Human sprouty 4, a new ras antagonist on 5q31, interacts
with the dual specificity kinase TESK1
Onno C. Leeksma,
1
Tanja A. E. van Achterberg,
1
Yoshikazu Tsumura,
4
Jiro Toshima,
4
Eric Eldering,
1
Wilma G. M. Kroes,
2
Clemens Mellink,
2
Marcel Spaargaren,
3
Kensaku Mizuno,
4
Hans Pannekoek
1
and Carlie J. M. de Vries
1
Departments of
1
Biochemistry,
2
Clinical Genetics and
3
Pathology, Academic Medical Center, University of Amsterdam,

and do not show substantial translocation to the plasma
membrane upon receptor tyrosine kinase stimulation.
Keywords: sprouty 4; ras; receptor tyrosine kinase; TESK 1.
Inducible signaling antagonists play a vital role in regulating
the strength, duration and range of action of cellular signals.
Along with the discovery of Drosophila melanogaster
sprouty as an inducible antagonist of FGF-receptor sign-
aling, three human orthologues, designated human sprouty
(hspry)1, 2 and 3, were identified [1]. Drosophila sprouty was
originally considered to be an extracellular fibroblast
growth factor (FGF)-inhibitor and owes its name to its
ability to prevent excessive airway branching [1]. Subse-
quent studies revealed that sprouty might fulfill a more
general, intracellular tyrosine kinase signaling inhibitory
role in fruit flies [2–4] and acts either upstream, via an
interaction with Drk (the Drosophila equivalent of the
human adaptor protein Grb2) and the GTPase-activating
protein GAP1 [2], or downstream of ras at the level of Raf/
MAP kinase [3]. Human sprouty family members are
assumed to exert a function similar to inhibitors of the ras/
MAP kinase signaling pathway that are induced by
activated ras itself, thus constituting a significant feed-back
inhibitory mechanism.
An evolutionary conservation of spry’s modulating role
in respiratory organogenesis has been demonstrated in mice,
in which orthologues of hspry1, 2 and 3 as well as a fourth
family member, designated mspry4, were described [5–7].
While a decrease in mspry2 expression was associated with
increased murine airway branching [5], overexpression of
mspry2 and 4 in chicken embryos both caused chon-

accepted 9 April 2002)
Eur. J. Biochem. 269, 2546–2556 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02921.x
various sprouty proteins. The recently reported interaction
of an N-terminal sequence of hspry2 with the RING finger
domain of the E3-ubiquitin ligase Cbl, a property presum-
ably shared by mspry1, but not by mspry4, suggests that
specificity relies on the respective N-terminal sequences [10].
There is increasing evidence however, that individual
sprouty family members do not act on their own, but
instead form a complex through hetero- and/or homo-
dimerization. Mutation of a single conserved tyrosine
residue to alanine in the N-terminal part of hspry2 creates
a protein that is dominant negative not only to its
corresponding wild-type but also to mspry4; in addition, a
similar mutation in mspry4 exerts dominant negative
activity on wild-type hspry2 [11].
In search of new genes involved in atherosclerosis, we
have used differential display of randomly primed mRNA
by reverse transcription polymerase chain reaction (DD/
RT-PCR) [12,13]. Umbilical artery smooth muscle cells
(SMCs) stimulated by the conditioned medium of oxidized
low-density lipoprotein (ox-LDL) activated monocytes
differentially expressed 30 new genes [13]. Here we describe
the cloning, sequencing and functional characteristics of one
of these genes, which turned out to be the human
homologue of murine spry4. Hspry4 was mapped to
5q31.3 and inhibited insulin- and EGF-receptor tyrosine
kinase-mediated ras activation. Moreover, we identified the
ubiquitously expressed dual specificity testicular protein
kinase 1 [14,15] as a partner of hspry4. TESK1 and its

