Src-Like adaptor protein (SLAP) binds to the receptor tyrosine kinase Flt3 and modulates receptor stability and downstream signaling

Abstract

Fms-like tyrosine kinase 3 (Flt3) is an important growth factor receptor in hematopoiesis. Gain-of-function mutations of the receptor contribute to the transformation of acute myeloid leukemia (AML). Src-like adaptor protein (SLAP) is an interaction partner of the E3 ubiquitin ligase Cbl that can regulate receptor tyrosine kinases-mediated signal transduction. In this study, we analyzed the role of SLAP in signal transduction downstream of the type III receptor tyrosine kinase Flt3. The results show that upon ligand stimulation SLAP stably associates with Flt3 through multiple phosphotyrosine residues in Flt3. SLAP constitutively interacts with oncogenic Flt3-ITD and co-localizes with Flt3 near the cell membrane. This association initiates Cbl-dependent receptor ubiquitination and degradation. Depletion of SLAP expression by shRNA in Flt3-transfected Ba/F3 cells resulted in a weaker activation of FL-induced PI3K-Akt and MAPK signaling. Meta-analysis of microarray data from patient samples suggests that SLAP mRNA is differentially expressed in different cancers and its expression was significantly increased in patients carrying the Flt3-ITD mutation. Thus, our data suggest a novel role of SLAP in different cancers and in modulation of receptor tyrosine kinase signaling apart from its conventional role in regulation of receptor stability.

Figure 1. SLAP interacts with Flt3 in response to FL-stimulation.

(A) COS-1 cells were co-transfected with pcDNA3-SLAP-FLAG and pcDNA3-Flt3-WT expression plasmids. Twenty four hours after transfection, cells were serum-starved overnight and stimulated by FL or not for 5 minutes before lysis. Cell lysates were immunoprecipitated with anti-Flt3 or anti-FLAG antibody followed by Western blotting analysis. (B) SLAP-FLAG and Flt3-ITD transfected COS-1 cells were stimulated for 5 minutes after overnight starvation. Cells were then processed for immunoprecipitation and Western blotting analysis. (C) SLAP-FLAG and Flt3-WT transfected COS-1 cells were stimulated for different time points after overnight starvation. Cells were then processed for immunoprecipitation with an anti-FLAG antibody and Western blotting analysis. Total Flt3 and SLAP phosphorylation was detected by 4G10 and the membrane was further probed with SLAP and Flt3 antibodies. (D) Structure of SLAP showing different domains and known tyrosine phosphorylation sites.

Figure 2. SLAP interacts with multiple Flt3 phosphotyrosine residues independent of the Src interaction sites.

(A) COS-1 cells were co-transfected with pcDNA3-SLAP-FLAG and pcDNA3-Flt3-Y589/591/599F expression vectors. Twenty four hours after transfection, cells were serum-starved overnight and stimulated by FL or not for 5 minutes before lysis. Cell lysates were immunoprecipitated with anti-FLAG antibody followed by Western blotting analysis. (B) Phospho-peptides corresponding to 12 tyrosine phosphorylation sites in Flt3 were immobilized on UltraLink. Peptide-bound slurry was incubated with SLAP-transfected COS-1 cell lysates and pulled-down proteins were then processed for Western blotting using an anti-SLAP antibody.

Figure 3. Expression of SLAP and Flt3 in Ba/F3 cells.

(A) Stably transfected Ba/F3 cells were labeled with isotyp-matched control or PE-conjugated Flt3 antibody and analyzed by flow cytometry. Gray area indicates cells labeled with isotype-matched control and black area indicates cells labeled with Flt3 antibody. (B) Lysates of stably transfected Ba/F3-Flt3-WT cells with SLAP shRNA or control shRNA were immunoprecipitated with an anti-SLAP antibody and analyzed by Western blotting. (C) Ba/F3 cells transfected with Flt3-WT and SLAP-FLAG were stained with fluorophore-conjugated primary antibodies. (D) Ba/F3 cells transfected with Flt3-WT and SLAP-FLAG were stimulated with FL for 5 minutes and then stained with fluorophore-conjugated primary antibodies. Blue: DAPI, green: SLAP-FLAG and red: Flt3. (E) Co-localization of SLAP and Flt3 was determined using CoLocalizer Pro 2.7.1.

Figure 4. Effects of SLAP on FL-induced Akt, Erk and p38 activation.

Ba/F3-Flt3 cells with SLAP shRNA or control shRNA were treated with 100 ng/ml FL or not for 5 minutes before lysis. (A) Total cell lysates were separated by SDS-PAGE and membranes were probed with phospho-specific antibodies. Membranes were then stripped and reprobed with respective antibodies to show the loading. (B) Signal intensities from three independent experiments of Fig. A were quantified using Multi-Gauge software.(C) Cell lysates were immunoprecipitated with respective antibodies and analyzed by Western blotting. (D) Signal intensities from three independent experiments of Fig. C were quantified using multi-gauge software.

Figure 5. SLAP depletion decreases ubiquitination and degradation of Flt3 receptor.

(A) Ba/F3-Flt3 cells with SLAP shRNA or control shRNA were serum-starved and stimulated with 100 ng/ml FL for 5 minutes. Cells were lysed and lysates were immunoprecipitated with an anti-Flt3 antibody followed by Western blotting analysis. (B) Ba/F3-Flt3 cells with SLAP shRNA or control shRNA were serum-starved and preincubated with cycloheximide for 4 hours. Cells were then stimulated with 100 ng/ml FL for 30 minutes before lysis and immunoprecipitated with an anti-Flt3 antibody. After Western blotting analysis signal intensities were quantified using Multi-Gauge software.

Figure 6. SLAP depletion decreases Cbl phosphorylation.

Ba/F3-Flt3 cells with SLAP shRNA or control shRNA were treated with 100 ng/ml FL or not for 5 minutes before lysis. (A) Cell lysates were immunoprecipitated with an anti-Cbl antibody and analyzed by Western blotting. (B) Signal intensities from three independent experiments were quantified using multi-gauge software.

Figure 7. SLAP mRNA expression in leukemia patient samples.

(A) SLAP mRNA expression was analyzed from microarray data of different patient samples and corresponding healthy donors. (B) SLAP mRNA expression was analyzed from the microarray dataset of acute promyelocytic leukemia (APL) patients carrying either wild-type FLT3 or FLT3-ITD mutation. Error bar shows SEM, and t-test was performed to determine significance. CML, Chronic myeloid leukemia; T-PLL, T-cell-prolymphocytic leukemia; WM, Waldenström's macroglobulinemia; CLL, Chronic lymphocytic leukemia.

Figure 8. Global protein interaction network of SLAP.

Global protein interaction network for SLAP was determined using PSICQUIC View, Gene Network Central and Cytoscape. Network was redrawn using Canvas. The Flt3-SLAP interaction is included from this study.

Figure 9. Schematic representation of SLAP signaling in response to FL stimulation.

SLAP interacts with Flt3 in response to FL stimulation and recruits Cbl leading to receptor degradation. Interaction of SLAP with Flt3 also leads to activation of Akt, Erk and p38 followed by cell proliferation.