A three-dimensional model of Suppressor Of Cytokine Signalling 1 (SOCS-1)

Fabrizio Giordanetto1 and Romano T. Kroemer1,2,3

1 Department of Chemistry, Queen Mary, University of London, Mile End Road, London E1 4NS, UK


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Suppressor Of Cytokine Signalling 1 (SOCS-1) is one of the proteins responsible for the negative regulation of the JAK-STAT pathway triggered by many cytokines. This important inhibition involves complex formation between SOCS-1 and JAK2, which requires particular structural domains (KIR, ESS and SH2) on SOCS-1. A three-dimensional theoretical model of SOCS-1 is presented here. The model was generated by the application of different modelling techniques, including threading, structure-based modelling, surface analysis and protein docking. The structure accounts for the interactions between SOCS-1 and two other key proteins in the JAK-STAT pathway, namely JAK2 and Elongin BC. The proposed model for the interaction between SOCS-1 and JAK2 suggests that the SOCS-1 suppress the kinase activity of JAK2 by obstructing the catalytic groove of the tyrosine kinase. Subsequent interaction of the JAK–SOCS complex with Elongin BC was also modelled. A sequence and structural comparison between the SH2 domain of SOCS-1 and the SH2 domains of other proteins highlights key residues that could be responsible for SOCS-1 specificity. Currently available mutational data are evaluated. The results are consistent with the experimental data and they provide deeper insights into the inhibitory function of SOCS-1 at a molecular level.

Keywords: homology modelling/JAK-STAT pathway/signal transduction/SOCS


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cytokines known to interact with cytokine type I and II receptors exert their physiological effects through receptor-associated tyrosine kinases (JAKs) and cytoplasmic transcription factors (STATs) (Imada and Leonard, 2000Go). The cytokines bind to the extracellular parts of the receptor chains and induce receptor oligomerization, which in turn activate JAKs in the cytoplasma. Activation of JAKs leads to specific phosphorylation of tyrosine residues in the receptor tails. STATs dock to the phosphorylated receptors via their SH2 domain and are activated by phosphorylation. Finally, the STATs dimerize and translocate into the cellular nucleus where they initiate gene transcription. This complex cascade is crucial for cell development, hematopoiesis and host defence and requires precise control and regulation (Diamond et al., 2000Go).

Suppressor Of Cytokine Signalling 1 (SOCS-1) is a protein that acts as a negative regulator of the JAK-STAT pathway. It inhibits signalling by a wide range of cytokines including interleukin 2 and 4 (IL-2, IL-4) (Losman et al., 1999Go; Sporri et al., 2001Go), interleukin 6 (IL-6) and leukaemia inhibitory factor (LIF) (Endo et al., 1997Go; Naka et al., 1997Go; Starr et al., 1997Go), interferon {gamma} (IFN-{gamma}) (Sakamoto et al., 1998Go), growth hormone (GH) (Adams et al., 1998Go; Hansen et al., 1999Go; Ram and Waxman, 1999Go) and prolactin (PRL) (Pezet et al., 1999Go; Tomic et al., 1999Go). SOCS-1 belongs to the SOCS (Suppressors Of Cytokine Signalling) (Starr et al., 1997Go) family, also known as the CIS (Cytokine Inducible SH2-containing) (Yoshimura et al., 1995Go), SSI (STAT-induced STAT Inhibitor) (Naka et al., 1997Go) or JAB (JAK Binding protein) (Endo et al., 1997Go) family. To date, this class of proteins includes eight different members with conserved structural features. All the SOCS members present an amino-terminal region that is variable both in length and in amino acid sequence, a central Src Homology 2 (SH2) domain and a conserved carboxy-terminal stretch of 40 amino acids. The latter region is alternatively referred to as SOCS box (Starr et al., 1997Go), CIS homology (CH) domain (Masuhara et al., 1997Go) or SSI COOH-terminal (SC) motif (Minamoto et al., 1997Go).

The SH2 region of SOCS-1 was shown to be essential for the binding to the kinase domain (JH1) of JAK2 (Yasukawa et al., 1999Go). In particular, mutation of the phosphotyrosine binding residue R105, on the SOCS-1 SH2 region, to a lysine (Yasukawa et al., 1999Go), a glutamic acid (Yasukawa et al., 1999Go) or to a glutamine (Narazaki et al., 1998Go) resulted in a protein unable to inhibit the cytokine signalling. This association, being SH2 dependent, required a specific phosphotyrosine on JAK2. Binding studies, employing different JAK2 phosphopeptides, indicated the phosphorylated tyrosine at position 1007 (pY1007), in the activation loop of JAK2, as the specific counterpart for SOCS-1 interaction (Yasukawa et al., 1999Go; Ungureanu et al., 2002Go). Moreover, it was demonstrated that an additional 24 residues amino-terminal to the SH2 domain were important for JAK2 binding and for the biological function of SOCS-1. More precisely, SOCS-1 mutants lacking the whole amino-terminal region (amino acids 3–75) or just the part adjacent to the SH2 domain (residues 47–75) failed to suppress IL-6-induced signalling (Narazaki et al., 1998Go). Mutational analysis of the F56–G79 segment on SOCS-1 highlighted specific residues for JAK2–SOCS-1 association. Twelve amino acids (I68–G79) were required for binding to different peptides containing pY1007. Mutation of I68 and L75 markedly reduced the interaction of SOCS-1 with the kinase domain of JAK2 and with pY1007, whereas mutations at F56 and F59 abolished the ability of SOCS-1 to inhibit the EPO/STAT5 signalling and had detrimental effects on JH1 binding (Yasukawa et al., 1999Go). Some of these residues appeared to be conserved on the region amino-terminal to the SH2 domain of STAT proteins (Yasukawa et al., 1999Go). These studies led to the identification of two additional functional domains on SOCS-1: the extended SH2 subdomain (ESS) (residues 68–79) necessary for binding to the pY1007 phosphopeptides and the kinase inhibitory region (KIR) (amino acids 56–67) responsible for the high-affinity binding to the kinase domain of JAK2 (Yasukawa et al., 1999Go). The kinase inhibitory region was compared with the activation loop of JAK2 and a possible interaction with the catalytic groove of JAK2 was suggested based on sequence similarity (Yasukawa et al., 1999Go).

The SOCS box represents the last distinct structural feature on SOCS-1. The amino acid sequence of this motif appears to be very conserved among the SOCS members, suggesting a similar role in each protein. Extensive database searches indicated that the C-terminal SOCS box was present on another 12 proteins belonging to four different structural classes (Hilton et al., 1998Go). These proteins shared only the SOCS box-like sequence with the SOCS proteins and they contained different structural features (WD-40 repeats, SPRY domains, ankyrin repeats and GTPase domains) on their amino-terminal regions. The SOCS box of SOCS-1 is not required for the suppression of cytokine signalling (Zhang et al., 2001Go). It has been demonstrated that SOCS-1 mutants lacking the carboxy-terminal region, including the SOCS box motif, retained the ability to inhibit STAT5 activation of transcription (Yasukawa et al., 1999Go) and IL-6-mediated signalling (Narazaki et al., 1998Go). However, the SOCS-box of SOCS-1 was essential for the association of SOCS-1 with the Elongin BC complex (Kamura et al., 1999; Zhang et al., 1999Go).