PSORT II
.
In vitro
transcription–translation
A PstI fragment of the 4.9-kb hspry4 cDNA (nucleotides
149–1225, encompassing the full-length coding sequence) in
pGEM4Z was used for in vitro transcription translation for
2 h at 30 °C in the presence of [
35
S]methionine, using a TnT-
coupled rabbit reticulocyte lysate system (Promega, Madi-
son, WI, USA). Radiolabeled proteins were analysed by
12% (w/v) SDS/PAGE under reducing conditions.
Eukaryotic expression plasmids
RasV12 [18] and Myc–ERK2 [19] plasmids were obtained
from J. L. Bos (University of Utrecht, Utrecht, the Neth-
erlands) and C. J. Marshall (Institute of Child Health,
London, UK), respectively. Hspry4 was provided at its
C-terminal end with a single hemagglutinin (HA) tag and
HA-spry4 cDNA was inserted into vector pcDNA3.1
(Invitrogen, Carlsbad, CA, USA). The construction was
done as follows: a 1076-bp PstI fragment of the 5-kb pUC18
insert was subcloned into pGEM4Z (Promega), digested
with SstIandHindIII, to yield a 1100-bp fragment and,
upon further digestion with NspI, a 710-bp 5¢ fragment. A
corresponding 3¢ fragment of 335 bp, containing the NspI
site at position 856 of hspry4 cDNA, was generated by PCR
with forward primer 5¢-CCAGACTCTGGTCAACTA
TGGCAC-3¢ and reverse primer 5¢-GTA
CCCGGGCTG

Umbilical artery SMC were isolated and cultured as
previously described [13]. A14 cells (NIH 3T3 cells, stably
expressing a human insulin receptor under a SV40
promotor [18]) were cultured in six-well plates (Nunc,
Roskilde, Danmark) in DMEM (Gibco-BRL, Paisley,
Scotland), supplemented with 10% (v/v) fetal bovine serum
(Gibco-BRL, Paisley, Scotland), 500 lgÆmL
)1
G418,
100 UÆmL
)1
penicillin and 100 UÆmL
)1
streptomycin.
Twenty-four hours post transfection by calcium phosphate
precipitation, cells were starved overnight in DMEM
without serum and subsequently used for experiments.
Ó FEBS 2002 Ras antagonist human sprouty 4 binds TESK1 (Eur. J. Biochem. 269) 2547
MAP kinase assay
Cells were transfected with the plasmid encoding Myc–
ERK2 and simultaneously with additional plasmids, as
indicated in the legend to Fig. 4. Following stimulation with
human recombinant insulin (Sigma, St Louis, MO, USA) or
EGF (Sigma), the transfected cells were washed once
with NaCl/P
i
(140 m
M
NaCl, 13 m
M

leupeptin,
0.1 l
M
aprotinin, 1 m
M
phenylmethanesulfonyl fluoride)
per well. Lysates were precleared for 45 min at 4 °Cwith
protein A–Sepharose and incubated for 2 h at 4 °Cwith
1 lg immunopurified anti-Myc monoclonal antibody 9E10.
Immune complexes bound to protein G–Sepharose were
washed twice with lysis buffer and once with kinase buffer
(30 m
M
Tris/HCl (pH 8.0), 20 m
M
MgCl
2
,2m
M
MnCl
2
,
10 l
M
ATP). Beads were resuspended in 100 lLkinase
buffer. Fifty microliters of this suspension were mixed with
sample buffer (0.125
M
Tris/HCl (pH 6.8), 4% (w/v) SDS,
17% (v/v) glycerol, 5 m

