Regulation of Sprouty2 stability by mammalian Seven‐in‐Absentia homolog 2

Abstract

Mammalian Sprouty (Spry) gene expression is rapidly induced upon activation of the FGF receptor signaling pathway in multiple cell types including cells of mesenchymal and epithelial origin. Spry2 inhibits FGFdependent ERK activation and thus Spry acts as a feedback inhibitor of FGF-mediated proliferation. In addition, Spry2 interacts with the ring-finger-containing E3 ubiquitin ligase, c-Cbl, in a manner that is dependent upon phosphorylation of Tyr55 of Spry2. This interaction results in the poly-ubiquitination and subsequent degradation of Spry2 by the proteasome. Here, we describe the identification of another E3 ubiquitin ligase, human Seven-in-Absentia homolog-2 (SIAH2), as a Spry2 interacting protein. We show by yeast two-hybrid analysis that the N-terminal domain of Spry2 and the ring finger domain of SIAH2 mediated this interaction. Co-expression of SIAH2 resulted in proteasomal degradation of Spry1, 2, and to a lesser extent Spry4. The related E3 ubiquitin-ligase, SIAH1, had little effect on Spry2 protein stability when coexpressed. Unlike c-Cbl-mediated degradation of Spry2, SIAH2-mediated degradation was independent of phosphorylation of Spry2 on Tyr55. Spry2 was also phosphorylated on Tyr227, and phosphorylation of this residue was also dispensable for SIAH2-mediated degradation of Spry2. Finally, co-expression of SIAH2 with Spry2 resulted in a rescue of FGF2-mediated ERK phosphorylation. These data suggest a novel mechanism whereby Spry2 stability is regulated in a manner that is independent of tyrosine phosphorylation, and provides an addition level of control of Spry2 protein levels.

The ubiquitin ligase Siah2 regulates tumorigenesis and metastasis by HIF-dependent and -independent pathways

“…Expression of the PHYL peptide competes with adaptor protein binding to both Siah2 and Siah1, thereby attenuating Siah's interaction with and degradation of PHD3. However, the PHYL peptide does not affect Spry2 levels because Spry2 does not contain the AXVXP motif found on other Siah2 substrates/adaptor proteins and because the Siah2/Spry2 interaction is mediated through a domain that is distant to the adaptor association region of Siah2 (3,22,26). Consistent with these conclusions are data obtained in in vitro protein binding experiments in which PHYL disrupts the Siah2/PHD3 interaction but has little effect on the Siah2/Spry2 interaction (data not shown).…”

“…26). Furthermore, Siah2 and Siah1 interact with PHD3 by means of adaptor proteins, whereas Siah2 interacts with Spry2 directly (26). Expression of the PHYL peptide competes with adaptor protein binding to both Siah2 and Siah1, thereby attenuating Siah's interaction with and degradation of PHD3.…”

“…4B). This difference may be attributed to direct interaction of SPRY2 and Siah2, apparently independent of the binding pocket that the PHYL peptide targets (26).…”

“…Because neither PHYL nor HIF-1␣ alters tumorigenesis in this mouse model and because S2RM alone does not alter levels of either PHD3 or HIF-1␣, we hypothesized that inhibition of tumorigenesis by S2RM may be mediated by a HIF-independent pathway. Among Siah2 targets is Sprouty2 (Spry2) (26), an inhibitor of the Ras/Raf signaling pathway shown to play an important role in tumorigenesis (27). Analysis of SW1 S2RM cells revealed a marked increase in Spry2 expression.…”

Sprouty proteins: modified modulators, matchmakers or missing links?

“…In a yeast two-hybrid screen, Nadeau et al (2006) identified human seven in absentia homolog 2 (SIAH2) as a SPRY2 interacting protein. The N-terminal half of Spry2 was demonstrated to interact with the ring finger domain of SIAH2 in a tyrosine phosphorylation-independent manner.…”