Báo cáo Y học: Human sprouty 4, a new ras antagonist on 5q31, interacts with the dual specificity kinase TESK1 potx

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,

the Netherlands;
4
Biological Institute, Graduate School of Science, Tohoku University, Sendai, Japan
The Drosophila melanogaster protein sprouty is induced
upon fibroblast growth factor (FGF)- and epidermal growth
factor (EGF)-receptor tyrosine kinase activation and acts as
an inhibitor of the ras/MAP kinase pathway downstream of
these receptors. By differential display RT-PCR of activated
vs. resting umbilical artery smooth muscle cells (SMCs) we
detected a new human sprouty gene, which we designated
human sprouty 4 (hspry4) based on its homology with
murine sprouty 4. Hspry4 is widely expressed and Northern
blots indicate that different isoforms of hspry4 are induced
upon cellular activation. The hspry4 gene maps to 5q31.3. It
encodes a protein of 322 amino acids, which, in support of a
modulating role in signal transduction, contains a prototypic
cysteine-rich region, three, potentially Src homology 3 (SH3)
binding, proline-rich regions and a PEST sequence. This
new sprouty orthologue can suppress the insulin- and
EGF-receptor transduced MAP kinase signaling pathway,
but fails to inhibit MAP kinase activation by constitutively
active V12 ras. Hspry4 appears to impair the formation of
active GTP-ras and exert its activity at the level of wild-type
ras or upstream thereof.
In a yeast two-hybrid screen, using hspry4 as bait, testi-
cular protein kinase 1 (TESK1) was identified from a human
fetal liver cDNA library as a partner of hspry4. The hspry4–
TESK1 interaction was confirmed by coimmunoprecipita-
tion experiments and increases by growth factor stimulation.
The two proteins colocalize in apparent cytoplasmic vesicles

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-

drodysplasia [7]. Moreover, mspry4 was shown to inhibit
vascular endothelial growth factor (VEGF)- and basic FGF
(bFGF)-dependent signaling in human endothelial cells
in vitro as well as angiogenesis in murine embryos [8].
All sprouty proteins have a characteristic, highly con-
served, cysteine-rich region in their C-terminal half. In
Drosophila, this region of sprouty was shown to be
responsible for targeting the protein to the plasma mem-
brane [2]. A conserved novel translocation domain within
this region was delineated in hspry2 and demonstrated to be
essential for relocating sprouty proteins to membrane ruffles
upon tyrosine kinase receptor activation [9]. Differences
between individual sprouty family members are greatest in
the N-terminal part of the proteins, suggesting that this part
of the protein may convey specificity to the activity of the
Correspondence to O. C. Leeksma, Department of Biochemistry,
Academic Medical Center, University of Amsterdam,
Meibergdreef 15, 1105 AZ Amsterdam, the Netherlands.
Fax: + 31 20 6915519, Tel.: + 31 20 5665140,
E-mail:
Abbreviations: hspry4, human sprouty 4; TESK1, testicular protein
kinase 1; DD/RT-PCR, differential display of randomly primed
mRNA by reverse transcription polymerase chain reaction; SMC,
smooth muscle cell; FGF, fibroblast growth factor; EGF, epidermal
growth factor; VEGF, vascular endothelial growth factor; PDGF,
platelet derived growth factor; ox-LDL, oxidized low-density
lipoprotein; HA, hemagglutinin; GST, glutathione S-transferase;
RBD, ras binding domain; EST, expressed sequence tag; EGFP,
enhanced green fluorescent protein.
(Received 31 August 2001, revised 25 February 2002,