with forward primer 5¢-CTA
GTCGACATGCTCAGCC
CCCTCCCC-3¢ and reverse primer 5¢-G
GAATTCCT
GTCAGAAAGGCTTGTCGG-3¢,creatingSalIand
EcoRI restriction sites (underlined), respectively, and ligated
in frame with a GAL4 DNA binding domain (BD) into
SalI–EcoRI digested pMD4. Vector pMD4 (generously
provided by M. van Dijk, Netherlands Cancer Institute,
Amsterdam, the Netherlands) was created by replacing the
GAL4 activation domain (AD) of pPC86 by the GAL4
DNA BD from pPC97 [22]. A human fetal liver pAct2
cDNA library, containing coding sequences that are in
frame with a GAL4 activation domain (Clontech), was
screened with full-length hspry4 in pMD4 as bait. Yeast
strain HF7c was simultaneously transformed with pMD4-
hspry4 and the pAct2 cDNA library, according to the
manufacturer’s instructions. Selection of positive interac-
tions occurred on agar plates in the presence of 15 m
M
3-amino-1,2,4-triazole and in the absence of the amino acids
leucine, tryptophan and histidine. Full-length human
TESK1 cDNA in pAct2, in frame with the GAL4 activation
domain and the HA-tag, was made by subcloning TESK1
cDNA from pBS-TESK1 by NcoI–EcoRI digestion into
pAct2.
Hspry4-TESK1 coimmunoprecipitation
COS-7 cells were cultured in DMEM supplemented with
10% (v/v) fetal bovine serum and transfected by calcium
phosphate precipitation. Thirty-six hours after transfection

M
dithiothreitol, 1% (w/v) SDS,
0.002% (w/v) bromophenol blue] and subjected to 8% (w/v)
SDS/PAGE. Proteins were transferred onto poly(vinylidene
difluoride) membranes (Bio-Rad, Hercules, CA, USA).
Membranes were blocked overnight with 3% (w/v) oval-
bumin in NaCl/P
i
with 0.05% (v/v) Tween 20 and incubated
for 1 h with the anti-HA Ig or anti-Myc Ig, respectively,
diluted in NaCl/P
i
containing 0.05% (v/v) Tween and 1%
(w/v) ovalbumin. After washing, membranes were probed
with horse-radish peroxidase-conjugated anti-(rabbit IgG)
Ig or goat anti-(mouse IgG) Ig and immunoreactive bands
were visualized by chemiluminescence (Amersham Phar-
macia Biotech).
Intracellular localization
Tissue-culture cells (A14, 293, and HeLa), used for subcel-
lular localization experiments, were grown on gelatin-coated
glass cover slips in 24-well plates in DMEM, with (A14) or
without G418 (293 cells), or in Iscove’s (HeLa cells),
supplemented with 10% (v/v) fetal bovine serum and
antibiotics, and transfected using Superfect (Qiagen, Hilden,
Germany), according to the manufacturer’s instructions.
Twenty-four hours post-transfection, culture media were
replaced by media without serum and subsequently cultured
overnight. After an incubation with or without EGF or
insulin, cells were washed once with ice-cold medium, fixed

COS-7 cells were transfected with pBOS-HA-sprouty4 and
pCAG-Myc-TESK1 (or empty vector pCAG), cultured for
24 hinDMEMplusfetalbovineserumandthenstarvedfor
24 h in DMEM. Following stimulation of transfected cells
with EGF for the indicated times, cells were lysed in 20 m
M
Hepes (pH 7.4), 1% NP-40, 10% glycerol, 50 m
M
NaF,
1m
M
phenylmethanesulfonyl fluoride, 1 m
M
Na
3
VO
4
and
21 l
M
leupeptin. Immunoprecipitation of HA-spry4 from
these lysates occurred essentially as described above except
that monoclonal anti-HA Ig 12CA5 was used. Precipitated
proteins were immunoblotted with anti-HA Ig and
anti-Myc Ig.
RESULTS
Induction of smag-84 mRNA and tissue distribution
One of the novel genes, provisionally designated smag
(smooth muscle activation gene)-84 [13], detected by DD/
RT-PCR analysis of activated vs. resting human umbilical

