Báo cáo khoa học: Secondary structure assignment of mouse SOCS3 by NMR defines the domain boundaries and identifies an unstructured insertion in the SH2 domain pdf

Secondary structure assignment of mouse SOCS3 by NMR
defines the domain boundaries and identifies an
unstructured insertion in the SH2 domain
Jeffrey J. Babon1, Shenggen Yao1, David P. DeSouza1,*, Christopher F. Harrison1,*, Louis J. Fabri2,
Edvards Liepinsh3, Sergio D. Scrofani2, Manuel Baca1,† and Raymond S. Norton1
1 Walter and Eliza Hall Institute, Parkville, Victoria, Australia
2 Amrad Corporation Ltd, Richmond, Victoria, Australia
3 Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden

Keywords
cytokine signalling; NMR; PEST sequence;
SOCS
Correspondence
J. J. Babon, Walter and Eliza Hall Institute,
1G Royal Parade, Parkville 3050, Victoria,
Australia
Fax: +61 3 93470852
Tel: +61 3 93452451
E-mail:
Present addresses
*School of Biochemistry, University of
Melbourne, Parkville 3050, Australia;
†Amrad Corporation Ltd, 576 Swan Street,
Richmond 3121, Victoria, Australia
(Received 31 July 2005, revised 22
September 2005, accepted 10 October 2005)
doi:10.1111/j.1742-4658.2005.05010.x

SOCS3 is a negative regulator of cytokine signalling that inhibits Janus
kinase-signal transduction and activator of transcription (JAK-STAT)
mediated signal tranduction by binding to phosphorylated tyrosine residues

on intracellular subunits of various cytokine receptors, as well as possibly
the JAK proteins. SOCS3 consists of a short N-terminal sequence followed
by a kinase inhibitory region, an extended SH2 domain and a C-terminal
suppressor of cytokine signalling (SOCS) box. SOCS3 and the related protein, cytokine-inducible SH2-containing protein, are unique among the
SOCS family of proteins in containing a region of mostly low complexity
sequence, between the SH2 domain and the C-terminal SOCS box. Using
NMR, we assigned and determined the secondary structure of a murine
SOCS3 construct. The SH2 domain, unusually, consists of 140 residues,
including an unstructured insertion of 35 residues. This insertion fits the
criteria for a PEST sequence and is not required for phosphotyrosine binding, as shown by isothermal titration calorimetry. Instead, we propose that
the PEST sequence has a functional role unrelated to phosphotyrosine
binding, possibly mediating efficient proteolytic degradation of the protein.
The latter half of the kinase inhibitory region and the entire extended SH2
subdomain form a single a-helix. The mapping of the true SH2 domain,
and the location of its C terminus more than 50 residues further downstream than predicted by sequence homology, explains a number of previously unexpected results that have shown the importance of residues close
to the SOCS box for phosphotyrosine binding.

Cytokine signalling acts through membrane-bound,
multisubunit receptor complexes that are phosphorylated by activated Janus kinases (JAKs), leading to subsequent activation and phosphorylation of members of
the signal transduction and activators of transcription
(STAT) family. The duration of the signalling response
is moderated by a classic negative feedback control
mechanism involving members of the suppressors of

cytokine signalling (SOCS) family (SOCS1–7) and
cytokine-inducible SH2-containing protein (CIS). The
SOCS family members share similar architecture, including an N-terminal region of varying size, a central
SH2 domain and a C-terminal SOCS box [1] (Fig. 1).
The SOCS SH2 domains are responsible for binding
to phosphorylated tyrosine residues on intracellular

domains of the cytokine receptors and ⁄ or the JAKs

Abbreviations
CIS, cytokine-inducible SH2-containing protein; ESS, extended SH2 subdomain; IPTG, isopropyl thio-b-D-galactoside; ITC, isothermal titration
calorimetry; JAK, Janus kinase; KIR, kinase inhibitory region; PtdIns, phosphatidylinositol; SOCS, suppressor of cytokine signalling; STAT,
signal transduction and activator of transcription.

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J. J. Babon et al.

