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: o.c.leeksma@amc.uva.nl 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. 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MAP kinase activation by constitutively active V12 ras was unaffected