frame with a premature stop codon, due to a single
nucleotide shift at position 494 (i.e. 998 in the smag-84
transcript), encodes a protein of 106 amino acids. Due to the
frameshift, this truncated protein contains a C-terminal
decapeptide sequence that is not present in the presumed
full-length smag-84 protein of 322 amino acids. The 2.5-kb
cDNA represented an aberrant transcript without any
substantial open reading frame. The longest transcript
(7 kb) harbors five polyadenylation sites (two AATAAA,
and three AATTAAA), nine ATTTA sequences [23], two
Alu-repeats and three CAGAC motifs [24].
BLAST
searches revealed the homology of the coding
sequences of the 4.9- and 7-kb cDNAs with the sprouty
(spry) gene family. Homology with murine spry4 (mspry4)
was especially striking, i.e. 87% at the DNA and 88% at the
protein level. Our novel gene was therefore named human
spry4 (hspry4). Because multiple tissue Northern blotting
revealed that the 4.9-kb transcript is the predominant
hspry4 mRNA in vivo, we decided to focus on the properties
of the 4.9-kb transcript and its corresponding protein.
In vitro transcription–translation of this hspry4 cDNA
confirmed our prediction of the open reading frame of 322
amino acids for hspry4, and yielded a protein with a
molecular mass of approximately 35 kDa (Fig. 2). In
agreement with observations from others showing expres-
sion induction of mammalian spry4 in an ERK activation
dependent manner [11,25], expression of hspry4 was
induced by growth factors and cytokines like VEGF, tumor
necrosis factor-a, and interleukin-1b. We developed a

Drosophila sprouty (Fig. 3B) reveals that the cysteine-rich
region and other motifs have been conserved. Proline-rich
regions are present in murine and human spry4, as well as in
Drosophila spry. Furthermore, the nuclear-export sequence
is similar between hspry2 and 4, but the nuclear localization
signal has not been conserved. While one or two SSXS
phosphorylation sequences are present in all sproutys,
PEST domains are unique to hspry4 and mspry4, according
to the
PEST
-
FIND
algorithm.
Inhibition of MAP kinase activation
Drosophila spry inhibits ras-mediated MAP kinase activa-
tion. To test whether hspry4 could similarly act as an
inhibitor of ras, a pcDNA3.1(+) eukaryotic expression
vector, containing HA-tagged hspry4 was constructed.
Kinase activity of cotransfected Myc-tagged MAP kinase
was measured by its ability to phosphorylate myelin basic
protein and was maximal 2 min after stimulation with
insulin or EGF; hspry4 inhibited MAP kinase activation by
either stimulus. This inhibition was most pronounced at
2 min and already lower at 5 min (Fig. 4A). MAP kinase
activation by constitutively active V12 ras was unaffected by
hspry4, indicating that the inhibition observed in insulin- or
EGF-stimulation occurred by interfering with the activation
of ras (Fig. 4B).
Ras inhibition
In order to demonstrate that hspry4 interfered with the

fetal liver cDNA library. Sequencing of DNA from
transformed Saccharomyces cerevisiae colonies, growing
on selective plates, revealed a partial cDNA of human
testicular protein kinase 1(TESK1), encoding the C-ter-
minal 167 amino acids (positions 459–626) fused to the
GAL4 activation domain (AD) (Fig. 6). The interaction
Fig. 2. In vitro transcription–translation of hspry4 cDNA. Analysis of
35
S-labeled protein by 12% (w/v) SDS/PAGE was carried out as
outlined under Experimental procedures. Lane 1, control DNA as
supplied by the manufacturer; lane 2, no DNA; lane 3, vector DNA;
lane 4, hspry4 cDNA.
2550 O. C. Leeksma et al. (Eur. J. Biochem. 269) Ó FEBS 2002
between GAL4 DNA BD-hspry4 and GAL4
AD-TESK1(459–626) was confirmed by b-galactosidase
staining. Cotransfection of full-length TESK1 cDNA,
cloned in frame with the GAL4 AD into vector pAct2,
with GAL4 DNA BD-hspry4 cDNA in vector pMD4 also
yielded colonies under selective conditions.
Fig. 3. Amino-acid sequence of hspry4 and
alignment with other spry family members. (A)
Sequence of hspry4. Proline-rich regions are
underlined. MAP kinase consensus sequence
phosphorylation site is given in italics. Arrows
indicate a putative nuclear export sequence.
An asterisk marks the functionally relevant
tyrosine [11]. Dash dot and underlined is a
possible nuclear localization signal. Double
underlined is a PEST sequence. The box
denotes a conserved cysteine-rich region:

insulin, 50 ngÆmL
)1
EGF or vehicle. After the indicated times, cells
were lysed and lysates from either unstimulated or insulin/EGF-sti-
mulated cells were immunoprecipitated with anti-Myc Ig 9E10. Kinase
activity of Myc–ERK2 was assessed by its ability to phosphorylate
myelin basic protein (MBP). Total ERK2 in immunoprecipitates was
quantitated by immunoblotting with an anti-ERK2 Ig. (B) MAP
kinase activation in V12 ras transfectants is unaffected by hspry4. A14
cells were transfected with 0.5 lg Myc–ERK2, different concentrations
of v12 ras plasmid and/or 2.0 lg hspry4 cDNA or pcDNA 3.1 vector
control as indicated. Expression of HA-hspry4 was analyzed by
anti-HA Ig immunoblotting of total lysates.
Fig. 7. In vi vo interactionofhspry4andTESK1asassessedbyimmu-
noprecipitation. COS-7 cells were cotransfected with different plasmids
as indicated. Cell lysates of these double transfectants were subjected to
immunoprecipitation with anti-Myc Ig or anti-HA Ig. Immunopre-
cipitated proteins, resolved by SDS/PAGE, were immunoblotted with
anti-HA Ig or anti-Myc Ig. Anti-Myc Ig coimmunoprecipitate
HA-spry4 and vice versa anti-HA Ig coimmunoprecipitate Myc-rat
TESK1 from COS-7 Myc-rat TESK1/HA-hspry4 double transfectants
(lane 4 of left panel of anti-HA Ig and anti-Myc Ig immunoblot,
respectively).
Fig. 5. Inhibition of ras. A14 cells were transfected with vector DNA,
or 2.0 lg HA-hspry4 cDNA and incubated for 2 min with either
vehicle, insulin or EGF as in Fig. 4A. GST Raf-RBD bead-associated
GTP-ras was quantitated by immunoblotting with anti-ras Ig. Phos-
phorylated ERK1/2 and total ERK1/2 were immunoblotted with anti-
(phospho-MAP kinase) Ig or anti-ERK1/ERK2 Ig, respectively.
Fig. 6. Schematic representation of hspry4 and TESK1 proteins, which

To determine whether the interaction between hspry4 and
TESK1 was affected by growth factor stimulation, COS
cells transfected with HA-hspry4- and Myc-TESK1 cDNAs
were stimulated with EGF for a maximum of 10 min. As
shown in Fig. 9, an increase in sprouty4-associated TESK1
was observed in time, with an apparent maximal interaction
occurring at 5 min.
DISCUSSION
Research in Drosophila melanogaster has led to the identi-
fication of many evolutionary conserved proteins, involved
in signal transduction. The sprouty protein family repre-
sents yet another example. We have identified a fourth
human member (hspry4) in a search for new genes involved
in atherosclerosis. In retrospect, it is not surprising, in view
of the methodology we employed, that we have detected a
Fig. 8. Colocalization of TESK1 and hspry4. HeLa cells, transiently transfected with HA-tagged human TESK1 cDNA and EGFP-tagged
hspry4cDNA, either unstimulated or stimulated for 2 min at 37 °C with EGF were visualized by confocal laser scanning microscopy. HA-TESK1
detected by indirect Cy3 immunofluorescent staining appears in bright red (A,D), hspry4-EGFP in green (B,E). Right panels (C,F) show merged
pictures, in which colocalizations of the two proteins in the cytoplasm appear in yellow. Note that there is some nonspecific Cy3 background
staining of nuclei from transfected and nontransfected cells.
Fig. 9. Effect of EGF stimulation on the interaction between TESK1 and
hspry4. COS-7 cells transfected with plasmids coding for HA-hspry4
and Myc-TESK1 were lysed after stimulation with EGF and analyzed
by immunoprecipitation with anti-HA Ig and immunoblotting with
anti-Myc Ig and anti-HA Ig. The amount of coimmunoprecipitated
TESK1 increases with time with an apparent maximum at 5 min. No
coimmunoprecipitation of anti-Myc Ig immunoreactive TESK1, as
detected by anti-Myc Ig immunoblotting, is observed in empty vector
cotransfected hspry4 transfectants and concentrations of Myc-TESK1
in cell lysates of TESK1 transfectants are equal.