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

orthologue in Drosophila, called CDI (Drosophila Center
Divider), were both suggested to be members of a novel
class of signaling proteins based on a unique sequence
within their substrate specificity determining kinase domain
[15,16]. In support of this suggestion, the kinase activity of
TESK1 is enhanced by fibronectin-mediated integrin sign-
aling, leading to phosphorylation of actin-binding cofilin
and actin reorganization [17], and, as shown in this paper,
the interaction of TESK1 with sprouty4 increases on growth
factor stimulation.
MATERIALS AND METHODS
DNA sequence analysis
DD/RT-PCR, Northern blotting, SMC cDNA library
construction and screening have been described in detail
previously [13]. Nucleotide sequences of SMC cDNAs,
identified from the activated umbilical artery SMC cDNA
library [13] by radioactive hybridization with EST W46239
(GenBank accession number), were determined from
both strands using a combination of vector- and cDNA-
specific primers on an ALF-express automatic sequencer
(Pharmacia, Uppsala, Sweden); the GenBank accession
number of hspry4 is AF227516. Predicted open reading
frames (ORF) were scanned against among others
PROSITE
(protein kinase C, casein kinase II, N-myristoylation sites),
TOP PRED
2 (transmembrane domain), the
PEST
algorithm
(Embnet; PESTfind), and

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

TCCGAAAGGCTTGTCGG-3¢,creatingaSmaIsite
(underlined) and relieving the stop codon at position 1156
by an A fi C substitution. The SstI/NspI 710-bp fragment
and 305-bp NspI/SmaI digest of the PCR product were
ligated together, in frame with the HA tag-encoding
sequence, into SstI/SmaI digested pGEM4Z-HA DNA.
pGEM4Z-HA DNA was made by ligating a 36-bp synthetic
oligonucleotide, encoding an 11 amino-acid HA sequence,
followed by a stop codon, into the SmaIandBamHI sites of
pGEM4Z DNA. HA-hspry4 cDNA was subcloned from
pGEM4Z-HA into pcDNA3.1 by PstI/XbaI digestion.
Plasmid EGFP–N2-hspry4, composed of vector EGFP-N2
(Clontech, Palo Alto, CA, USA) and hspry4 cDNA, was
constructed with primers: 5¢-TTA
GGATCCATGCT
CAGCCCCCTCCCC-3¢ forward and 5¢-G
GAATTC
TCCGAAAGGCTTGTCGG-3¢reverse, creating BamHI
and EcoRI restriction sites (underlined) for ligation in
frame into BglII/EcoRI digested EGFP-N2. The expression
plasmid, coding for N-terminally Myc epitope-tagged
TESK1, was constructed by inserting a NcoI–NotIfragment
of rat TESK1 cDNA (nucleotides 1129–3600) into the NotI
site of vector pCAG-Myc, containing Myc-epitope
sequence EQKLISEEDL [20]. HA-tagged human TESK1
was obtained by subcloning a BglII fragment of TESK1-
pAct2 (see below under yeast two-hybrid screen) into
BamHI digested pcDNA3.1. Orientation and integrity of
inserts was verified by DNA sequencing.
Cell culture and transfection

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

Na
2
HPO
4
,2m
M
NaH
2
PO
4
, pH 7.4) and lysed for 10 min at 4 °Cin
250 lL lysis buffer (50 m
M
Tris/HCl, pH 7.5, 100 m
M
NaCl, 50 m
M
NaF, 5 m
M
EDTA, 40 m
M
2-glycerophos-
phate, 200 l
M
Na
3
VO
4
, 1% Triton X-100, 1 l
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