Domain characterization of SOCS3

N-terminal

SH2

SOCS
box
P

1

45

185

socs1
socs2
socs4
socs5
socs6
socs7

P

P

socs3

225

P

P
P

CIS

Fig. 1. The suppressor of cytokine signalling (SOCS) family of proteins. The eight members of the SOCS family [SOCS1–7 and cytokine-inducible SH2-containing protein (CIS)] are shown schematically. All eight members of the SOCS family contain a C-terminal SOCS box (black), a
central SH2 domain (dark grey) and an N-terminal domain of varying lengths (light grey). CIS also contains a small insertion of  60 residues
between the SH2 domain and the SOCS box (light grey). The SH2 domain boundaries shown for SOCS3 are as identified in this study. The
position of potential PEST motifs in the SOCS family, as suggested by this work, are indicated by a boxed ‘P’; note that they are not shown
to scale. SOCS4–7 have much longer N-terminal domains than the other SOCS family members (300–400 residues), the dotted lines indicate
that these regions are not drawn to scale. The residue numbering refers to SOCS3 only.

themselves [2]. They act therefore by directly blocking

signal transduction or by interfering with STAT access
to the phosphorylated receptor subunits.
SOCS3, in particular, has a 22-residue N-terminal
segment, followed by a 12-residue kinase inhibitory
region (KIR). Mutation of essential residues in the
KIR, or its deletion, affects kinase inhibition without
affecting phosphotyrosine binding [3,4]. One model
proposed to explain the KIR action is that it can
mimic the activation loop found in kinases such as
JAK2 and FGF receptor kinase [5] and prevent substrate access to the catalytic groove of the kinase [6].
In support of this, the sequences of the SOCS3 and
SOCS1 KIRs share some homology with that of the
JAK1 and JAK2 activation loops [6]. There is no
structural information available for the KIR, but if
this mechanism operates it implies that the KIR is an
extended loop or unstructured.
Immediately following the KIR in SOCS3 is the
extended SH2 subdomain (ESS), an 11-residue segment
preceding the true SH2 domain, which can affect phosphotyrosine binding via an unknown mechanism [3].
There is no direct structural information available for
the ESS, but sequence analysis, modelling [7] and the
slight homology shared between the ESS and similar
regions on Stat1 [8] and Stat3b [9] suggest that it may
consist of one or two a-helices.
The SH2 domain of SOCS3 is immediately downstream from the ESS. The SH2 domain is a common
motif, present in proteins capable of binding to phos-

photyrosine residues. It typically contains around 100
residues, and adopts a fold consisting of a central
b-sheet flanked on each face with an a-helix. The SH2

domain of murine SOCS3 has been mapped previously
by sequence comparison to residues 46–142 [10], but
mutagenesis experiments have shown that residues as
far away as Leu182 are important for phosphotyrosine–peptide binding [3]. There is therefore some uncertainty about the extent of the SH2 domain, depending
on whether it is predicted by sequence homology or
functional analysis.
In addition to their role in blocking the activation
of downstream signalling intermediates, the SOCS proteins may also act by directing the degradation of
bound signalling molecules [11]. As the C-terminal
SOCS box is capable of interacting with an E3–ubiquitin ligase complex by binding directly to elongins B
and C [12], SOCS proteins can recruit bound signal
transduction proteins, such as activated kinases or the
cytokine receptors themselves, for proteasome-mediated degradation [11,13,14]. Although there is no structural information on the SOCS box, sequence and
functional homologies suggest that it will adopt a similar structure to the corresponding region in the VHL
protein [15], which is also responsible for binding to
elonginB ⁄ C. Reports differ as to whether the interaction between elonginB ⁄ C and SOCS stabilizes [16,17]
or destabilizes [12,18] the SOCS proteins themselves.
Unambiguous secondary structure assignment, whether by NMR or other spectroscopic techniques, can

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J. J. Babon et al.

be a powerful tool in determining domain architecture.
In this study we show that the N-terminal portion of

the KIR of murine SOCS3 is unstructured, but the
C-terminal half of the KIR and the entire ESS form
one single a-helix. In addition, we show that the SH2
domain is a 140-residue domain that contains a
35-residue unstructured PEST motif insertion which is
not required for phosphotyrosine binding but may
have an important functional role.

Results

hydrophilic in PtdIns 3-kinase but not in SOCS3 were
identified and mutated to match the PtdIns 3-kinase
residue. The six mutants (A50D, G53R, L58E, A62E,
A65E, G99D) were all cloned and expressed in E. coli as
part of a 22–185 construct. All six constructs again
expressed in inclusion bodies and required refolding.
The maximum concentration obtained by any of the six
point mutants was  3 mgỈmL)1, in the presence of
peptide, no higher than the wild-type SOCS3(22–185)
construct. As SOCS3(22–225) was too poorly soluble
to obtain any meaningful structural data, the wild-type
SOCS3(22–185) construct was pursued.