The Elongin BC complex was discovered as an activator of RNA polymerase II elongation factor Elongin A (Bradsher et al., 1993Go; Aso et al., 1995Go). It was also identified as a component of the multiprotein von Hippel–Lindau (VHL) tumor suppressor complex (Duan et al., 1995bGo; Kibel et al., 1995Go). Interestingly, Elongin A, VHL protein and SOCS-1 SOCS box shared a single conserved region in their amino acid sequences that has been defined as a consensus Elongin BC binding site (BC box) (Kamura et al., 1998Go). Deletion of the whole motif, or mutations in it, unpaired the protein–Elongin BC association (Aso et al., 1995Go, 1996Go; Duan et al., 1995aGo,bGo; Kibel et al., 1995Go; Kishida et al., 1995Go; Kamura et al., 1998Go; Zhang et al., 1999Go). Solution of the structure of VHL–Elongin BC complex (Stebbins et al., 1999Go) confirmed those results and underlined specific hydrophobic interactions involving the segment on the VHL protein that is conserved in both Elongin A and SOCS-1. Additionally, employing fold recognition methods, Stebbins et al. proposed a sequence structure alignment between the Elongin BC binding site and the SOCS box of SOCS-1 (Stebbins et al., 1999Go). The alignment displayed strong conservation of the exposed hydrophobic residues of VHL protein known to bind the Elongin BC complex.

Elongins B and C were shown to lead proteins to proteasomal destruction through the Elongin B–proteasome or the Elongin BC–Cullin-2 interaction (Conaway et al., 1998Go; Kaelin and Maher 1998Go; Maxwell et al., 1999Go). Kamura et al. recently suggested that Cullin-5 associates with SOCS box-containing proteins, including SOCS-1, via the Elongin BC complex (Kamura et al., 2001Go). In a similar manner, the SOCS box bound to Elongin BC could assist the SOCS proteins in leading JAK tyrosine kinases to degradation, thereby interrupting cytokine signalling. However, the role of the SOCS box is still a matter of debate. It was reported that SOCS-1 was protected from proteolytic degradation by the SOCS box, because treatment with proteasome inhibitors increased the levels of expression of SOCS-1 mutants, lacking the SOCS box motif (Narazaki et al., 1998Go). Zhang et al. proposed a different model for the functional role of SOCS box, based on the fact that SOCS-3 was degraded in a proteasome-dependent manner (Zhang et al., 1999Go). According to this model, SOCS proteins first bind to JAK tyrosine kinase, followed by binding of the SOCS box to the Elongin BC complex. Finally, either through direct interaction of Elongin B with the proteasome or through associated Cullin-5-induced ubiquitination of substrates and subsequent proteasomal association, the JAK and the SOCS proteins are destroyed. Kamizono et al. provided experimental data supporting this model (Kamizono et al., 2001Go). They discovered that co-expression of SOCS-1 drastically accelerated the degradation of TEL–JAK2 fusion proteins and that this rapid degradation was significantly delayed by treatment of the cells with proteasome inhibitors. In line with these results, De Sepulveda et al. reported that SOCS-1 associated with the hematopoietic-specific nucleotide guanine nucleotide exchange factor (VAV) and led VAV to the ubiquitination machinery and final proteasomal degradation (De Sepulveda et al., 2000Go). Moreover, Ungureanu et al. indicated that the ubiquitin–proteasome pathway negatively regulates tyrosine-phosphorylated JAK2 in cytokine receptor signalling and that this model requires SOCS-1–JAK2 complexation (Ungureanu et al., 2002Go).

The correct signal blocking carried out by SOCS-1 is a fundamental process for the preservation of vital processes inside the cell. The molecular mechanisms involved in this important event are still unknown, because no experimentally derived structure of SOCS-1 is available to date. We therefore decided to predict the structure of SOCS-1 by computer. The models of the three-dimensional structures of SOCS-1 and of its association with both JAK2 and Elongin BC are presented here and the available experimental information is evaluated using those structures.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The amino acid sequences used in the present studies were retrieved from the SWISSPROT database (Bairoch and Apweiler, 2000Go). Multiple sequence alignment was accomplished employing CLUSTALW (Thompson et al., 1994Go) and residue conservation was calculated exploiting phylogenetic relationships (Clamp, 1998Go). Sequence-to-structure alignments were obtained using the following fold recognition methods: GenThreader and Threader (Jones, 1999bGo), TopLign (Thiele et al., 1999Go), 3D-PSSM (Kelley et al., 2000Go) and UCLA–DOE (Fischer and Eisenberg, 1996Go). Secondary structure assignments were carried out using PSIpred V2.0 (Jones, 1999aGo) and Predict Protein (Rost and Sander, 1993Go, 1994Go; Rost et al., 1994Go).

Coordinate files of template proteins were downloaded from the Protein Data Bank (Berman et al., 2000Go). Structural neighbor searching and comparison were performed with DALI (Holm and Sander, 1993Go), FSSP (Holm and Sander, 1996Go), SCOP (Murzin et al., 1995Go), VAST (Gibrat et al., 1996Go), CATH (Orengo et al., 1997Go) and CE (Shindyalov and Bourne, 1998Go).

The three-dimensional models of the SOCS-1 SOCS box and the KIR-ESS-SH2 domain were generated by application of restraint-based homology modelling as implemented in the program MODELLER V.4 (Sali and Blundell, 1993Go). Loop fragments were chosen according to the best score for homology and r.m.s. fit of the anchor residues as calculated by loop database search algorithms (Jones and Thirup, 1986Go; Claessens et al., 1989Go).

The orientation of the SOCS box domain with respect to the remainder of the SOCS-1 molecule was determined using the program 3D_Dock (Aloy et al., 1998Go). The SOCS box model was docked employing an angular increment of 3' during the search. The resulting complexes were sorted according to surface complementarity and electrostatic score values. An empirical scoring of the complexes, using residue level pair potentials (Aloy et al., 1998Go), was also performed. The same procedure was followed in generating the complex between SOCS-1 and JAK2 JH1. However, in this case experimental information, based on site-directed mutagenesis, was employed for filtering the results. The JH1 domain of JAK2 used in the docking with SOCS-1 was built in its active state, with the activation loop in the open conformation. This model had previously been generated (Lindauer et al., 2001Go) based on the three-dimensional structure of the phosphorylated Insulin Receptor (IR) Tyrosine Kinase (Hubbard, 1997Go).

Docking of different phosphopeptides, belonging to the JAK2 JH1 activation loop, to the SH2 domain of SOCS-1 was accomplished using the QXP program (McMartin and Bohacek, 1997Go). During docking the different peptides were treated as completely flexible, as well as the side chains present on SOCS-1. SOCS-1 side chains were allowed to move freely for up to 0.5 Å and then subjected to a quadratic 20 kJ/mol.Å2 penalty. Each docking run lasted for 20 000 steps of Monte Carlo perturbation, subsequent fast search step and final energy minimization. The results were evaluated in terms of total estimated binding energy.

