Mammalian Sprouty4 suppresses Ras-independent ERK activation by binding to Raf1

Abstract

The signalling cascade including Raf, mitogen-activated protein kinase (MAPK) kinase and extracellular-signal-regulated kinase (ERK) is important in many facets of cellular regulation. Raf is activated through both Ras-dependent and Ras-independent mechanisms, but the regulatory mechanisms of Raf activation remain unclear. Two families of membrane-bound molecules, Sprouty and Sprouty-related EVH1-domain-containing protein (Spred) have been identified and characterized as negative regulators of growth-factor-induced ERK activation. But the molecular functions of mammalian Sproutys have not been clarified. Here we show that mammalian Sprouty4 suppresses vascular epithelial growth factor (VEGF)-induced, Ras-independent activation of Raf1 but does not affect epidermal growth factor (EGF)-induced, Ras-dependent activation of Raf1. Sprouty4 binds to Raf1 through its carboxy-terminal cysteine-rich domain, and this binding is necessary for the inhibitory activity of Sprouty4. In addition, Sprouty4 mutants of the amino-terminal region containing the conserved tyrosine residue, which is necessary for suppressing fibroblast growth factor signalling, still inhibit the VEGF-induced ERK pathway. Our results show that receptor tyrosine kinases use distinct pathways for Raf and ERK activation and that Sprouty4 differentially regulates these pathways.

Spreds, inhibitors of the Ras/ERK signal transduction, are dysregulated in human hepatocellular carcinoma and linked to the malignant phenotype of tumors

“…Immunoblot analysis was carried out by using antibodies for c-Myc, GAPDH (Santa Cruz Biotechnology, Delaware, CA, USA), phospho-ERK1,2 (Thr 202, Tyr 204), ERK-1,2, phospho-Akt (Ser 473), Akt (Cell Signaling, Beverly, MA, USA) and Spred-1 (Wakioka et al, 2001) as described (Sasaki et al, 2003). Immunohistochemistry was performed on 5 mm sections of formalin-fixed, paraffin-embedded tissue.…”

Epigenetic inactivation of the human sprouty2 (hSPRY2) homologue in prostate cancer

“…Phosphorylated Spry can either bind to Grb2, thus competitively inhibiting the recruitment of Grb2-Sos complex (Hanafusa et al, 2002) or sequester c-Cbl, which would otherwise mediate internalization of activated RTKs Rubin et al, 2003). In addition, Spry4 may act downstream of Ras resulting in suppression of VEGFRmediated ERK activation in an Ras-independent manner by binding to Raf1 directly (Sasaki et al, 2003). This function appears not to require the conserved Tyr 55 and binding to Raf1 is through its highly conserved carboxy-terminal cysteine-rich domain (commonly called the Sprouty box).…”

The Sprouty-related protein, Spred, inhibits cell motility, metastasis, and Rho-mediated actin reorganization

“…The physiological functions of mSproutys, however, have not been well established. We and others have shown that mSprouty2 and mSprouty4 inhibit vascular endothelial growth factor-induced extracellular signalregulated kinase (ERK) activation and vascular branching and sprouting of vessels (Impagnatiello et al, 2001;Lee et al, 2001;Cabrita and Christofori, 2003;Sasaki et al, 2003). Since mSproutys inhibit not only the proliferation but also the migration of endothelial cells (Lee et al, 2001) as well as Hela cells (Yigzaw et al, 2001), mSproutys may modulate cytoskeletal organization.…”