SOCS3 phosphotyrosine peptide complex
Two initial constructs of mouse SOCS3 [SOCS3(22–
225) and SOCS3(22–185)] were cloned and expressed
in Escherichia coli. Both contain the KIR and the
extended SH2 domain, but SOCS3(22–185) lacks the
C-terminal SOCS box. Both constructs expressed in
inclusion bodies in E. coli and required refolding. A

phosphotyrosine peptide from gp130 (STASTVEpYSTVVHSG) has been shown previously to bind
with high affinity to mouse SOCS3 [19,20]. The addition of a molar excess of peptide significantly increased
<
the solubility in NaCl ⁄ Pi from < 1 mgỈmL)1 to
 1 mgỈmL)1 for SOCS3(22–225) and to 3 mgỈmL)1
for SOCS3(22–185).
As SOCS3(22–185) in the presence of the tyrosinephosphorylated peptide could not be concentrated
beyond  0.2 mm, seven constructs of shorter length
were expressed in E. coli and their solubility examined.
All constructs contained the SH2 domain, as defined by
sequence homology [10], but included differing lengths
of sequence outside this region. All seven constructs
(22–142, 22–128, 22–126, 44–185, 44–142, 44–128 and
44–126), and the control 22–185 and 22–225 fragments,
were expressed in inclusion bodies in E. coli and
required refolding. The construct showing the highest
solubility was SOCS3(22–185). Constructs shorter than
this at the C-terminal end did not bind tightly to the
gp130 peptide (data not shown). All of the other constructs had equal or lower solubility, even in the presence of the tyrosine-phosphorylated peptide, including
the predicted SH2 domain alone (44–142). This implied
that the SH2 domain itself was a cause of poor solubility, as was the SOCS box. The sequences of the SOCS3
SH2 domain and the phosphatidylinositol (PtdIns)
3-kinase (N-terminal) SH2 domain (the SH2 domain
with the highest sequence identity in the PDB) were
therefore aligned and hydrophobic residue substitutions
in SOCS3 that were surface-exposed in the PtdIns 3-kinase structure were considered as candidates for point
mutagenesis. Six residues that were solvent-exposed and
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NMR assignments for murine SOCS3(22–185)

After buffer optimization, SOCS3(22–185) was soluble
to  0.5 mm, but UV-visible spectra of the protein
showed that significant aggregation was occurring at
this concentration, indicated by a high apparent absorption at 320 nm as a result of scattering. Many NMR
experiments required for full protein assignment therefore did not yield acceptable results, in particular
HNCACB, HCCH-TOCSY and 13C-NOESY-HSQC.
Nevertheless, near-complete backbone resonance assignments were made for SOCS3(22–185). Apart from five
missing spin systems (Ser25–Ser28 and Gly170), 100%
of 1HN, 100% of 15N (excluding 18 proline residues),
96% of 13Ca, 84% of 13Cb, 87% of 13C¢ and 84% of 1Ha
were assigned unambiguously (Fig. 2). HNCO experiments were used to obtain 13C¢ resonances and therefore
all 13C¢ N-terminal to proline residues remain unassigned. The majority of side-chain assignments were
determined, but because of the poor spectral quality of
HCCH-TOCSY and 13C NOESY-HSQC experiments,
no hydrophilic or polar c, d or e carbon assignments
were made. Secondary structure elements were determined by analysis of backbone and 13Cb chemical shifts
(supplementary figure Fig. S1), from characteristic
NOE patterns in the 15N-edited NOESY-HSQC and by
using talos [21]. Assignments revealed that SOCS3 had
an aabbbbbabbb topology, with the ESS and the C-terminal end of KIR forming the first a-helix (Fig. 2). Significantly, there was a large unstructured region between
Met128 and Arg163 that contained a high proportion of
proline residues (12 out of 35). The chemical shifts of
mouse SOCS3 have been deposited in BioMagRes-Bank
() with accession number
6580.
Murine SOCS3 contains a PEST region
The sequence of the unstructured region of murine
SOCS3 is highly conserved in mammalian SOCS3, as

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J. J. Babon et al.

A

Domain characterization of SOCS3

shown in Fig. 3. This region displays all of the common features of PEST sequences [22], namely a high
proportion of Pro, Glu, Ser and Thr residues, the
absence of Lys, His and Arg except at the termini, and
the fact that it is completely unstructured based on the
absence of medium- and long-range NOEs and the
observation of intense backbone amide peaks (Fig. S1).
The primary sequence of SOCS3 was analysed for the
presence of a PEST sequence by using the pestfind
program ( />PESTfind) [23]. This analysis identified the likely presence (PESTfind score +11.11 [23]) of a single PEST
sequence in SOCS3 spanning residues His126–Lys162.
The unstructured region of SOCS3 spans Met128–
Arg163 and therefore matches almost exactly the predicted PEST region. Residues from Met128–Arg163
showed no inter-residue NOEs other than sequential
connectivities, did not have restrained / ⁄ w angles
according to TALOS, had amide resonances in the
random coil region of the 15N-HSQC spectrum and
showed significantly narrower line-widths than any
other residues in the protein. This indicates that the
PEST sequence is an unstructured, highly mobile
region within SOCS3.
The PEST sequence is an insertion in the SH2
domain