The predicted interfaces of SOCS-1 with both the Elongin BC complex and the JAK2 JH1 domain were energy minimized for a total of 5000 steps (200 of steepest descent and 4800 of conjugate gradient) with AMBER (Pearlman et al., 1994); 80 ps of molecular dynamics simulation served to optimize side chain geometry and the energy minimization was repeated. In the case of SOCS-1–JH1 complex, the activation loop of JAK2 was left completely unconstrained during the molecular dynamics run.

The final three-dimensional models were analysed with the software Procheck (Laskowski et al., 1993Go).

Molecular interaction fields were calculated using GRID (Goodford, 1985Go) and electrostatic potentials were evaluated with Delphi (Nicholls and Honig, 1991Go).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
An overall model of SOCS-1 is presented in Figure 1AGo. The model comprises the SH2 domain, the KIR-ESS region and the SOCS Box domain. In the following these domains are described in detail.



View larger version (55K):
[in this window]
[in a new window]
 
Fig. 1. (A) Overall model of SOCS-1 comprising the KIR-ESS region (black), the SH2 (grey) and the SOCS box (white) domain. (B) Close-up of the SOCS box domain of SOCS-1. (C) Interface between the KIR-ESS region (black) and the SH2 domain (grey) in the proposed SOCS-1 model. (D) Close-up of the predicted interface between the SH2 (grey) and the SOCS box domain (white) of SOCS-1. Figure prepared with Molscript (Kraulis, 1991Go).

 
SH2 domain

Application of fold recognition methods indicated several experimental structures as suitable templates for the central part of the SOCS-1 amino acid sequence (L85–A167). All matches found displayed rather high values. The selection of the templates was based on pairwise energy sum and solvation energy sum scores, as implemented in Threader (Jones et al., 1999bGo). The SH2 domains belonging to the growth factor receptor binding protein (Rahuel et al., 1998Go) and the amino-terminal SH2 domain of Syp tyrosine phosphatase (Lee et al., 1994Go) were chosen and the sequence alignment with SOCS-1 is shown in Figure 2Go.



View larger version (75K):
[in this window]
[in a new window]
 
Fig. 2. Alignment for the KIR-ESS-SH2 regions of SOCS-1 (G52–L167). 1bg1, 1bf5, 1aya and 1zfp are the PDB identifiers for the three-dimensional structures of STAT-3ß (Becker et al., 1998Go), STAT-1 (Chen et al., 1998Go), Syp tyrosine phosphatase (Lee et al., 1994Go) and growth factor receptor binding protein (Rahuel et al., 1998Go), respectively. Secondary structure prediction for JAK2 generated with PSIpred. Secondary structure assignments for the template structures are indicated as follows: H, helix; E, extended strand; C, coil; B, isolated beta-bridge; G, 3/10 helix; S, bend; T, hydrogen-bonded turn. Black shading indicates amino acid identities and grey shading represents conserved physico-chemical properties. Start (>) and end (<) of the domains are indicated.

 
The fold of the two structures is very similar, as indicated by a root mean square deviation (r.m.s.d.) value of 0.609 Å when superimposed using the {alpha}-carbon atoms of residues in the secondary structure elements. SOCS-1 aligned well with both structures: the physico-chemical properties of the residues appeared to be conserved and there was a good agreement between the observed and predicted secondary structure features. The proposed SH2 model of SOCS-1 folds into a three-stranded ß-sheet (T101–D106, F114–M120 and G123–F131) flanked by two {alpha}-helices (V87–R95 and L148–A157) and one additional ß-strand (R135–H137).

The conserved arginine responsible for the interaction with phosphotyrosines corresponds to R105 in SOCS-1. The phosphotyrosine-binding (PTB) pocket is made up by the coil segment before the first {alpha}-helix and its terminus (L8–V87), the last residues of the first ß-strand and the following loop fragment (R105–Q109), the middle part of the second ß-strand (A115–S117) and a segment on the third ß-strand (S126–R128). The edges of the PTB groove are delineated by the side chains of V87, R108, Q109, A115, S126 and R128. The side chain conformation of R105 is stabilized in its geometry by H-bond interactions with the backbone carbonyl oxygen of L85 and the side chain oxygen of S117. Additionally, the side chains of R128 and Q109 are linked by two H-bonds.

Electrostatic potential calculations highlighted that the side chains of R105, R108 and R128 create a strong positive electrostatic field surrounding the phosphotyrosine-binding pocket (Figure 3AGo).



View larger version (75K):
[in this window]
[in a new window]
 
Fig. 3. (A) Molecular electrostatic potential of the phosphotyrosine peptide-binding site of SOCS-1 (left) and most favourable hydrophobic potential on the SOCS box domain (right). Potentials were calculated with DelPhi and GRID, respectively and were displayed with GRASP (Nicholls et al., 1991Go). (B) Close-up of the interface between the SOCS-1 SOCS box (green) and the Elongin C (yellow). (C) Close-up of the SOCS-1 SH2 domain (green)–JAK2 activation loop (red) interface. (D) Interface between SOCS-1 KIR region (green) and the catalytic core of JAK2 (red). Parts (B), (C) and (D) drawn with Molscript (Kraulis, 1991Go).

 
KIR-ESS

The region amino-terminal to the SH2 domain of SOCS-1 was found to be homologous to a comparable segment located before the SH2 domain of both STAT-1 (Chen et al., 1998Go) and STAT-3ß (Becker et al., 1998Go). The two parts of the crystal structures are practically identical (r.m.s. 0.341 Å) with many amino acid identities and high conservation of physico-chemical properties. The corresponding sequence on SOCS-1 aligned well with the templates, including a good agreement between the secondary structure predictions and the experimentally derived assignments (Figure 2Go).

The predicted model for SOCS-1 KIR-ESS contains two {alpha}-helices (R57–R69 and L74–A77) linked by a small coil segment. The two helices are orthogonal to each other and they display an ion pair between R67 on the first helix and D76 on the second.

SOCS box

The SOCS box motif of SOCS-1 displayed strong homology with the corresponding amino acid stretch of the crystal structure of VHL protein in the complex with Elongin BC (Stebbins et al., 1999Go). The obtained alignment (Figure 4Go) displays several amino acid identities; physico-chemical properties of the amino acids are very conserved, and also secondary structure elements. The resulting model consists of three {alpha}-helices (L175–A185, R189–R194 and P199–S207) connected by short coil segments. The relative helix–helix orientation is stabilized by two H-bonds between the side chain of R180 and the backbone carbonyl oxygen atoms of both S207 and Y204, whose side chains in turn interact with the side chain of Q176 (Figure 1BGo).



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4. Sequence–structure alignment of the SOCS-1 SOCS box. 1vcb is the PDB accession number for the VHL–Elongin BC complex (Stebbins et al., 1999Go). Colour shadings as in Figure 1Go.

 
SH2–KIR-ESS interface

The interface between the SH2 domain and the KIR-ESS region was derived using the structures of STAT-1 (Chen et al., 1998Go) and STAT-3ß (Becker et al., 1998Go) and the alignment shown in Figure 2Go. The overlap region with the template structures encompasses the initial part of their SH2 domains (first {alpha}-helix and the first two ß-strands). The whole SH2 domains of STAT proteins were not included in the alignment because their confidence values for fold assignment were lower than those obtained employing only the first part.