M
dithiothreitol, 0.01% (w/v)
bromophenol blue), heated for 5 min at 95 °C, and used
for anti-ERK2 Ig (Santa Cruz, CA, USA) immunoblotting.
The remaining 50 lLwereusedforthein vitro kinase assay
of 7.5 lg myelin basic protein (Sigma) in the presence of
3 lCi [c
32
-P]ATP (Amersham Pharmacia Biotech, Buck-
inghamshire, UK) for 30 min at room temperature. The
reaction was stopped by adding sample buffer and analyzed
by 15% (w/v) SDS/PAGE, followed by autoradiography.
Raf-RBD GST pulldown
Detection of GTP–ras was performed as described previ-
ously [21], except that murine anti-ras monoclonal antibody
R2021 (Transduction Laboratories, Lexington, KY, USA)
instead of rat monoclonal antibody Y 13-259 was used in
combination with horse-radish peroxidase-conjugated goat
anti-(mouse IgG) Ig (Jackson Laboratories, Westgrove, PA,
USA) for immunoblotting. Rabbit anti-(phospho-MAP
kinase) 42/44 Ig (New England Biolabs) was used to assess
the level of phosphorylation of ERK1 and ERK2 in the
lysates used for GTP-ras pull down, whereas total ERK1
and ERK2 were quantitated using a 1 : 1 mixture of rabbit
anti-ERK1Igandanti-ERK2Ig(SantaCruz).Lysate
volumes used for the pull down assays and total lysate
analysis were adjusted to ensure identical total protein
concentrations as determined by BCA assay (Bio-Rad).
Yeast two-hybrid assay
Full-length human sprouty 4 cDNA was amplified by PCR

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

cells were washed three times with ice-cold NaCl/P
i
,
suspended in RIPA buffer [50 m
M
Tris/HCl (pH 8.0),
150 m
M
NaCl, 1 m
M
dithiothreitol, 10% (v/v) glycerol, 1%
(v/v) NP40, 1 m
M
phenylmethanesulfonyl fluoride, 21 l
M
leupeptin] and incubated for 30 min on ice. After centrif-
ugation, lysates were precleared for 2 h at 4 °C with protein A
–Sepharose. Precleared supernatants were incubated over-
night at 4 °C with anti-Myc monoclonal 9E10 or rabbit
polyclonal anti-HA serum and protein A–Sepharose. Im-
munoprecipitates were washed three times with wash buffer
[50 m
M
Tris/HCl (pH 8.0), 150 m
M
NaCl, 0.5% (v/v) NP-
40], suspended in sample buffer [50 m
M
Tris/HCl (pH 6.8),
10% (v/v) glycerol, 1 m

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

for 30 min at 4 °C with 4% (w/v) paraformaldehyde in
NaCl/P
i
, washed twice with NaCl/P
i
and permeabilized for
5 min at room temperature with 0.2% (v/v) Triton-X-100
(Sigma) in NaCl/P
i
. Cover slips were then washed with
NaCl/P
i
, incubated for 1 h in blocking solution [2% (v/v)
2548 O. C. Leeksma et al. (Eur. J. Biochem. 269) Ó FEBS 2002
normal goat serum in NaCl/P
i
]andfor1hwithanti-HAIg
HA.11 (BAbCO, Richmond,CA), diluted 1 : 200 in block-
ing solution. After three washes with 0.05% (v/v) Tween in
NaCl/P
i
, cells were stained for 1 h with Cy3-labeled goat
anti-(mouse IgG) Ig (Jackson Laboratories), diluted 1 : 300
in blocking solution, washed again three times and mounted
in mowiol embedding solution (Calbiochem, La Jolla, CA,
USA) on glass slides. Intracellular localization was analyzed
with a confocal laser scanning microscope (Bio-Rad), using
LASERSHARP
software.
Effect of EGF on hspry4–TESK1 interaction

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

artery SMC was represented by a number of expressed
sequence tags (ESTs), assembled in UniGene cluster Hs.
6553 in the NCBI database. Expression of this gene was
maximal 4 h after stimulation of the SMC with the
conditioned medium of monocytes activated by ox-LDL
(Fig. 1A). This stimulation was associated with a 14-fold
induction of a 4.9-kb mRNA and the appearance of less
abundant transcripts of approximately 7.9, 11.3 and 13 kb.
The 4.9-kb transcript of this gene was expressed by all
tissues examined on a multiple tissue Northern blot
(Fig. 1B).
Characteristics of the
hspry4
gene
We identified three cDNAs of about 2.5, 4.9 and 7 kb, using
a cDNA library constructed with mRNA isolated from
cultured, activated human SMCs [13]. These cDNA
sequences could be aligned with EST W46239 and were
different transcripts of the same novel gene. The 4.9-kb
cDNA contained the largest predicted open reading frame,
encoding a protein of 322 amino-acid residues. Alternative
splicing and different polyA site usage probably gave rise to
the 7-kb cDNA. It has an extended 3¢ UTR and lacks two
exons within the coding sequence, based on a comparison
with the 4.9-kb cDNA and alignments, using the Basic
Logical Alignment Search Tool (
BLAST
) program, with high
throughput genomic sequences and human genome chro-
mosome 5 sequences in GenBank. A smaller open reading