B

Fig. 2. 15N-1H HSQC spectrum and secondary structure assignment of SOCS3(22–185). (A) The 15N-1H HSQC spectrum is shown
of 0.1 mM SOCS3 at 500 MHz and 298 K in 50 mM sodium-phosphate buffer (pH 6.7) containing 2 mM dithiothreitol. The assigned
residues are labelled with their residue number in the HSQC; some
assignment labels are omitted for clarity. (B) The secondary structure of SOCS3 was assigned by examining NOE patterns, analyses
of backbone and 13Cb chemical shifts, and TALOS predictions [26],
and is shown schematically with residue numbers marking the
boundaries of each motif. The PEST motif is shown as a thick black
line. The relevant secondary structure motifs are indicated at the
top of the figure with the nomenclature used by Grucza et al. [30].
The topology of the b-sheet and two b-hairpins was determined by
examining long-range backbone–backbone NOEs (supplementary
table Table S1). ESS, extended SH2 subdomain; PEST, PEST motif.

Analysis of the secondary structure of SOCS3, and
sequence alignments with SH2 domains, reveal that the
PEST sequence begins immediately after the last residue
of helix B in the SH2 domain. However, most SH2
domains do not end with this helix, but contain further
structural elements at their C termini, including the ‘BG
loop’ and the ‘G’-strand (Fig. 2) [24]. Hortner et al. [25]
have modelled the structure of the SOCS3 SH2 domain
and suggest that the BG loop and bG strand are formed
from residues Gly132–Val148, which we have shown to
be unstructured and part of the PEST region. We examined the sequence of the 19 structured residues immediately downstream of the PEST region and found a high
likelihood that they constitute the BG loop and bG
strand of the SH2 domain of SOCS3 (supplementary
figure Fig. S2). In particular, Leu176–Leu182 aligned

well with the seven C-terminal residues of a number of
SH2 domains, supporting this hypothesis. In agreement
with this scenario, deletion of residues 182–185 had
been shown previously to affect phosphotyrosine peptide binding [3]. Although the sequence between Tyr165
and Pro175, which would form the ‘BG loop’, was not
significantly similar to other SH2 domains, the SHP-2
[26], grb7 [27] and, in particular, STAT3b [9] SH2
domains contain extended loops in this region that

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J. J. Babon et al.

Fig. 3. PEST sequence conservation in SOCS3. Sequence alignment of the region of SOCS3 containing the PEST motif for a number of
mammalian species is shown, with conserved residues in the unstructured PEST motif shown hatched in grey. The numbering refers to
mouse SOCS3. The unstructured residues defined by this study are shown in bold.

structurally resemble a b-hairpin. Analysis of the secondary structure of SOCS3 shows that it forms a b-hairpin
in this region. Thus, it appears that the PEST sequence
constitutes an insertion in the true SOCS3 SH2 domain.
Murine SOCS3(D129–163) binds to a
phosphotyrosine peptide from gp130
In order to determine whether deleting the PEST
region would have an impact on the function of the
SH2 domain, binding studies and isothermal titration

calorimetry (ITC) were performed using a 22–185 construct lacking the PEST region [SOCS3(22–185)(D129–
163)] and the tyrosine phosphorylated peptide from
gp130. SOCS3(D129–163) was constructed by replacing
Pro129–Arg163 inclusive, with an eight residue [(Gly–
Ser) · 4] linker in the 22–185 construct. As shown in
Fig. 4, the construct lacking the PEST region binds to
the gp130 peptide. ITC analyses showed that the titration curve could be fitted using a single binding site
mode with a Kd of 74 ± 7 nm. The Kd of wild-type
SOCS3(22–185) binding was 152 ± 25 nm.
PEST sequences in other SOCS family proteins
In order to determine whether other members of the
SOCS family contained PEST motifs, their sequences
were analysed using the PESTfind algorithm [23].
Of the eight members of the murine SOCS family,
SOCS1, -3, -5 and -7, and CIS, show a probable
PEST motif with a PESTfind score of > 5 (Table 1).
CIS and SOCS3 have the PEST motif within the
SH2 domain, while SOCS1, -5 and -7 contain PEST
motifs in the N-terminal domain. The PEST sequence
in the CIS SH2 domain is located eight residues
downstream from the terminus of the predicted aB
helix. Whether those eight residues are also unstructured, thus placing the unstructured insertion at an
identical position to the PEST sequence in SOCS3,
could not be determined. Secondary structure prediction by sequence analysis gives no prediction for
those eight residues.
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The KIR ⁄ ESS consists of a single a-helix
Based on observed NOEs, chemical shift deviations and
TALOS predictions, residues Glu29–Ser44 form a single a-helix, whilst residues 22–28 are unstructured. The

helix encompasses the entire ESS and the four residues
at the C terminus of the KIR. The remaining residues
that comprise the KIR appear to be unstructured.