Several amino acid identities were found between the template structures and the r.m.s. values for the relative superimposition were very low (0.626 Å in the worst case).

The SH2 domain of SOCS-1 participated in the interface with a turn segment (F80–Y81), the end of the first ß-strand, the following coil region and the first residues of the second ß-strand (D106–F114), the coil region between the third and fourth ß-strands (F131–F136) and the initial part of the second {alpha}-helix (C147–F149). KIR-ESS contributed with the whole structure to the interface. Between the two moieties several interactions were found. The side chain of R60 was hydrogen-bonded to the backbone carbonyl oxygen of D106 and showed a salt bridge with D64. The side chain of R66 was linked with two H-bonds to both the side chain and backbone atoms of N111. R67 was involved in electrostatic interactions with D76 and D106, the latter of which in turn interacted via an H-bond with the hydroxyl group of Y81 (Figure 1CGo). Hydrophobic interactions also contributed to the interface. In particular, the side chain of S72 was closely packed to that of L148, whereas L75 was in close contact with the side chains of L148 and F149 as part of an extensive hydrophobic network involving F80, V104, F114 and L152.

SH2–SOCS box interface

The interface between the SH2 domain and the SOCS-1 box motif was derived by docking the SOCS box on to the SH2 region using 3D_Dock (Aloy et al., 1998Go). The best solutions, according to surface complementarity scores and electrostatic and pair potential values, were further filtered according to the distance between the two ends of the proteins, as it was necessary to accommodate a linker of four amino acids (R168–R171). In the best complex, the SOCS box bound to the loop region between the fourth ß-strand and the second {alpha}-helix (G140–R142), alongside the coil segment after the second {alpha}-helix (A158–P166) of the SH2 domain. Specific interactions between the two proteins involved H-bonds between the side chain of N198 and the main chain carbonyl group of S141, and also H-bonds between the backbone atoms of the side chains of A121 and Q211. Two ion pairs linked the side chain of R160 with E177 and the carboxy group of terminal I212 (Figure 1DGo). Hydrophobic contacts were displayed by the pyrrolidine rings of P159 and P174 and by the branched side chains of L163 and V200. The short coil segment between the SH2 domain and the SOCS box motif (R168–R171) was modelled using loop-database searching techniques (Jones and Thirup 1986Go; Claessens et al., 1989Go).

JAK2–SOCS-1–Elongin BC complex

The overall complex formed by JAK2, SOCS-1 and Elongin BC is depicted in Figure 5Go. SOCS-1 displays extensive interactions with both JAK2 and Elongin BC. According to the model, there are no molecular contacts between JAK2 and Elongin BC. The two interfaces involving SOCS-1 are described in the following sections.



View larger version (52K):
[in this window]
[in a new window]
 
Fig. 5. Overall model of the complex between JAK2 (red), SOCS-1 (yellow) and Elongin BC (green). Figure prepared with Molscript (Kraulis, 1991Go).

 
SOCS box–Elongin BC interface

The predicted model of the SOCS-1 SOCS box was superimposed on the corresponding region of the VHL protein (Stebbins et al., 1999Go), in order to create a three-dimensional model for the interaction between SOCS-1 and the Elongin BC complex. Fitting the {alpha}-carbon atoms, an r.m.s. value of 0.459 Å was obtained. In order to account for possible inaccuracies during the superimposition the whole complex was first energy-minimized, subsequently the side chains were subjected to a 60 ps molecular dynamics run and the final coordinates were energy-minimized again.

The resulting complex showed that Elongin BC interacts with the SOCS box employing Elongin C’s {alpha}-helices and coil segment [V73–C112 equivalent to H3, H4 and L5 following the notation used by Stebbins et al. (Stebbins et al., 1999Go)]. Additionally, another part of SOCS-1 (R142–M161, comprising the second {alpha}-helix of the SH2 domain) was found in contact with T38–P49, a short {alpha}-helix (H2) and flanking residues (L2 and L3) and also with H4 on the Elongin C structure. The interactions between SOCS-1 and Elongin BC are displayed in Figure 3BGo and summarized in Table IGo.


View this table:
[in this window]
[in a new window]
 
Table I. Interacting amino acids in the proposed SOCS-1–Elongin BC interface
 
The SOCS-1–Elongin BC interface was also analysed with respect to its hydrophobic properties. To this end, a molecular interaction field analysis was performed with the DRY probe implemented in GRID. This analysis indicated a wide hydrophobic patch on the SOCS box domain (Figure 3AGo), which is in contact with the lipophilic counterpart on Elongin C in the predicted complex.

SOCS-1/JAK2 JH1 interface

The orientation between the tyrosine kinase domain (JH1) of JAK2 and the SOCS-1 protein was predicted using docking simulations, as described in the Methods section. The final complex displayed different areas of interactions between the two partners.

SOCS-1 participated in the complex with the KIR-ESS region and with the first amino acids of the SH2 domain (G52–H88), the end of the first ß-strand, the following turn and half of the second ß-strand (R105–L116), and also with the region preceding the second {alpha}-helix on the SH2 domain (S126–D146). The JAK2 JH1 domain contributed to the complex with part of its activation loop (D994–W1020), the nucleotide binding loop and flanking residues (Q854–G861), part of the third {alpha}-helix (Y934–Q942), the second half of the catalytic loop (H974–N981), the end of the sixth {alpha}-helix and the subsequent coil stretch (V1042–V1075).

A number of stabilizing interactions were found both in the interface between the activation loop of JAK2 JH1 and the SH2 domain of SOCS-1 (Table IIGo and Figure 3CGo) and also in the interface formed by the KIR-ESS region of SOCS-1 and the JH1 catalytic core (Table IIIGo and Figure 3DGo).


View this table:
[in this window]
[in a new window]
 
Table II. Interacting amino acids in the proposed SOCS-1 SH2–JAK2 activation loop interface
 

View this table:
[in this window]
[in a new window]
 
Table III. Interacting amino acid in the proposed SOCS-1 KIR-ESS–JAK2 JH1 interface
 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
KIR-ESS

The predicted model of SOCS-1 displayed three essential structural features: an amino-terminal region, an SH2 domain and a SOCS box motif. The amino-terminal region showed homology with the region preceding the SH2 domain in the crystal structures of STAT-1 (Chen et al., 1998Go) and STAT-3ß (Becker et al., 1998Go), as indicated earlier by sequence alignment (Yasukawa et al., 1999Go). This part of the STAT proteins packs closely against part of the SH2 domain and makes contact with the three-stranded-antiparallel ß-sheet. In the SOCS-1 model the same kind of contacts could be preserved.

Electrostatic interactions were found between R60 and D106, R66 and N111, and R67 and D106. The amino acids L75, L148 and F149 were engaged in hydrophobic contacts. The network of interactions displayed could therefore account for the stability of the inter-domain orientation.

