Identification of a Dominant Negative Mutant of Sprouty That Potentiates Fibroblast Growth Factor- but Not Epidermal Growth Factor-induced ERK Activation

Abstract

Various mitogenic stimuli such as epidermal growth factor (EGF), fibroblast growth factor (FGF), and phorbol 12,13-dibutyrate (PDBu) activate the Ras-Raf-MEK-ERK pathway, but the regulatory mechanism of this pathway remains to be investigated. Here we found that in 293 cells, mammalian Sprouty2 and Sprouty4 were rapidly induced by EGF, FGF, and PDBu in an ERK pathway-dependent manner. Forced expression of Sprouty2 and Sprouty4 inhibited FGF-induced ERK activation but did not affect EGF- or PDBu-induced ERK activation. To examine whether endogenous Sproutys were also selective inhibitors, we generated a dominant negative form of Sprouty2 (Y55A) and Sprouty4 (Y53A) in which conserved tyrosine residues were mutated. These mutants reverted the suppressive effect of both Sprouty2 and Sprouty4 but not that of RasGAP or SPRED (Sprouty-related EVH1 domain-containing protein), another Sprouty-related Ras suppressor. Expression of dominant negative Sprouty2 and Sprouty4 enhanced and prolonged FGF- but not EGF-induced ERK activation in 293 cells. In PC12 cells, endogenous Sprouty4 was also induced by FGF. Overexpression of wild-type Sprouty4 blocked FGF-induced differentiation, whereas Y53A-Sprouty4 enhanced it. These observations suggest that endogenous Sprouty2 and Sprouty4 are physiological negative feedback regulators of growth factor-mediated ERK pathway and that there are Sprouty-sensitive and -insensitive ERK activation pathways. Finding a dominant negative form of Sproutys will facilitate the study of the molecular mechanism and physiological function of Sproutys.

Negative regulation of receptor tyrosine kinases: unexpected links to c-Cbl and receptor ubiquitylation

“…Spry2 appears to physically interact with multiple components of the Ras-MAPK pathway, including Grb2, FRS2, Raf1 the Ras GTPase activating protein (Ras-GAP; [32]) and c-Cbl [33]. Several regulatory mechanisms have been proposed, including inhibition at the levels of Grb2 [34], GAP [4], or the Raf1 kinase [35,36]. In contrast, the interactions with c-Cbl may positively impact on EGFR signaling [26,37].…”

“…First, the activation of RTKs triggers an induction in spry2 transcription in epithelial cells [35]. Regulation of the mature protein is mediated by ligandinduced phosphorylation of Spry2 on a tyrosine residue located at position 55 [24,26,34,38].…”

Sprouty4 negatively regulates protein kinase C activation by inhibiting phosphatidylinositol 4,5-biphosphate hydrolysis

“…In addition, three Sprouty-related genes, Spreds were identified (Sprouty-related Ena/VASP homology 1 domain-containing proteins) . Mammalian Sproutys and Spreds inhibit growth factor-induced cellular responses by inhibiting the RTK-dependent ERK signaling pathway (Gross et al, 2001;Impagnatiello et al, 2001;Lee et al, 2001;Sasaki et al, 2001Sasaki et al, , 2003Yigzaw et al, 2001;Hanafusa et al, 2002;Yusoff et al, 2002). Sproutys are considered to be a candidate of anti-oncogenes, as reduced expression of Sproutys has been shown in several cancers Lo et al, 2006).…”

“…Similarly, Sprouty4 inhibits vascular endothelial growth factor (VEGF)-A-induced PKC-mediated ERK activation, whereas Sprouty4 does not inhibit EGF as well as VEGF-C-mediated ERK activation (Sasaki et al, 2001(Sasaki et al, , 2003Taniguchi et al, 2007). Molecular basis for the suppression of the PKC pathway remains to be solved.…”

“…Sprouty2 and Sprouty4 mutants, in which tyrosine residues (Y55 for Sprouty2 and Y53 for Sprouty4) in the N-terminal region are replaced by a non-phosphorylatable residue, have been shown to act as a dominant-negative form for the FGF-induced ERK kinase activation. Therefore, the phosphorylation of these N-terminal tyrosine residues seems to be essential for the inhibitory activity of Sproutys on FGF-induced ERK activation (Sasaki et al, 2001;Hanafusa et al, 2002). However, in the case of VEGF-A signaling, the N-terminal region including Y53 of Sprouty4 is not necessary for suppression, whereas the CR domain is indispensable (Sasaki et al, 2003).…”