Human sprouty 4, a new ras antagonist on 5q31, interacts with the dual specificity kinase TESK1

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.