Yasukawa et al., (1999)Go divided the region amino-terminal to the SH2 domain of SOCS-1 (F56–G79) into two further subdomains: F56–R67 and I68–G79. The first one was re-named Kinase Inhibiting Region (KIR) and the second Extended SH2 Subdomain (ESS).

Detailed mutational analysis revealed that KIR and ESS regions, alongside the SH2 domain, were required for JAB activity (Yasukawa et al., 1999Go). Moreover, substitution of I68 and L75 with glutamic acids reduced the interaction of SOCS-1 with the phosphopeptides belonging to the activation loop of JAK2 (Yasukawa et al., 1999Go).

In the proposed SOCS-1 model, I68 and L75 are located on the first and second {alpha}-helix of the KIR-ESS region, respectively. The side chain of I68 shows a van der Waals (vdW) contact with the phenyl group of Y81 on the SH2 domain, while L75 is involved in a network of hydrophobic interactions with F80, L148 and F149. I68 is partially buried in the interface between the two {alpha}-helices of KIR and ESS and is close to an acidic residue (D76). It would therefore be possible that an I68D mutation disrupts the fold in that area because of electrostatic repulsion. The side chain of L75 is situated opposite to the phenyl ring of F149. This zone is part of the hydrophobic core that stabilizes the relative orientation between the first and second helix of the ESS region. Also in this case mutation to a charged residue should have destabilizing effects.

SH2 domain

The SH2 domain of SOCS-1 stretches from residues F80 to L167. Many of its amino acids are conserved when compared with the protein used for the model building, especially in the first ß-strand and flanking residues (G100–S106). This structural element forms the basis for the phosphotyrosine-binding (PTB) domain on SOCS-1. The importance of the SOCS-1 SH2 region stems from the fact that its correct interaction with the phosphotyrosine at position 1007 (pY1007) in the activation loop of JAK2 JH1 is a necessary step for inhibition of the cytokine-mediated signalling (Nicholson et al., 1999Go; Ungureanu et al., 2002Go).

In the model R105 is the basic amino acid, identical in all the SH2 domains (Figure 6Go), that primarily interacts with the phosphate group on a phosphotyrosine. This is in line with the experimental observation that mutation of R105 (Narazaki et al., 1998Go; Yasukawa et al., 1999Go) compromised the interaction with the phosphotyrosine, unpaired the SOCS-1–JAK2 JH1 association and resulted in a loss of SOCS-1 inhibiting activity.



View larger version (54K):
[in this window]
[in a new window]
 
Fig. 6. Multiple sequence alignment of different SH2 domains for which experimental three-dimensional structures are currently available. The sequence of the SOCS-1 SH2 domain is shown at the top as a reference. Symbols *, # and @ indicate SOCS-1 R105, R108 and R128, respectively.

 
In addition to R105, other positively charged amino acids (R108 and R128) are found on the edges of the PTB and they could assist in coordinating the phosphotyrosine. The physico-chemical properties of R128 appear to be conserved throughout the alignment (Figure 6Go). A structural analysis of the SH2–phosphopeptide complexes currently available highlighted the importance of a basic and long residue at that position on the third ß-strand of common SH2 domains. Most of the complexes displayed vdW contacts between the phenyl ring of the phosphotyrosine and the side chain carbon atoms, whilst electrostatic interactions occurred between the phosphate group and the residue basic function.

R108 is not conserved (Figure 6Go). Many SH2 domains show an acidic residue at the same position in the loop between the second and third ß-strands. The only protein displaying a positively charged amino acid at the same position is the Syp tyrosine phosphatase (Lee et al., 1994Go). Moreover, the phospholipase C-{gamma}-1 (PLC-{gamma}-1) contains an arginine next to that position. Interestingly, in the three-dimensional complex between a high-affinity phosphopeptide and the SH2 domain of PLC-{gamma}-1 (Pascal et al., 1994Go), that arginine formed a hydrogen bond with the phosphate group of the phosphopeptide.

The PTB pocket of SOCS-1 differs from others also with respect to the presence of Q109 and S126. In other SH2-containing proteins those residues are often replaced by a serine and by a histidine, respectively. Therefore, the differences in the amino acids surrounding the PTB pocket of SOCS-1 could be exploited for the specific inhibition of this enzyme.

SOCS-1–JAK2 JH1 complex

Experimental results demonstrated that the binding between SOCS-1 and JAK2 depends on two SOCS-1 structural elements: the Kinase Inhibiting Region (KIR) and the PTB pocket. In particular, Yasukawa et al. indicated in the KIR of SOCS-1 a number of mutations that diminished the affinity of binding between SOCS-1 and JH1 (Yasukawa et al., 1999Go). It has also been demonstrated that the phosphorylation of JAK2 is an important step for the SOCS-1-mediated inhibition of cytokine signalling (Endo et al., 1997Go). Furthermore, it has been shown that the phosphorylated Y1007, located on the activation loop of JAK2, binds to the SH2 domain of SOCS-1 (Ungureanu et al., 2002Go). For these reasons, a rigid docking simulation was first performed between the JH1 domain of JAK2, modelled in its active state (Lindauer et al., 2001Go), and the model of SOCS-1. Starting from the best-obtained complex, a subsequent flexible docking simulation was carried out in order to accommodate the activation loop of JAK2 on to the SH2 domain of SOCS-1.

The final model for the complex between SOCS-1 and JAK2 JH1 domain consists of two main interfaces: the first is made up by the SH2 domain of SOCS-1 and the activation loop of JAK2 (Figure 3CGo), whereas the second is formed by the KIR region of SOCS-1 and the catalytic core of JAK2 (Figure 3DGo).

The complex showed the first {alpha}-helix (R57–R59) of the SOCS-1 model packed tightly against the first ß-strand of JH1 (L855–N859) and the coil segment between H974 and N981. The segment containing residues L855–N859 is part of the nucleotide-binding loop (NBL), necessary for the coordination of the ATP molecule. D976, R980 and N981 are part of the second half of the catalytic loop (C-loop), required for phosphotransfer reaction and coordination of Mg2+. In the proposed model of the SOCS-1–JAK2 JH1 complex there are therefore two regions of interaction with segments of the JH1 domain that are crucial for kinase activity. According to the model, the SOCS-1–JH1 interactions lead also to a potential obstruction of the ATP binding pocket of JH1. When an ATP analog taken from the insulin receptor crystal structure was fitted into the ATP binding site of JH1, it became evident that in the predicted SOCS-1–JAK2 JH1 complex part of the ATP binding pocket of JH1 is occupied by part of the {alpha}-helix of KIR, including H62–R66, thus making it impossible to accommodate the triphosphate group of ATP.

Based on sequence similarity, it was suggested that KIR resembled somewhat the activation loop of JAK2 and that it could function as a non-phosphorizable ‘pseudosubstrate’ or mimic the activation loop regulating the access to the catalytic groove (Yasukawa et al., 1999Go). To test the first hypothesis, a comparison was made with the IR structure since it also contained a peptide substrate. In the predicted complex the KIR region did not occupy the position of the peptide substrate but was shifted towards the catalytic groove. Interestingly, the loop between the first and second ß-strands on SOCS-1 SH2 domain resided in the substrate location.