Discussion
In this study we defined the secondary structure elements of the SOCS3 protein, apart from the first 21
residues and the SOCS box. The true SH2 domain
boundaries were also defined for the first time, and an
unstructured insertion therein was identified. Residues
29–128 and 164–185 of SOCS3 are structured, but the
N-terminal half of the KIR, and 35 residues in the
SH2 domain, were shown by NMR to be unstructured. This was evinced by the lack of any nonadjacent inter-residue NOEs in those regions, as well as
the significantly sharper line-widths, characteristic of
mobile, unstructured sections of polypeptide. That the
KIR is mostly unstructured when SOCS3 is in isolation is perhaps not surprising in view of the hypothesis
for its mechanism proposed by Yasukawa et al. [6].
This requires the KIR to structurally mimic the activation loop of JAK2, so that in the absence of JAK2
the KIR would consist of an extended loop structure or be completely unstructured and separate from
the globular core of the protein, so it can access, and
block, the catalytic groove of the JAK2 kinase
domain.
Mutagenesis studies have shown that a number of
residues [3,4,25,28], important in binding phosphotyrosine-containing peptides or proteins, lie outside the
SH2 domain predicted by sequence homology. This led
to the 12 residues immediately upstream of the SH2
domain being designated the ESS. Our secondary
structure assignment of SOCS3 shows that the entire
ESS forms a single a-helix. Giordanetto & Kroemer [7]
modelled the structures of the ESS and KIR of

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Domain characterization of SOCS3

Fig. 4. SOCS3 lacking the PEST motif binds
a gp130 peptide with high affinity. (A) Titration of 80 lM gp130 peptide into 10 lM
SOCS3(D101–133). The integrated heats
from which the heat of dilution has been
subtracted are shown, as well as the fit to a
single site binding isotherm that yielded Kd
78 nM and DH )6.4 kcal mol)1. (B) Titration
of 160 lM gp130 peptide into 13 lM wild
type SOCS3. The fit to a single site binding
isotherm is shown, yielding a Kd of 168 nM
and a DH of )6.2 kcalỈmol)1. Both the wildtype and (D101-133) proteins used spanned
residues 22–185 and not 22–225 because of
the higher solubility of the former.

SOCS1, based on the similarity of the ESS sequence to
a similar region in Stat1 [8] and Stat3b [9], and suggested that the ESS and KIR form two short orthogonal
helices. This differs from the single a-helix found in
SOCS3, but a comparison of the ESS sequences from
SOCS1 and SOCS3 shows that there are several divergent residues in this region, including a leucine (Leu32)
in SOCS3 in place of an arginine (Arg67) in SOCS1,
predicted in their model to make a critical ion pair
with Asp76.
The identification, by deletion mutagenesis, of residues affecting phosphotyrosine binding but located

> 50 residues downstream of the predicted C terminus
of the SH2 domain suggested that the functional SH2
domain was longer than originally suggested by
sequence comparison [3]. However, subsequent
attempts to determine key residues important for the
binding specificity of SOCS3 by structural modelling
were hampered by the logical, yet incorrect, assumption that the SH2 domain consisted of c. 100 contiguous residues. In this report we have shown that the
true SH2 domain is disrupted in murine SOCS3 by a
35-residue unstructured insertion that is predicted to
form a PEST motif [22]. This results in residues 164–
185 forming the BG loop and bG strand of a classic
SH2 domain [24], rather than residues 129–147, as
commonly assumed [25]. This information is crucial
for future attempts to alter the specificity of the
SOCS3 SH2 domain by point mutation.
The PEST sequence identified in SOCS3 does not
occur, according to sequence analysis, in the same
location in any other members of the SOCS family,
apart from CIS. Analysis of the sequence of CIS using

the PESTfind algorithm [23] shows a probable PEST
sequence in residues 172–187, a region suggested by
sequence homology to be located in a similar site in
the SH2 domain as in SOCS3. Other members of the
SOCS family contain putative PEST sequences, but
these are all located in the N-terminal region, upstream
of the SH2 domain. The conservation of the PEST
motif of SOCS3 in mammals (Fig. 3), and the presence
of probable PEST regions in most SOCS family members, suggests that it has an important functional role.
The appropriate duration of the cellular response to

