR experiments.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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