Sprouty2 acts at the Cbl/CIN85 interface to inhibit epidermal growth factor receptor downregulation

Abstract

The ubiquitin ligase Cbl mediates ubiquitination of activated receptor tyrosine kinases (RTKs) and interacts with endocytic scaffold complexes, including CIN85/endophilins, to facilitate RTK endocytosis and degradation. Several mechanisms regulate the functions of Cbl to ensure the fine-tuning of RTK signalling and cellular homeostasis. One regulatory mechanism involves the binding of Cbl to Sprouty2, which sequesters Cbl away from activated epidermal growth factor receptors (EGFRs). Here, we show that Sprouty2 associates with CIN85 and acts at the interface between Cbl and CIN85 to inhibit EGFR downregulation. The CIN85 SH3 domains A and C bind specifically to proline-arginine motifs present in Sprouty2. Intact association between Sprouty2, Cbl and CIN85 is required for inhibition of EGFR endocytosis as well as EGF-induced differentiation of PC12 cells. Moreover, Sprouty4, which lacks CIN85-binding sites, does not inhibit EGFR downregulation, providing a molecular explanation for functional differences between Sprouty isoforms. Sprouty2 therefore acts as an inducible inhibitor of EGFR downregulation by targeting both the Cbl and CIN85 pathways.

Figure 1

Complex between Sprouty2 and CIN85 in mammalian cells. (A) Endogenous Sprouty2 from C2C12 cell lysates was immunoprecipitated (IP) with antisprouty2 antibodies. Immunoblotting (IB) was performed using specific antibodies against Sprouty2 and CIN85. Pre-immune serum (Pre-im) was used as a negative control. TCL, total cell lysate. (B) Immunoprecipitates of Myc–Sprouty2 and FLAG–CIN85 were separated by SDS–polyacrylamide gel electrophoresis and analysed by immunoblotting with antibodies against the Myc epitope and CIN85. (C) Glutathione-S-transferase (GST) pull-down of Myc–Sprouty2 with isolated SH3 domains of CIN85 (SH3A, SH3B, SH3C) or GST alone. Levels of GST fusion protein were determined by Ponceau staining.

Figure 2

Identification of CIN85-binding sites in Sprouty2. (A) List of proline–arginine motifs in Sprouty2 and c-Cbl/Cbl-b. The amino-acid position of the conserved arginine is indicated. (B) Glutathione-S-transferase (GST) pull-down of Myc-tagged Sprouty2 or Sprouty2 mutants, performed as in Fig 1C. IB, immunoblotting; TCL, total cell lysate; WT, wild type.

Figure 3

CIN85-binding motifs in Sprouty in vivo. (A) Serumstarved human embryonic kidney (HEK) 293T cells overexpressing CIN85 and Myc-tagged Sprouty2 or indicated Sprouty2 mutants were mock treated or stimulated with 50 ng/ml epidermal growth factor (EGF) for 10 min. Levels of CIN85, Sprouty2 and its mutants in immunoprecipitates (IP) and total cell lysate (TCL) were determined by immunoblotting (IB). WT, wild type. (B) Table listing the CIN85-binding motifs in the Sprouty isoforms in different species. d, Drosophila; h, human; m, mouse. (C) Immunoprecipitation of CIN85 to analyse its interaction with FLAG-tagged Sprouty1, Sprouty4 or the Sprouty4/2 chimaera (S4/2). Sprouty1 is found in complex with endogenous CIN85 that is only detected on longer exposures.

Figure 4

Sprouty2 blocks epidermal growth factor receptor (EGFR) downregulation by acting on both Cbl and CIN85. (A) Serumstarved human embryonic kidney (HEK) 293T cells overexpressing EGFR, Cbl, haemagglutinin (HA)–ubiquitin (Ub), alone or in combination with Sprouty2 wild type (WT), Sprouty2-Y55A or Sprouty2-R64,72,309A, were mock treated or stimulated with 50 ng/ml EGF for 10 min. Immunoprecipitated EGFR was assayed for ubiquitination and association with Cbl. IB, immunoblotting; IP, immunoprecipitation; TCL, total cell lysate. (B) Chinese hamster ovary (CHO) cells were transfected with constructs for EGFR, Cbl and Sprouty2, Sprouty2-R72A, Sprouty2-R309A, Sprouty2-R72,309A, Sprouty2-R64,72,309A, Sprouty2-Y55A, Sprouty2-R64,72,309A,Y55F or green fluorescent protein (GFP) as a control and subjected to an EGFR downregulation assay, as described in the Methods. (C) CHO–EGFR cells were untransfected or transfected with Myc–Sprouty2 as indicated, serum starved and stimulated with 50 ng/ml EGF for 0, 5, 15 or 60 min. Total cell lysates (TCL) and immunoprecipitates of endogenous Cbl were analysed as shown. (D) CHO–EGFR cells were transfected with Myc–Sprouty2 and stimulated as in (C). Immunoprecipitates of endogenous CIN85 and overexpressed Sprouty2 were analysed by immunoblotting, as indicated. (E) In a ternary complex, Cbl interacts in an EGF-inducible way with both Sprouty2 and CIN85 by means of the tyrosine kinase-binding (TKB) domain and the PKPFPR motif (R829), respectively. Simultaneously, CIN85 associates constitutively with Sprouty2 by means of its SH3A and SH3C domains. Sprouty2-R64,72,309A is impaired in binding to CIN85, but interacts with Cbl. Conversely, Cbl-G306E-70Z does not bind to Sprouty2, but still retains the ability to interact directly with CIN85. Bottom left panel: HEK 293T cells were transfected with Myc–Sprouty2-R64,72,309A, alone or together with Cbl, as indicated. Bottom right panel: HEK 293T cells were transfected with HA–Cbl-G306E-70Z and Myc–Sprouty2, alone or together with FLAG–CIN85. Cells were serum starved and mock treated or stimulated for 5 min with 50 ng/ml EGF. Total cell lysates and immunoprecipitates of Myc–Sprouty2 were analysed by immunoblotting. RF, ring finger domain.

Figure 5

Sprouty2 requires binding to both Cbl and CIN85 to promote neurite outgrowth. (A) PC12 cells grown on poly-D-lysine-coated coverslips were transfected with Myc-tagged plasmids encoding Sprouty2, Sprouty2-R72A, Sprouty2-R309A, Sprouty2-R72,309A, Sprouty2-R64,72,309A, Sprouty2-R64,72,309A,Y55F or Sprouty2-Y55A. Cells were fixed after incubation for 4 days in lowserum medium without added growth factor (Sprouty2 ‘Control') or with 100 ng/ml epidermal growth factor. Cells were subjected to immunofluorescence, as described in the Methods. (B) Experimental sets in (A) were quantified in three independent experiments. Cells with neurite extensions of one to two times the length of the cell body were scored as neurite-containing cells. The graph shows the average percentage of cells showing neurite outgrowth from the three experiments. The error bars represent the standard deviation.