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

fluorescent in situ hybridization probe, using the genomic
BAC CTC463A16 clone. This BAC contains the hspry4
Fig. 1. Induction of hspry4 mRNA in SMCs as shown by Northern
blotting. (A) Stimulation of SMCs by conditioned medium of ox-LDL
activated monocytes supernatant analyzed by Northern blotting, using
a radiolabeled probe for hspry4 [13]. (B) Multiple tissue Northern
blotting that shows expression of the 4.9-kb hspry4 mRNA in all
tissues represented.
Ó FEBS 2002 Ras antagonist human sprouty 4 binds TESK1 (Eur. J. Biochem. 269) 2549
gene, as shown by PCR, and hybridizes to 5q31.3 (data not
shown).
Amino-acid sequence of hspry4
The amino-acid sequence of hspry4 harbors three poten-
tially SH3-binding proline-rich regions, a feature compatible
with a modulating role in signal transduction [26] (Fig. 3A).
Hspry4 also contains a PEST sequence [27], with two SSXS
sequences [28], which may be involved in regulating a timely
degradation of the protein. The N-terminal end contains an
extra 23 amino acids compared with mspry4. Hence the
functionally relevant conserved tyrosine is at position 75
instead of 52 [11]. There are six predicted casein kinase
II- and four protein kinase C-phosphorylation sites (not
shown), a single MAP kinase consensus sequence phos-
phorylation site [29], a possible nuclear localization signal
and nuclear-export sequence [30]. The conserved cysteine-
rich region harbors a putative N-myristoylation site, a trans-
membrane domain and a zinc-binding RING finger motif
[31].
Alignment of the available sequences of the murine and
human sprouty family members with the sequence of

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

activation of ras we initially performed experiments in
which HA-tagged H-ras was cotransfected with hspry4 or
empty vector, analogous to the experiments with Myc-
tagged MAP kinase, to essentially limit the analysis of ras
activation to transfected cells. In these experiments hspry4
coexpression reduced the amount of GTP-ras pulled down
by a GST-fusion protein, containing the ras-binding domain
(RBD) of Raf, which preferentially binds active GTP-ras
[21]. Introduction of HA–H-ras by transfection however,
led to the presence of activated ras in unstimulated cells,
which we failed to prevent by either prolonging the
starvation period to 40 h [21] or reducing the HA–H-ras
plasmid concentration from 0.5 lgto0.1lg (not shown).
We therefore decided to look at the effect of introducing
hspry4 on endogenous GTP-ras formation in A14 cells.
While endogenous GTP-ras was negligible in nonstimulated
cells, stimulation with insulin or EGF for 2 min led to
readily detectable GTP-ras. Overexpression of hspry4 was
reproducibly associated with a reduction in Raf RBD
bound GTP-ras (Fig. 5) Transfection efficiencies in these
experiments were in the order of 35–40% and we did not
observe a similar reduction by hspry4 of phosphorylated
endogenous ERK1 and ERK2 as determined by immuno-
blotting, suggesting that residual ras activation in nontrans-
fected cells was still sufficient to activate Raf.
Hspry4 interacts with testicular protein kinase 1
In search of partners, which might reveal its mechanism of
action, hspry4 cDNA was inserted in vector pMD4, in
frame with a GAL4 DNA binding domain (BD), and used
as bait in a yeast two-hybrid screen with a pAct2 human,

