Elsevier

Cellular Signalling

Volume 19, Issue 11, November 2007, Pages 2277-2285
Cellular Signalling

Sprouty2 binds Grb2 at two different proline-rich regions, and the mechanism of ERK inhibition is independent of this interaction

https://doi.org/10.1016/j.cellsig.2007.07.008Get rights and content

Abstract

Sprouty2 has been widely implicated in the negative regulation of the fibroblast growth factor receptor–extracellular regulated kinase (ERK) pathway. Sprouty2 directly interacts with the adapter protein Grb2, member of the receptor tyrosine kinase-induced signaling pathways. In considering the functional role of Grb2, we investigated whether the interaction with this protein was responsible for ERK pathway inhibition. We found that the binding between Sprouty2 and Grb2 is constitutive, independent of Sprouty2 tyrosine phosphorylation, although it is increased when fibroblast growth factor receptor is activated. This connection is mediated by the N-terminal SH3 domain of Grb2 and two Sprouty2 proline-rich stretches (residues 59–64 and 303–307). Most importantly, a double Sprouty2 mutant (hSpry2 P59AP304A), which is unable to bind Grb2, developed at a similar inhibition level of fibroblast growth factor receptor–ERK pathway than that which originated from Sprouty2 wt. These results are evidence that the Sprouty2 mechanism of ERK inhibition is independent of Grb2 binding.

Introduction

Mammalian Sprouty (Spry) is a family of proteins that repress the receptor tyrosine kinase (RTK)–extracellular regulated kinase (ERK) pathway [1], [2]. Forced expression of Sprouty2 (Spry2) inhibits fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF), but not epidermal growth factor-(EGF) induced ERK activation [3]. Spry proteins have also been implicated in the negative feedback regulation of FGF signaling in embryogenesis [1], [4], angiogenesis [5] and myogenesis [6]. The ERK pathway is critical to tumorigenic processes, and it has been suggested that some members of the Spry family could have a role as tumor suppressors because of their capacity to inhibit ERK activation. We recently detected hSpry2 promoter hypermethylation in 37% of B-cell diffuse lymphoma cases, and found that it was associated with a significant decrease in the five-year survival rate (Sánchez A et al., unpublished results).

However some aspects of the molecular mechanism of the action of Spry2 still remain controversial. Several reports have proposed that the Ras-GTP loading occurring when FGFR is activated could be blocked by in vivo Spry2–Grb2 complexes that would reduce the Sos–Grb2 levels that are recruited to FGFR [7], [8]. By contrast, it has also been suggested that Spry2 inhibits Raf but not Ras activation [3].

Another contentious point concerning Spry2 is its molecular interaction with Grb2, which elicits in vivo Spry2–Grb2 complexes. Grb2 is an adaptor protein with one SH2 domain flanked by two SH3 domains. The SH3 domains of Grb2 bind to specific proline-rich sequences, such as the Grb2-Binding Motifs (Grb2-BM) (PΨΨPPR) and the SH3-Minimal Binding Sites (SH3-MBS) (ΨPXΨP), which are located in the C-terminal region of Sos [9], [10], [11] and adopt a left-handed polyproline type II helix conformation [12]. In general, the SH2 domains recognize specific protein motifs containing a phospho-tyrosine residue, usually pTyr-X1-X2-X3 (where X1–3 are variable peptide units and X3 is a peptide residue containing a lipophilic side chain such as Leu or Ile) [13]. The minimal peptide sequence recognized by the Grb2 SH2 domain would be pTyr-Ile-Asn [14], which is located in the cytoplasm of activated RTKs, or at phospho-tyrosine proteins that are not RTKs, such as Shc, IRS-1, and Syp [15], [16], [17], [18]. Data from several sources provide evidence of the in vivo interaction between Spry2 and Grb2 [7], [8], [19]. Spry2 contains different putative Grb2-binding sites, such as the proline-rich stretches located either in its N-terminal portion (residues 1–177), or in its C-terminal end (residues 178–315) with potential binding affinity for SH3 domains, and the tyrosine 55 (phosphorylated upon RTK activation) with putative SH2 binding capacity. However, several aspects still remain the subject of considerable debate, including (1) the identity of the Spry2 regions responsible for this binding (proline-rich regions or phospho-tyrosine 55) [8], [19], [20], (2) the Grb2 domains involved (SH3 domains N-, C-terminal, both, or the SH2 domain) [8], [19], [20], (3) the type of Spry2–Grb2 complex (constitutive or inducible upon FGFR activation) [7], [8], (4) the capacity to impair the recruitment of Grb2–Sos complex to the cell membrane [7], [8], [20], and (5) the ability to block Ras activation [3], [5], [7], [19].

This study was carried out to identify the hSpry2 regions and Grb2 domains involved in the hSpry2–Grb2 complex, and to ascertain whether the capacity of hSpry2 to block the FGFR–ERK pathway depends on its binding to Grb2.

Section snippets

Cell lines

NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 10% calf serum (CS, Invitrogen). The human 293T (kidney keratinocyte) cell line was maintained in DMEM supplemented with 10% fetal calf serum (FCS, Invitrogen).

In vitro mapping of the hSpry2 Grb2-binding sites

We carried out pull-down assays to evaluate the hSpry2 binding affinity of different Grb2 domains (Fig. 1A). Cytoplasmic extracts from 293T cells overexpressing AU5-tagged full-length hSpry2 were incubated with different GST-Grb2 chimerical-proteins coupled to glutathione-sepharose beads (Fig. 1A). Whereas purified GST alone did not bind any hSpry2, similar amounts of hSpry2 were associated with GST-Grb2 independently of RTK activation. Here we used serum as a stimulus, although the same

Acknowledgments

NM, CAGD, BD, JLO, NZ, and AS were recipients of fellowships from the Instituto de Salud Carlos III (NM, NZ, and AS), FIS-BEFI (CAGD and JLO), and the Consejería de Sanidad, Junta de Comunidades de Castilla-La Mancha (BD). This work was supported by grants SAF2006-04247 from the Ministerio de Educación y Ciencia, Spain, FMMA-2005 from the Fundación Mutua Madrileña Automovilista, from the Fundación Científica de la Asociación Española Contra el Cáncer, and RETICS from the Instituto de Salud

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