Sprouty was originally discovered in Drosophila as an inducible antagonist of FGF receptor signaling and subsequently shown to act as an intracellular inhibitor of the ras/ MAPkinase pathway. Three human orthologues.sprouty 1,2 and 3,have been described. We have cloned and sequenced a fourth human sprouty gene from an umbilical artery smooth muscle (SMC) cDNA library. Expression of this gene was not restricted to these SMC,but observed in pancreas, kidney skeletal muscle, liver, lung, placenta, brain, heart, prostate, stomach, small intestine, colon, uterus, bladder as well as leukemic cell lines. The predicted ORF of the gene encodes a protein of 322 amino acid residues. Apart from the conserved prototypic cysteine rich region, human sprouty 4 (hspry 4) contains in support of a modulatory role in signal transduction 3 potentially SH3 binding proline rich regions and a PEST sequence. Overexpression of the protein in NIH3T3 cells with a human insulin receptor was associated with inhibition of MAPkinase activation downstream of insulin and EGF receptor tyrosine kinases. MAPkinase activity induced by constitutively active v12 ras was unaffected by hspry4. These data suggest that the inhibitor exerts its activity at the level of normal ras or upstream thereof. In line with this hypothesis sprouty transfectants showed a reduction in active OTP-ras formed upon insulin or EGF stimulation as determined by pull down experiments using a ras binding domain of raf GST fusion protein. In a yeast two hybrid screen of a fetal liver cDNA library,using hspry4 as bait.TESKl (testicular kinase 1) was identified as one of sprouty's partners. The two proteins co-localize in apparent intra-cytoplasmic dot-like structures in cotransfection experiments in Hela cells and co-immunoprecipitate. While the precise mechanism of action of hspry 4 requires additional study, 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 modulatory mechanisms. In view of the pleiotropic in vivo effects of ras, sprouty is likely to exert its activity at different levels. As 5q deletions are strongly implicated in (secundary) acute myeloid leukemia and myelodysplasia, the question emerges if hspry 4, of which a constituent EST was mapped between D5S500 and D5S436,is perhaps one of the long sought tumor suppressor genes in this region.
|Issue number||11 PART I|
|Publication status||Published - 2000 Dec 1|
ASJC Scopus subject areas
- Cell Biology