Src-like adaptor protein 2 (SLAP2) binds to and inhibits FLT3 signaling

Abstract

Fms-like tyrosine kinase (FLT3) is a frequently mutated oncogene in acute myeloid leukemia (AML). FLT3 inhibitors display promising results in a clinical setting, but patients relapse after short-term treatment due to the development of resistant disease. Therefore, a better understanding of FLT3 downstream signal transduction pathways will help to identify an alternative target for the treatment of AML patients carrying oncogenic FLT3. Activation of FLT3 results in phosphorylation of FLT3 on several tyrosine residues that recruit SH2 domain-containing signaling proteins. We screened a panel of SH2 domain-containing proteins and identified SLAP2 as a potent interacting partner of FLT3. We demonstrated that interaction occurs when FLT3 is activated, and also, an intact SH2 domain of SLAP2 is required for binding. SLAP2 binding sites in FLT3 mainly overlap with those of SRC. SLAP2 over expression in murine proB cells or myeloid cells inhibited oncogenic FLT3-ITD-mediated cell proliferation and colony formation in vitro, and tumor formation in vivo. Microarray analysis suggests that higher SLAP2 expression correlates with a gene signature similar to that of loss of oncogene function. Furthermore, FLT3-ITD positive AML patients with higher SLAP2 expression displayed better prognosis compared to those with lower expression of SLAP2. Expression of SLAP2 blocked FLT3 downstream signaling cascades including AKT, ERK, p38 and STAT5. Finally, SLAP2 accelerated FLT3 degradation through enhanced ubiquitination. Collectively, our data suggest that SLAP2 acts as a negative regulator of FLT3 signaling and therefore, modulation of SLAP2 expression levels may provide an alternative therapeutic approach for FLT3-ITD positive AML.

High expression of FLT3 is a risk factor in leukemia

“…Several interacting proteins are also reported to interact with FLT3 to negatively or positively regulate FLT3 pathways. Spleen tyrosine kinase (SYK) and the mucin 1-C-terminal subunit (MUC1-C) oncoprotein are reported to directly bind to and activate FLT3-related pathways ( 50 , 51 ), whereas suppressor of cytokine signaling 2 (SOCS2) and src-like adaptor protein 2 (SLAP2) interact with FLT3 protein to inhibit its signaling ( 52 , 53 ). In addition, the transcription of FLT3 can be regulated in AML.…”

The Src family kinase LCK cooperates with oncogenic FLT3/ITD in cellular transformation

“…These findings suggest that the role of protein kinases or phosphatases cannot be simplified and specific kinase or phosphatase can act as negative or positive regulators of FLT3 signaling. Furthermore, although several E3 ubiquitin ligases such as SOCS2 18 , SOCS6 19 , SLAP 20 and SLAP2 9 accelerate ubiquitination-directed degradation of FLT3, signaling molecules play diverse roles in regulating mitogenic signaling. For instance, SLAP depletion partially blocked activation of FLT3 downstream signaling cascades 20 while depletion of SOCS6 accelerated mitogenesis 19 .…”

SRC-like adaptor protein 2 (SLAP2) is a negative regulator of KIT-D816V-mediated oncogenic transformation

“…A recent study has shown that SLAP, a close homolog of SLAP2, associates with wild-type KIT as well as the oncogenic mutant KIT-D816V 11 . Since SLAP2 has also been shown to interact with other type III RTKs such as CSF1R 9 and FLT3 10 , we sought to investigate the possible role of SLAP2 in KIT signaling. In order to determine whether SLAP2 can interact with KIT, we overexpressed FLAG-tagged SLAP2 and wild-type KIT in COS-1 cells and incubated cells in the absence or presence of SCF for 5 min.…”

“…In order to investigate the possible role of the SH2 domain, we generated a mutant of the SLAP2 SH2 domain that fails to bind phosphotyrosine (SLAP2-R121E-FLAG). In this mutant, the positively charged arginine residue in the phosphotyrosine binding pocket of the SH2 domain was replaced by a negatively charged glutamic acid, thus blocking binding to phosphotyrosine residues 10 . COS-1 cells were co-transfected with either wild-type KIT or KIT-D816V and wild-type SLAP2 or SLAP2-R121E or an empty vector (EV) and incubated in the absence or presence of SCF for 5 min.…”

“…Specifically, a study from 2007 showed that SLAP2 negatively regulates signaling through the type III receptor tyrosine kinase colony-stimulating factor-1 receptor (CSF1R) by recruiting CBL to the activated receptor, which results in enhanced ubiquitination and degradation of the receptor 9 . Furthermore, we have recently shown that SLAP2 binds to and negatively regulates another type III receptor tyrosine kinase, Fms like tyrosine kinase 3, FLT3 10 . Therefore, we hypothesized that SLAP2 might play a role in the RTK KIT.…”