The predicted SOCS-1–JH1 complex therefore suggests that the KIR region inhibits JAK2 tyrosine kinase activity by obstructing the access of both ATP and substrate to their respective binding sites. The proposed complex could therefore serve for the design of specific JAK2 inhibitors. N859 and K857 on the nucleotide-binding loop and R980 on the catalytic loop display positive interactions with the KIR-ESS region of the SOCS-1 protein. These specific residues on JAK2 JH1 could thus represent interesting targets for structure-based design strategies, in order to inhibit this enzyme.

The KIR region of SOCS-1 has been extensively mutated by Yasukawa et al. (Yasukawa et al., 1999Go). A D64R mutation reduced the ability of SOCS-1 to bind to JH1. In the predicted complex D64 is closely packed against the JH1 model and displays a hydrogen bond with the side chain of N859. The introduction of a long arginine side chain could possibly disrupt this close contact and would generate electrostatic repulsion due to the presence of K857 on JH1.

The most detrimental effects on SOCS-1 binding to JH1 were caused by mutations of F56 and F59. However, when the hydrophobic properties of these two residues were conserved in the mutations, SOCS-1 mutants were as effective as the wild-type (Yasukawa et al., 1999Go). In the complex the side chains of F56 and F59 are in direct contact with JH1. F59 is deeply buried and displays hydrophobic contacts with I1018 and Y1021. F56 in turn is engaged in interactions with both L997 and L1001. This region represents the centre of a hydrophobic patch that involves also Y972, I973, L1026 and F1031. The model therefore indicates that this hydrophobic part of the interface is important for the stability of the SOCS-1–JH1 complex and agrees well with mutational data. It would therefore be interesting to mutate the hydrophobic amino acids of JAK2 indicated above in order to test the proposed interaction model.

Sasaki et al. showed that another SOCS family member, SOCS-3, exploits its KIR region to bind the kinase domain (JH1) of JAK2 (Sasaki et al., 1999Go). Interestingly, the sequence identity between SOCS-1 and SOCS-3 within the KIR region is very high. Therefore, based on the sequence alignments, one may conclude that the SOCS-3 KIR region binds to JAK2 in a similar way to KIR of SOCS-1.

More interactions between SOCS-1 and JH1 can be found in the interface between the SH2 domain of SOCS-1 and the activation loop of JH1 (Table IIGo). In the model obtained, the phosphate group of phosphorylated tyrosine (pY1007) in the activation loop is deeply buried in the phosphotyrosine-binding pocket (PTB) of SOCS-1 (Figure 3CGo). Specifically, pY1007 interacts with R105 and R128. The phenyl moiety of the tyrosine is packed against the peptide bond between R108 and Q109. Several other interactions between the SH2 domain of SOCS-1 and the activation loop of JH1 were found in the proposed complex (Table IIGo and Figure 3CGo). Additionally to the phosphotyrosine interactions, a strong electrostatic complementarity between the activation loop of JAK2 and the SH2 domain of SOCS-1, supported mainly by three different ion pairs (Table IIGo), seems to be the driving force of the binding event. Specifically, E1006, E1012 and E1015 could represent interesting targets for mutational experiments. According to the results, the side chains of N111 and S126 are buried in the interface with SOCS-1 as well, whereas residues at position pY+1 to pY+3 appeared to be more solvent exposed and they do not participate directly in the binding. Interestingly, many of these residues are not conserved in the SOCS protein family and could therefore contribute to the specificity of SOCS-1. Using this information, it should be possible to design phosphopeptides selectively interacting with SOCS-1.

The proposed model for SOCS-1-mediated JAK2 inhibition would therefore involve multiple stages: phosphorylation of Y1007 on the activation loop of JAK2 serves to recruit SOCS-1 through the interaction with its SH2 domain. This event then allows the correct positioning of the KIR-ESS domain of SOCS-1 on to the catalytic groove. Both interactions between the two proteins, namely the interaction of SOCS-1 SH2 with the activation loop of JH2 and also the interaction of SOCS-1 KIR-ESS with the catalytic groove of JH2, are required for a stable association between JAK2 and SOCS-1. This would also explain why mutants of SOCS-1 with a defective or absent KIR are unable to inhibit JAK2 activity. As already indicated above, this hypothesis could be tested by introducing mutations on the JH2 amino acids of JAK2 that are predicted to interact with F56 and F59 on the KIR-ESS of SOCS-1, while leaving SOCS-1 unchanged.

SOCS box and SOCS-1–Elongin BC complex

The SOCS box domain of SOCS-1 was first reported to protect SOCS-1 from proteolytic degradation (Narazaki et al., 1998Go). Later, however, several groups (Zhang et al., 1999Go; Kamizono et al., 2001Go; Ungureanu et al., 2002Go) supported the hypothesis that the SOCS box is responsible for the binding of SOCS-1 and its binding partner JAK2 to the Elongin BC and for their subsequent proteosomal degradation.

The results presented here suggest that the binding between SOCS-1 and Elongin BC is highly stabilized by several interactions and that hydrophobic forces play a major role in the association (Figure 3BGo and Table IGo). Molecular interaction field calculations further support this model: a wide lipophilic cleft on the SOCS box domain is involved in complex formation (Figure 3AGo). The amino acids that create the predicted pocket are strongly conserved in the VHL amino acid sequence (Stebbins et al., 1999Go), as shown in Figure 4Go. Interestingly, the corresponding VHL residues are involved in extensive contacts with Elongin C in the crystal structure of the complex between VHL and Elongin BC (Stebbins et al., 1999Go).

According to the proposed model, amino acid conservation between the exposed hydrophobic residues of VHL protein and the SOCS box is important in order to retain the function of SOCS-1.

Conclusions

A theoretical model of SOCS-1 comprising the KIR-ESS region, the SH2 and the SOCS box domain has been built using comparative modelling and rigid docking.

The predicted model has been evaluated against currently available experimental data and is consistent with those data.

Multiple sequence alignments and structural comparison between the SH2 domain of SOCS-1 and the SH2 domains belonging to different protein classes indicated a number of amino acids that are divergent in the phosphotyrosine peptide binding site among the SH2 domains. These molecular differences could be exploited for ligand specificity towards SOCS-1.

A model for the molecular interaction between SOCS-1 and JAK2 has been proposed. According to the results, the KIR region of SOCS-1 protrudes towards the catalytic region of JAK2 and occupies the ATP binding site. This suggests that SOCS-1 inhibits the kinase activity of JAK2 avoiding substrate binding. The molecular interactions between SOCS-1 and JAK2 suggest new approaches for the design of selective JAK2 inhibitors.

A theoretical model for the SOCS-1–Elongin BC complex has also been built. A highly conserved hydrophobic pocket on the SOCS box domain of SOCS-1 is responsible for the association with Elongin BC. The proposed interactions could be tested by introducing point mutations in the predicted interface and by evaluating the biological activity of the mutants.

Taken together, SOCS-1 is able to abolish cytokine-mediated signalling in two different ways: the interaction between SOCS-1 and JAK2 causes inhibition of the tyrosine kinase activity of JAK2; in addition, JAK2 is led to proteosomal degradation by binding the JAK2–SOCS-1 complex to Elongin BC using the SOCS box of SOCS-1.