cytokine signalling will be determined, in large part, by
the rate of turnover of the SOCS proteins. Expression
of the SOCS proteins is induced directly by STAT
binding to the appropriate promoters. Rapid destruction of the SOCS protein is also necessary, once signalling has ceased, to allow for subsequent cytokine
stimulation. The level of SOCS1 and SOCS3 protein
in vivo appears to be strongly regulated by protein degradation, and the short half-life of SOCS proteins
intracellularly appears to be the result primarily of
proteolytic degradation [28,29]. This may be important
mechanistically, as the efficient turnover of SOCS proteins, and their induction of degradation of associated
signalling molecules via the SOCS box, allows cells to
respond to cytokine stimulation, quickly inhibit any
prolonged activation and rapidly return to basal SOCS
levels, ready for another round of stimulation. There
appear to be a number of features important for effective degradation of SOCS proteins, even apart from
any role the SOCS box may play in this process. Sasaki
et al. [28] have shown that a naturally occurring
alternative transcript of SOCS3, lacking the first 11

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J. J. Babon et al.

Table 1. Predicted PEST motifs in suppressor of cytokine signalling (SOCS) family proteins. Sequences of predicted PEST motifs in the
SOCS family members are shown with their PESTfind score [29] and domain location. NA indicates, for SOCS-2, -4 and -6, that no PEST
motif was predicted for these proteins.

Protein
SOCS1
SOCS2
SOCS3
SOCS4
SOCS5
SOCS6
SOCS7
CIS

PESTfind
score

Sequence

Location

14.2
NA
11.1
NA
7.4
11.3
NA
11.9
9.0

22 RSEPSSSSSSSSPAAPVR 39
NA

126 HYMPPPGTPSFSLPPTEPSSEVPEQPPAQALPGSTPK 162
NA
96 KDSDSGATPGTR 107
243 HSTFFDTFDPSLVSTEDEEDR 263
NA
74 KTAGGGCCP CPCPPQPPPPQPPPPAAAPQAGEDPTETSDALLVLEGLESEAESLETNSCSEEELSSPGR 142
172 RSDSPDPAPTPALPMSK 188

N terminus
NA
SH2
NA
N terminus
N terminus
NA
N terminus
SH2

residues, has a prolonged half-life in Ba ⁄ F3 haemopoietic cells owing to the absence of Lys6, a major ubiquitination site of SOCS3. Chen et al. [18] found that the
N-terminal region of SOCS 1 (upstream of the SH2
domain) contained a site for pim-1 kinase phosphorylation that significantly increased SOCS1 stability.
SOCS3 has also been shown to interact, and be phosphorylated by, pim-1 kinase, at an unknown site,
which also confers stability [30].
Proteasome-induced proteolysis is catalysed by the
presence of regions of unstructured sequence in a protein [31]. The PEST sequence is one such sequence
commonly found in intracellular proteins of extremely
short half-life [22,32]. PEST sequences are hydrophilic,
contain a high proportion of proline, glutamic acid,
serine and threonine residues, and do not contain
lysine, arginine or histidine. The X-ray structures of

several proteins containing PEST sequences have been
determined, but in each case the electron density of the
PEST sequence is missing (e.g. NF-jb [33] and ornithine decarboxylase [34]), presumably because this region
is unstructured and mobile. They can act in a modular
manner, as transplanting PEST sequences from unstable proteins into stable proteins has been shown to
reduce the half-life of the resulting chimaeras
[32,35,36]. The presence of a PEST sequence has been
shown to be important in the proteolysis ⁄ degradation
of a number of proteins with diverse functions, such as
the glutamate receptor [37], proto-Dbl [38], and c-Fos
[39]. Biophysical characterization of the NF-jb PEST
sequence [40] has shown it to be solvent-exposed and
probably unstructured.
The PEST sequence in SOCS3 is located between
two secondary structural elements, namely the aB
helix and the BG loop. In all SH2 domains these are
located on the opposite face of the protein to the
phosphotyrosine-binding site, so the PEST sequence is
6126

not expected to interfere with phosphotyrosine binding
by the SH2 domain. Indeed, replacing the entire
35-residue PEST sequence with GSGSGSGS had little
effect upon binding a phosphorylated gp130 peptide,
as shown by ITC. In fact, the construct lacking the
PEST motif bound slightly more tightly to the phosphotyrosine containing gp130 peptide than did wildtype SOCS3(22–185). Whether the twofold change in
Kd is significant is difficult to determine as the construct lacking the PEST motif shows significantly less
aggregation than wild-type SOCS3, which could alter
the binding kinetics without representing a truly
enhanced Kd. The similarity of the two Kd values

implies that the PEST sequence does not significantly
affect phosphotyrosine binding. Structurally, the PEST
sequence is a benign insertion that may nevertheless
play a critical functional role in regulating cellular
SOCS3 levels.
Our identification of the secondary structure and correct domain boundaries of SOCS3 will enable manipulation of SOCS3 function by rational mutagenesis. This
includes mutagenesis of the PEST motif, either by complete removal or by point mutagenesis, to determine
the effect it has on the biological function of SOCS3
in vivo, as well as mutagenesis of the SH2 domain to
alter substrate specificity. These approaches will allow
a more thorough dissection of SOCS3 activity.