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:

underlined residues within this box corres-
pond to zinc-binding RING finger motif.
A wave underline represents a putative
N-myristoylation motif. The predicted trans-
membrane domain is shaded grey. (B) Align-
ment of amino-acid sequences of spry family
members, using
CLUSTAL W
. Identical or
similar residues in the majority of the aligned
sequences are shaded black or grey, respect-
ively. Fully conserved cysteine residues are
marked with an asterisk. Gaps have been
introduced to maximize alignment. dspry is
Drosophila melanogaster spry; mspry is murine
spry; hspry is human spry.
Ó FEBS 2002 Ras antagonist human sprouty 4 binds TESK1 (Eur. J. Biochem. 269) 2551
Hspry4 and TESK1 coimmunoprecipitate
HA-tagged hspry4 and Myc-tagged rat TESK1 were
coexpressed in COS cells to validate the interaction observed
in the yeast two-hybrid screen. Anti-HA Ig co-immunopre-
cipitated Myc-TESK1. Conversely, anti-Myc Ig precipitated
the HA-tagged hspry4 protein from COS cells, that were
transfected with both Myc-TESK1 cDNA and HA-hspry4
cDNA (Fig. 7). Consequently, both from the data obtained
with the yeast two-hybrid screen and the coimmunoprecip-
Fig. 4. MAP kinase inhibition by hspry4. (A) A14 cells, transfected
with 0.5 lg Myc-tagged ERK2 and either 2.0 lg pcDNA 3.1, or
pcDNA 3.1-HA-hspry4 were incubated at 37 °Cwith5lgÆmL
)1

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

interact in yeast two-hybrid assay. The hspry4 protein, fused to the
GAL4 DNA binding domain (BD), contains three potentially SH3
binding sequences (PXXP), a MAP kinase consensus sequence phos-
phorylation site (PLTP), a PEST sequence and a cysteine-rich (c-rich)
region. The TESK1 protein, fused to the GAL4 activation domain
(AD), harbors a kinase domain and a proline-rich (pro-rich) region.
The partial TESK1 cDNA fragment, selected by yeast two-hybrid
screen, spans amino acids 459 till the C-terminus at 626.
2552 O. C. Leeksma et al. (Eur. J. Biochem. 269) Ó FEBS 2002
itation experiments, we conclude that hspry4 and TESK1
are associated and, conceivably, functionally interact.
Colocalization of TESK1 and hspry4
We constructed HA-tagged TESK1 cDNA and fused
hspry4 cDNA with DNA encoding (enhanced) green
fluorescent protein (EGFP) to establish the intracellular
localization of the hspry4 and TESK1 proteins. HA-TESK1
was expressed, in agreement with previous observations, in
the cytoplasm, where it colocalized with EGFP- hspry4
(Fig. 8). A similar colocalization was observed with hspry4,
fused to HA (HA-hspry4), and TESK1 fused to the Myc-
tag as determined by indirect immunofluorescence, using
rabbit anti-HA Ig and murine anti-Myc Ig (data not
shown). The colocalization of hspry4 and TESK1 remained
primarily in peri- and para-nuclear dots upon stimulation
with either EGF or insulin. An identical intracellular
localization was seen of TESK1 and hspry4 in TESK1 or
hspry4 single transfectants, respectively, suggesting an effect
of hspry4 on the localization of TESK1 and vice versa is
unlikely.
Hspry4/TESK1 interaction is increased by EGF

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.