On the whole, the results provide a comprehensive picture of the different biological functions of SOCS-1 and can therefore be used to guide future experiments, in order to elucidate the role of SOCS-1 further.

The coordinates of the SOCS-1 model are available upon request from the authors via E-mail and can be downloaded from http://www.chem.qmul.ac.uk/ccs/SOCS1.pdb.gz.


    Notes
 
2 Present address: Molecular Modelling and Design, Discovery Research Oncology, Pharmacia, Viale Pasteur 10, 20014 Nerviano (MI), Italy Back

3 To whom correspondence should be addressed. E-mail: romano.kroemer{at}pharmacia.com Back


    Acknowledgments
 
The authors gratefully acknowledge support for this work from the Nuffield Foundation and from the National Foundation for Cancer Research (USA).


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Adams,T.E., Hansen,J.A., Starr,R., Nicola,N.A., Hilton,D.J. and Billestrup,N. (1998) J. Biol. Chem., 273, 1285–1287.[Abstract/Free Full Text]

Aloy,P., Moont,G., Gabb,H.A., Querol,E., Aviles,F.X. and Sternberg. M.J.E. (1998) Proteins, 33, 535–549.[CrossRef][ISI][Medline]

Aso,T., Lane,W.S., Conaway,J.W. and Conaway,R.C. (1995) Science, 269, 1439–1443.[ISI][Medline]

Aso,T., Haque,D., Barstead,R.J., Conaway,R.C. and Conaway,J.W. (1996) EMBO J., 15, 5557–5566.[Abstract]

Bairoch,A. and Apweiler,R. (2000) Nucleic Acids Res., 28, 45–48.[Abstract/Free Full Text]

Becker,S., Groner,B. and Muller,W. (1998) Nature, 394, 145–151.[CrossRef][ISI][Medline]

Berman,H.M., Westbrook,J., Feng,Z., Gilliland,G., Bhat,T.N., Weissig,H., Shindyalov,I.N. and Bourne,P.E. (2000) Nucleic Acids Res., 28, 235–242.[Abstract/Free Full Text]

Bradsher,J.N., Jackson,K.W., Conaway,R.C. and Conaway,J.W. (1993) J. Biol. Chem., 268, 25587–25593.[Abstract/Free Full Text]

Chen,X., Vinkemeier,U., Zhao,Y., Jeruzalmi,D., Darnell,J.E.,Jr and Kuriyan,J. (1998) Cell, 93, 827–839.[ISI][Medline]

Claessens,M., Van Cutsem,E., Lasters,I. and Wodak,S. (1989) Protein Eng., 2, 335–345.[Abstract]

Clamp,M. (1998) Jalview, http://www.ebi.ac.uk/~michele/.

Conaway,J.W., Kamura,T. and Conaway,R.C. (1998) Biochim. Biophys. Acta, 1377, M49–M54.[CrossRef][ISI][Medline]

De Sepulveda,P., Ilangumaran,S. and Rottapel,R. (2000) J. Biol. Chem., 275, 14005–14008.[Abstract/Free Full Text]

Diamond,P., Doran,P., Brady,H.R. and McGinty A. (2000) J. Nephrol., 13, 9–14.[CrossRef][ISI][Medline]

Duan,D.R., Humphrey,J.S., Chen,D.Y.T., Weng,Y., Sukegawa,J., Lee,S., Gnarra,J.R., Linehan,W.M. and Klausner,R.D. (1995a) Proc. Natl Acad. USA, 92, 6459–6463.[Abstract]

Duan,D.R., Pause,A., Burgess,W.H., Aso,T., Chen,D.Y.T., Garrett,K.P., Conaway,R.C., Conaway,J.W., Linehan,W.M. and Klausner,R.D. (1995b) Science, 269, 1402–1406.[ISI][Medline]

Endo,T.A., Masuhara,M., Yokouchi,M. and Yoshimura,A. (1997) Nature, 387, 921–924.[CrossRef][ISI][Medline]

Fischer,D. and Eisenberg,D. (1996) Protein Sci., 5, 947–955.[Abstract/Free Full Text]

Gibrat,J.F., Madej,T. and Bryant,S.H. (1996) Curr. Opin. Struct. Biol., 6, 377–385.[CrossRef][ISI][Medline]

Goodford,P.J. (1985) J. Med. Chem., 28, 849–857.[ISI][Medline]

Hansen,J.A., Lindberg,K., Hilton,D.J., Nielsen,J.H. and Billestrup,N. (1999) Mol. Endocrinol., 13, 1832–1843.[Abstract/Free Full Text]

Hilton,D.J., Richardson,R.T., Alexander,W.S. and Nicola,N.A. (1998) Proc. Natl Acad. Sci. USA, 95, 114–119.[Abstract/Free Full Text]

Holm,L. and Sander,C. (1993) J. Mol. Biol., 233, 123–128.[CrossRef][ISI][Medline]

Holm,L. and Sander,C. (1996) Science, 273, 595–602.[Abstract/Free Full Text]

Hubbard,S.R. (1997) EMBO J., 18, 5572–5581.[CrossRef]

Imada,K. and Leonard,W.J. (2000) Mol. Immunol., 37, 1–11.[CrossRef][ISI][Medline]

Jones,D.T. (1999a) J. Mol. Biol., 292, 195–202.[CrossRef][ISI][Medline]

Jones,D.T. (1999b) J. Mol. Biol., 287, 797–815.[CrossRef][ISI][Medline]

Jones,T.A. and Thirup,S. (1986) EMBO J., 5, 819–822.[Abstract]

Kaelin,W.G.,Jr and Maher,E.R. (1998) Trends Genet., 14, 423–427.[CrossRef][ISI][Medline]

Kamizono,S., Hanada,T., Yasukawa,H., Minoguchi,S. and Kato,R. (2001) J. Biol. Chem., 16, 12530–12538.[CrossRef]

Kamura,T., Sato,S., Haque,D., Liu,L., Kaelin,W.G., Conaway,R.C. and Conaway,J.W. (1998) Gen. Dev., 12, 3872–3881.[Abstract/Free Full Text]

Kamura,T., Burian,D., Yan,Q., Schmidt,S.L., Lane,W.S., Querido,E., Branton,P.E., Shilatifard,A., Conaway,R.C. and Conaway,J.W. (2001) J. Biol. Chem., 276, 29748–29753.[Abstract/Free Full Text]

Kelley,L.A., MacCullum,R.M., Sternberg,M.J.E. (2000) J. Mol. Biol., 299, 501–522.[CrossRef]

Kibel,A., Iliopoulos,O., DeCaprio,J.A. and Kaelin,W.G.,Jr. (1995) Science, 269, 1444–1446.[ISI][Medline]

Kishida,T., Stackhouse,T.M., Chen,F., Lerman,M.I. and Zbar,B. (1995) Cancer Res., 20, 4544–4548.

