The Src-like Adaptor Protein 2 Regulates Colony-stimulating Factor-1 Receptor Signaling and Down-regulation

Benjamin Pakuts,Christophe Debonneville,Larissa M Liontos,Michael P Loreto,C Jane Mcglade

doi:10.1074/jbc.m701182200

Abstract

Src-like adaptor protein 2 (SLAP-2) is a hematopoietic adaptor protein previously implicated as a negative regulator of T-cell antigen receptor (TCR)-mediated signaling. SLAP-2 contains an SH3 and an SH2 domain, followed by a unique carboxyl-terminal tail, which is important for c-Cbl binding. Here we describe a novel role for SLAP-2 in regulation of the colony-stimulating factor 1 receptor (CSF-1R), a receptor tyrosine kinase important for growth and differentiation of myeloid cells. SLAP-2 co-immunoprecipitates with c-Cbl and CSF-1R in primary bone marrow-derived macrophages. Using murine myeloid cells expressing CSF-1R (FD-Fms cells), we show that SLAP-2 is tyrosine-phosphorylated upon stimulation with CSF-1 and associates constitutively with both c-Cbl and CSF-1R. In addition, we show that expression of a dominant negative form of SLAP-2 impairs c-Cbl association with the CSF-1R and receptor ubiquitination. Impaired c-Cbl recruitment also correlated with changes in the kinetics of CSF-1R down-regulation and trafficking. CSF-1-mediated differentiation of FD-Fms cells and activation of downstream signaling events was also enhanced in cells stably expressing dominant negative SLAP-2. Together, these results demonstrate that SLAP-2 plays a role in c-Cbl-dependent down-regulation of CSF-1R signaling.

Highlights

The colony-stimulating factor 1 receptor (CSF-1R)4 is a member of the type III receptor tyrosine kinase (RTK) family, which includes c-Kit, platelet-derived growth factor receptor ␣ and ␤, and Flt3 [1]
The non-myristoylated Src-like adaptor protein 2 (SLAP-2) mutant (G2A) showed similar percentages of SSChigh- and CD11b-positive cells as the HSC control cell line. These results indicate that interfering with SLAP-2 function potentiates colony-stimulating factor-1 (CSF-1) signaling and suggest that SLAP-2 acts as a negative regulator of CSF-1R
As described for other RTKs, the RING-finger protein c-Cbl is involved in CSF-1R internalization and degradation [11]

Summary

EXPERIMENTAL PROCEDURES

Bacterial and Mammalian Expression Constructs—Wildtype SLAP-2 and SLAP-2 mutants have been previously described [17]. Subcellular Fractionation—FD-Fms cells (2.5 ϫ 107) were washed with phosphate-buffered saline (PBS) and lysed in 1 ml of hypotonic lysis buffer (10 mM Tris-HCl (pH 8.0), 1 mM MgCl2) containing complete protease inhibitors and 1 mM sodium orthovanadate. Cells were washed twice with cold PBS, blocked for 5 min in DMEM (10% FBS, 100 mM glycine) at 4°C, washed twice in cold PBS, and lysed in PLC lysis buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 10% (v/v) glycerol, 1% (v/v) Triton-X-100, 1 mM EGTA (pH 8.0) freshly supplemented with complete protease inhibitors (Roche Applied Science), 1 mM sodium orthovanadate, and 10 mM NaF).

RESULTS

DISCUSSION

Because the dominant negative