Experimental procedures
Cloning and expression
Fragments of mouse SOCS3 were subcloned by PCR into a
ligation-independent cloning vector constructed by one of
us (JJB). The vector encodes constructs with the leader
sequence MASYHHHHHHDYDIPTTENLYFQGAHDGS,
which consists primarily of a His6-tag and a TEV protease

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J. J. Babon et al.

cleavage site. For unlabelled protein, expression was performed in baffled flasks, with cells grown to an attenuance
(D) at 600 nm of 0.6 in superbroth containing 50 lgỈmL)1
kanamycin. Expression was induced with 1 mm isopropyl
thio-b-d-galactoside (IPTG). Cells were harvested, 3 h after
induction, by centrifugation (6200 g, 4 °C, 30 min). For

15
N labelling, cells were grown to a D at 600 nm of 0.6 in
Neidhardt’s medium [41] containing 1.0 gỈL)1 15NH4Cl as
the sole nitrogen source. For 15N ⁄ 13C-labelled samples,
1.0 gỈL)1 15NH4Cl and 2 gỈL)1 13C glucose were the sole
sources of nitrogen and carbon, respectively. Cells were
harvested 8 h after IPTG induction by centrifugation
(6200 g, 4 °C, 30 min). All SOCS3 clones express as insoluble inclusion bodies.

Domain characterization of SOCS3

were screened again at pH 6.7. At this pH only the
presence of 50 mm arginine plus 50 mm glutamate [43]
yielded an increase in protein solubility, from 3 to
10 mgỈmL)1, although UV-visible spectroscopy suggested
that there was significant aggregation at that concentration.
SDS ⁄ PAGE analysis of the protein after storage revealed
that disulphide bond-linked multimers formed slowly over
time in the absence of a reducing agent. In the presence of
dithiothreitol and EDTA, the protein was stable at 4 °C for
at least 2 months. The final buffer conditions chosen for
SOCS3 were therefore: 20 mm sodium phosphate, 20 mm
NaCl, 50 mm arginine, 50 mm glutamate, 2 mm dithiothreitol, 1 mm EDTA, pH 6.5. Concentration to
10 mgỈmL)1 was performed using centrifugal concentration
devices (Amicon Inc., Beverly, MA, USA).

Protein purification and buffer screening
Inclusion bodies were prepared via cell homogenization and
centrifugation at 20 000 g, and solubilized using 6 m guanidine hydrochloride. The soluble inclusion body preparation
was then purified using Ni-nitrilotriacetic acid resin

(Qiagen, Valencia, CA, USA). Protein binding was performed at pH 8.0, washing at pH 6.3, and elution at
pH 4.5. The eluted protein was quantified by absorbance at
280 nm, diluted to 0.1 mgỈmL)1, then refolded by extensive
dialysis against 25 mm sodium phosphate, 50 mm sodium
chloride, 5 mm 2-mercaptoethanol, pH 6.7. The refolded
protein was tested for correct conformation by binding an
aliquot to a column with immobilized phosphorylated
gp130 peptide (STASTVEpYSTVVHSG; pY ¼ phosphotyrosine [19,20]). The refolded protein was limited in its
solubility, but addition of a 1.5· molar excess of the gp130derived phosphopeptide increased the solubility to
c. 3 mgỈmL)1. As this concentration was still too low for
structure determination by high-resolution NMR, a thorough screen of buffer conditions was undertaken in an
attempt to improve the maximum solubility obtainable for
SOCS3(22–185). The buffer screen was performed in microdrop format [42] and studied the pH range from 4 to 9 in
0.5 unit intervals, the salt concentration from 0 to 500 mm
in 50 mm intervals, and temperatures of 4, 25 and 37 °C.
Both constructs of SOCS3 showed highest solubility in
buffers of low salt and high pH, and at low temperature.
The buffer conditions chosen for further additive screening
were 20 mm Tris, pH 8.5, 20 mm NaCl, at 25 °C. This
starting condition was used to test the effect of 14 different
additives, most at several concentrations. The additive
screen yielded promising results, and SOCS3 was shown to
be soluble to  10 mgỈmL)1 ( 0.5 mm) in buffers containing > 10% glycerol, > 0.5 m non detergent sulfobetaine
(NDSB), > 0.5 m trehalose or 50 mm arginine plus 50 mm
glutamate [43]. However, initial NMR analysis showed that
under these conditions, many amide cross-peaks were
missing from 15N HSQC spectra. As the high pH was
judged to be the cause of this, the most promising additives

