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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, testicular 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 coimmunoprecipitation 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.
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 signaling, three human orthologues, designated human sprouty (hspry)1, 2 and 3, were identified . 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 . Subsequent 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 , or downstream of ras at the level of Raf/MAP kinase . 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 , overexpression of mspry2 and 4 in chicken embryos both caused chondrodysplasia . 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 .
All sprouty proteins have a characteristic, highly conserved, 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 membrane . 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 . 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 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 presumably shared by mspry1, but not by mspry4, suggests that specificity relies on the respective N-terminal sequences . 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 .
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 . 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 signaling, leading to phosphorylation of actin-binding cofilin and actin reorganization , and, as shown in this paper, the interaction of TESK1 with sprouty4 increases on growth factor stimulation.
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- Materials and methods
Research in Drosophila melanogaster has led to the identification of many evolutionary conserved proteins, involved in signal transduction. The sprouty protein family represents 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 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 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  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  or to WW domains, which mimic SH3 sequences . 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 , it is generally agreed upon that cysteine- and histidine-rich RING-like regions are instrumental in ubiquitination. Studies on sprouty's function have indicated a role for Drosophila spry and mspry as inhibitors of the ras/MAP kinase signaling pathway downstream 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 . hspry4 apparently exerts a similar function as Drosophila sprouty in acting as an intracellular inhibitor of ras . 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 . Our observation that hspry4 overexpression 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 . 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 . 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  and the ability of sprouty-related molecules called spreds to uncouple ras activation from Raf activation . 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 . 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 , 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 phosphorylation and palmitoylation . 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 signaling components.
A particularly relevant question is whether TESK1 can phosphorylate the conserved functionally important tyrosine residue in the N-terminus of spry2 and spry4 . 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 polyclonal anti-TESK1 Ig and anti-mspry4 Ig probably account for our inability so far to unequivocally demonstrate binding of endogenous TESK1 to hspry4.
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.