receptor tyrosine kinases [1–8,11,36]. Based on our data
with hspry4, the insulin receptor can now be added to this
growing list. Furthermore, it has been recently reported that
mspry1 is a downstream target of Wilms Tumor 1 (Wt1),
providing additional evidence for involvement of spry
proteins in atherogenesis and hematopoiesis [36]. hspry4
apparently exerts a similar function as Drosophila sprouty in
acting as an intracellular inhibitor of ras [2]. The inability of
hspry4 to inhibit constitutively active V12 ras argues in
favor of an effect upstream of this GTPase, but does not
preclude an effect at the level of (normal) ras. These findings
are in agreement with a study in endothelial cells, showing
inhibition by mspry4 of MAP kinase activation induced by
VEGF and bFGF, which could be rescued by constitutively
active L61 ras [8]. Our observation that hspry4 overexpres-
sion causes a reduction in GTP-ras on stimulation with
insulin and EGF is in agreement with that of others showing
a similar effect of mspry1 and mspry2 on bFGF induced
GTP-ras [36]. Intriguingly, we were able to demonstrate a
reduction in Raf-RBD associated endogenous GTP-ras
molecules/proteins in transient transfection experiments.
Because sprouty was originally believed to be a secreted
inhibitor, we looked for its presence in the medium. We
failed to detect any HA-hspry4 using anti-HA Ig, which
should have detected the protein unless it had been partially
(i.e. C-terminally) degraded. Overexpression of hspry2 has
been shown to lead to the appearance in the conditioned
medium of an as yet unidentified inhibitor of FGF2
signaling [37]. Our data are compatible with a similar
paracrine effect of hspry4, primarily affecting GTP-ras.

cells (data not shown). In view of the presence of H- and
N-ras in the Golgi [39], this observation raises the question
as to whether the inhibitory effect of hspry4 on ras
activation is (solely) due to an activity of hspry4 at the
inner plasma membrane. spry1 and spry2 were recently
shown to associate with caveolin-1 in perinuclear and
vesicular structures and undergo post-translational phos-
phorylation and palmitoylation [40]. Only a small subset of
spry1 was recruited to the plasma membrane as part of lipid
rafts upon cellular activation by VEGF, also casting doubt
as to whether spry1 would exert its activity at the plasma
membrane via contact with receptor tyrosine kinase sign-
aling components.
A particularly relevant question is whether TESK1 can
phosphorylate the conserved functionally important tyro-
sine residue in the N-terminus of spry2 and spry4 [11]. In
preliminary experiments we were unable to demonstrate
hspry4 phosphorylation by TESK1 or a modulating effect
of hspry4 on the kinase activity of TESK1.
Studies in our laboratory are ongoing to test whether the
cysteine-rich region with its potential RING finger may
enable hspry4 to ubiquitinate itself and/or target TESK1 or
other proteins for degradation by the proteasome.
Other questions needing to be addressed include whether
hspry4 can be phosphorylated and palmitoylated similar to
spry1 and spry2, and if TESK1 can interact with other spry
proteins (directly). Finally, in view of the strength and
specificity of the interaction between TESK1 and hspry4 in
yeast, their intracellular colocalization and increased
interaction on growth factor stimulation, it is reasonable

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