Kraulis,P.J. (1991) J. Appl. Crystallogr., 24, 946–950.[CrossRef][ISI]

Laskowski,R.A., McArthur,M.W., Moss,D.S. and Thornton,J.M. (1993) J. Appl. Crystallogr., 26, 283–291.[CrossRef][ISI]

Lee,C.H., Komrinos,D., Jacques,S., Margolis,B., Schlessinger,J., Shoelson,S.E. and Kuriyan,J. (1994) Structure, 15, 423–438.

Lindauer,K., Loerting,T., Liedl,K.R. and Kroemer,R.T. (2001) Protein Eng., 14, 27–37.[Abstract/Free Full Text]

Losman,J.A. Chen,X.P., Hilton D. and Rothman,P. (1999) J. Immunol., 162, 3770–3776.[Abstract/Free Full Text]

Masuhara,M., Sakamoto,H., Matsumoto,A. and Yoshimura,A. (1997) Biochem. Biophys. Res. Commun., 239, 439–446.[CrossRef][ISI][Medline]

Maxwell,P.H., Wiesener,M.S., Chang,G.W., Clifford,S.C., Vaux,E.C., Cockman,M.E., Wykoff,C.C., Pugh,C.W., Maher,E.R. and Ratcliffe,P.J. (1999) Nature, 399, 271–275.[CrossRef][ISI][Medline]

McMartin,C. and Bohacek,R. (1997) J. Comput. Aid. Mol. Des., 11, 333–344.[CrossRef][ISI]

Minamoto,S., Ikegame,K., Ueno,K. and Kishimoto,T. (1997) Biochem. Biophys. Res. Commun., 237, 79–83.[CrossRef][ISI][Medline]

Murzin,A.G., Brenner,S.E., Hubbard,T. and Chothia C. (1995) J. Mol. Biol., 247, 536–540.[CrossRef][ISI][Medline]

Naka,T., Narazaki,M., Hirata,M. and Kishimoto,T. (1997) Nature, 387, 924–929.[CrossRef][ISI][Medline]

Narazaki,M., Fujimoto,M., Matsumoto,T., Morita,Y., Saito,H., Kajita,T., Yoshizaki,K., Naka,T. and Kishimoto,T. (1998) Proc. Natl Acad. Sci. USA, 95, 13130–13134.[Abstract/Free Full Text]

Nicholls,A. and Honig,B. (1991) J. Comput. Chem., 12, 435–445.[ISI]

Nicholls,A., Sharp,K. and Honig,B. (1991) Proteins, 11, 281–287.[ISI][Medline]

Nicholson,S.E., Willson,T.A., Farley,A., Starr,R., Zhang,J.G., Baca,M., Alexander,W.S., Metcalf,D., Hilton,D.J. and Nicola,N.A. (1999) EMBO J., 18, 375–385.[Abstract/Free Full Text]

Orengo,C.A., Michie,A.D., Jones, S, Jones,D.T., Swindells,M.B. and Thornton,J.M. (1997) Structure, 5, 1093–1108.[ISI][Medline]

Pascal,S.M., Singer,A.U., Gish,G., Yamazaki,T., Shoelson,S.E., Pawson,T., Kay,L.E. and Forman-Kay,J.D. (1994) Cell, 77, 461–472.[ISI][Medline]

Pearlman,D.A., Case,D.A., Caldwell,J.W., Ross,W.S., Cheatham,T.E., DeBolt,S., Ferguson,D., Seibel,G. and Kollman,P. (1995) Comput. Phys. Commun., 91, 1–41.[CrossRef][ISI]

Pezet,A., Favre,H.,. Kelly,P.A and Edery,M. (1999) J. Biol. Chem., 274, 24497–24502.[Abstract/Free Full Text]

Rahuel,J., Garcia-Echeverria,C., Furet,P., Strauss,A., Caravatti,G., Fretz,H., Schoepfer,J. and Gay,B. (1998) J. Mol. Biol., 279, 1013–1022.[CrossRef][ISI][Medline]

Ram,P.A. and Waxman,D.J. (1999) J. Biol. Chem., 274, 35553–35561.[Abstract/Free Full Text]

Rost,B. and Sander,C. (1993) J. Mol. Biol., 232, 584–599.[CrossRef][ISI][Medline]

Rost,B. and Sander C. (1994) Proteins: Struct. Funct. Genet., 19, 55–77.[ISI][Medline]

Rost,B., Sander,C. and Schneider,R. (1994), CABIOS, 10, 53–60.[Medline]

Sakamoto,H. et al. (1998) Blood, 92, 1668–1676.[Abstract/Free Full Text]

Sali,A. and Blundell,T.L. (1993) J. Mol. Biol., 234, 779–815.[CrossRef][ISI][Medline]

Sasaki,A., Yasukawa,H., Suzuki,A., Kamizono,S., Syoda,T., Kinjyo,I., Sasaki,M., Johnston,J.A. and Yoshimura,A. (1999) Genes Cells, 4, 339–351.[Abstract/Free Full Text]

Shindyalov,I.N. and Bourne,P.E. (1998) Protein Eng., 11, 739–747.[Abstract]

Sporri,B., Kovanen,P.E., Yoshimura A. and Leonard,W.J. (2001), Blood, 97, 221–226.[Abstract/Free Full Text]

Starr,R., Wilson,T.A., Viney,E.M. and Hilton,D.J. (1997) Nature, 387, 917–921.[CrossRef][ISI][Medline]

Stebbins,C.E., Kaelin,W.G. and Pavlevich,N.P. (1999) Science, 284, 455–461.[Abstract/Free Full Text]

Thiele,R., Zimmer,R. and Lengauer,T. (1999) J. Mol. Biol., 290, 757–779.[CrossRef][ISI][Medline]

Thompson,J.D., Higgins,D.G. and Gibson,T.J. (1994) Nucleic Acids Res., 22, 4673–4680.[Abstract]

Tomic,S., Chugtai,N. and Ali,S. (1999) Mol. Cell. Endocrinol., 158, 45–54.[CrossRef][ISI][Medline]

Ungureanu,D., Saharinen,P., Junttila,I., Hilton,D.J. and Silvennoinen,O. (2002) Mol. Cell. Biol., 22, 3316–3326.[Abstract/Free Full Text]

Yasukawa,H., Misawa,H., Sakamoto,H., Masuhara,M. and Yoshimura,A. (1999) EMBO J., 18, 1309–1320.[Abstract/Free Full Text]

Yoshimura,A., Ohkubo,T., Kiguchi,T. and Miyajima,A. (1995) EMBO J., 14, 2816–2826.[Abstract]

Zhang,J., Farley,A., Nicholson,S.E. and Baca,M. (1999) Proc. Natl Acad. Sci. USA, 96, 2071–2076.[Abstract/Free Full Text]

Zhang,J. et al. (2001) Proc. Natl Acad. Sci. USA, 98, 13261–13265.[Abstract/Free Full Text]

Received September 30, 2002; revised December 9, 2002; accepted December 17, 2002.





This Article
Abstract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (11)
Request Permissions
Google Scholar
Articles by Giordanetto, F.
Articles by Kroemer, R. T.
PubMed
PubMed Citation
Articles by Giordanetto, F.
Articles by Kroemer, R. T.