NMR spectroscopy

Spectra were recorded at 298 K on a Bruker Avance 500
(using a cryoprobe), DRX-600, DMX-600 (using a cryoprobe) and Varian Unity INOVA 800 spectrometers. Conventional 2D TOCSY and NOESY spectra were obtained
using 2048 complex data points in the directly detected
dimension and typically 200–400 t1 increments. A TOCSY
spin-lock time of 60 ms and a NOESY mixing time of
120 ms were used. Spectra were processed using xwinnmr
(Bruker AG, Karlsruhe, Germany) or nmr-pipe [44], and
were analysed using xeasy (version 1.3.13) [45] or
nmrdraw [44]. Spectra were referenced to the H2O signal
at 4.77 p.p.m. (298 K) or a small impurity at 0.15 p.p.m.
Ca, Cb, Ha, C¢, and N chemical shifts were used in the
program TALOS [21] to obtain backbone torsion angle predictions. Sequence-specific resonance assignments for the
backbone were accomplished using HNCA, HN(CO)CA,
CBCA(CO)NH, HN(CA)CO and HNCO experiments [46].
Side-chain assignments were accomplished by combining
the data from the following experiments: 15N-edited
TOCSY-HSQC and NOESY-HSQC, HCCH-TOCSY and
HCCH-COSY [46].

ITC
Isothermal calorimetric titrations were performed using a
Microcal omega VP-ITC (MicroCal Inc., Northampton,
MA, USA). SOCS3(22–185) was dialysed against buffer
(50 mm NaCl, 50 mm arginine, 50 mm glutamate, 5 mm
2-mercaptoethanol, pH 6.7) and the dialysis buffer was
used to dissolve the tyrosine-phosphorylated gp130 peptide.
Experiments were performed at 298 K. Solutions of 10–
25 lm SOCS3 in the cell were titrated by injection of a
total of 290 lL of 80–200 lm of the gp130 peptide. The
heat of dilution of the gp130 peptide into buffer was determined in control experiments and subtracted from the raw

data of the binding experiment. The data were analysed
using the evaluation software, Microcal Origin, version

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J. J. Babon et al.

5.0, provided by the manufacturer. The binding curve fitted
a single-site binding mode in all cases, and Kd values were
determined from experiments repeated at least twice.

11

Acknowledgements
We thank Gottfried Otting for generously recording
NMR spectra on SOCS3. We thank the Knut and
Alice Wallenberg Foundation for the cryoprobe used
to record NMR spectra at 600 MHz and access to the
800 MHz NMR spectrometer at Biovitrum AB.

12

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Supplementary material
The following supplementary material is available for
this article online:
Fig. S1. Chemical shifts and peak intensities for
mSOCS3(22–185). The chemical shift differences from
random coil are shown for the (A) CO, (B) Ca, (C)
Ha, and (D) Cb atoms of mSOCS3 plotted against residue number. (D) The average chemical shift index
values for each residue are shown. (E) 1H-15N HSQC
peak intensities (arbitrary units) for each backbone
amide. Peak intensity in HSQC experiments is correlated approximately with T2 relaxation times and hence
flexibility. Note that the PEST insertion displays
abnormally large peak intensities, suggesting that it is
truly flexible.
Fig. S2. The SOCS3 SH2 domain sequence and secondary structure alignment. As SOCS3 and SHP2 bind
to the same site on the gp130 receptor [1–3], the N-terminal SH2 domain of SHP2 and its eight closest

6130

sequence neighbours were aligned with SOCS3. Residues shown in upper case are those conserved amongst
SH2 domains according to the conserved domain database [4]. The secondary structure of the N-terminal
SH2 domain of SHP2, as determined by Lee et al. [5],
is shown above in black, with the structural elements
labelled according to classical SH2 domain nomenclature. The secondary structure of SOCS3, as determined
in the present study, is shown below in grey. For
sequence comparison, the PEST region has been omitted and its site of omission shown with a black triangle. Residues 176–183 in SOCS3 align well with the
bG strand in SHP2 and other SH2 domains. The BG
loop in SOCS3 is a short b-hairpin rather than a true
‘loop’ region. * Growth factor receptor binding protein.
Table S1. Backbone–backbone NOEs that allowed

helix and b-sheet topology determination. Pairs of residues with either NH–NH or NH–Ha NOEs are listed.

FEBS Journal 272 (2005) 6120–6130 ª 2005 The Authors Journal Compilation ª 2005 FEBS