Ó FEBS 2002 Ras antagonist human sprouty 4 binds TESK1 (Eur. J. Biochem. 269) 2553
protein induced by ras activation. Although we did not
analyze the composition of the supernatant derived from
monocytes stimulated with ox-LDL, such a supernatant
may contain a cocktail of growth factors and cytokines e.g.
VEGF [32] capable of promoting via activation of ras the
expression of a feedback inhibitor such as hspry4, which in
SMC may serve to limit cellular proliferation. The hspry4
gene is localized relatively near a region of chromosome 5 in
which deletions [33] and translocations are associated with
acute myeloid leukemia and myelodysplasia. Such deletions
are assumed to encompass a long sought-after tumor
suppressor gene. We are currently using fluorescence in situ
hybridization to screen for 5q31 translocations involving the
hspry4 gene.
As to the mechanism of action of hspry4, a number of
features may indicate its potential functional interactions.
Proline-rich sequences in the N-terminal part of hspry4 can
be envisaged to interact with SH3-containing proteins,
analogous to the observed binding of Drosophila spry to the
adaptor protein Drk [2] or to WW domains, which mimic
SH3 sequences [34]. The cysteine-rich region of hspry4
appears to fulfill criteria for a zinc-binding RING-finger.
Although sequences can vary significantly from the accepted
RING consensus sequences [35], it is generally agreed upon
that cysteine- and histidine-rich RING-like regions are
instrumental in ubiquitination. Studies on sprouty’s func-
tion have indicated a role for Drosophila spry and mspry as
inhibitors of the ras/MAP kinase signaling pathway down-
stream of FGF-, EGF-, VEGF-, PDGF-, NGF- and c-Kit

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.

Others have provided arguments for a sprouty sensitive and
insensitive ERK activation pathway [11] and the ability of
sprouty-related molecules called spreds to uncouple ras
activation from Raf activation [38]. Yet, our data differ
from theirs in that we do find inhibition (by hspry4) of
EGF-induced MAP kinase activation. This discrepancy
could reflect differences in timing EGF responses (i.e. 2 vs.
10 min) or properties of hspry4 vs. mspry4 [11]. Unraveling
the precise molecular mechanism of action of endogenous
sproutys clearly requires additional studies.
By performing a yeast two-hybrid analysis, using a
human fetal liver cDNA library and hspry4 as bait, we
aimed at identifying (a) partner(s) of the hspry4 protein.
Surprisingly, we did not select any of the established
components of the ras/MAP kinase signaling pathway, but
instead encountered TESK1. The interaction between
hspry4 and TESK1 is apparently constitutive, increases on
growth factor stimulation and is conserved among rat and
human TESK1. Preliminary experiments with a hspry4
variant, lacking the cysteine-rich region, indicate that this
domain is required for the interaction with TESK1 (data
not shown).
As for the intracellular localization of hspry4 and
TESK1, we failed to observe massive membrane association
in ruffles of hspry4 irrespective of whether cells were
cotransfected with TESK1 cDNA or stimulated by EGF or
insulin. Although some membrane association was
observed, most of the colocalization was peri- and para-
nuclear and in cytoplasmic dots even after 10 min of
stimulation. This picture did not differ in HeLa, A14 or 293

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

to assume that both proteins interact in vivo, although
further proof of a functional interaction is required. The
relatively low levels of expression of the two proteins and the
limited sensitivity/specificity of currently available polyclon-
al anti-TESK1 Ig and anti-mspry4 Ig probably account for
our inability so far to unequivocally demonstrate binding of
endogenous TESK1 to hspry4.
2554 O. C. Leeksma et al. (Eur. J. Biochem. 269) Ó FEBS 2002
It is evident that the discovery of the sprouty protein
family as ras inhibitors, induced by ras itself, contributes to
the seemingly ever increasing complexity of ras signal
modulating mechanisms. In view of the pleiotropic in vivo
effects of ras, ras/MAP kinase-inhibiting hspry4 is likely to
exert its activity at different levels. Additional insight into
the mechanism of action of a natural ras inhibitor like
hspry4, may eventually contribute to the development of
novel ras inhibitory, antiatherogenic and antioncogenic
strategies.
ACKNOWLEDGEMENTS
Dr Johan van Es (University of Utrecht, Department of Immunology,
Utrecht, the Netherlands) is gratefully acknowledged for technical
assistance with the yeast two-hybrid procedure. We thank Ruud
Fontijn for excellent technical assistance with the confocal laser
scanning microscopy. This work was supported by Molecular Cardi-
ology program grant M93.007 of the Netherlands Heart Foundation,
The Hague, the Netherlands and the Fonds National de la Recherche
Scientifique Bekales, Brussels, Belgium.
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