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.
References
[1]
Masson K, Ronnstrand L (2009) Oncogenic signaling from the hematopoietic growth factor receptors c-Kit and Flt3. Cell Signal 21: 1717–1726.
[2]
Roche S, Alonso G, Kazlauskas A, Dixit VM, Courtneidge SA, et al. (1998) Src-like adaptor protein (Slap) is a negative regulator of mitogenesis. Curr Biol 8: 975–978.
[3]
Sosinowski T, Pandey A, Dixit VM, Weiss A (2000) Src-like adaptor protein (SLAP) is a negative regulator of T cell receptor signaling. J Exp Med 191: 463–474.
[4]
Myers MD, Sosinowski T, Dragone LL, White C, Band H, et al. (2006) Src-like adaptor protein regulates TCR expression on thymocytes by linking the ubiquitin ligase c-Cbl to the TCR complex. Nat Immunol 7: 57–66.
[5]
Reindl C, Quentmeier H, Petropoulos K, Greif PA, Benthaus T, et al. (2009) CBL exon 8/9 mutants activate the FLT3 pathway and cluster in core binding factor/11q deletion acute myeloid leukemia/myelodysplastic syndrome subtypes. Clin Cancer Res 15: 2238–2247.
[6]
Dragone LL, Myers MD, White C, Gadwal S, Sosinowski T, et al. (2006) Src-like adaptor protein (SLAP) regulates B cell receptor levels in a c-Cbl-dependent manner. Proc Natl Acad Sci U S A 103: 18202–18207.
[7]
Gilliland DG, Griffin JD (2002) The roles of FLT3 in hematopoiesis and leukemia. Blood 100: 1532–1542.
[8]
Levis M, Small D (2003) FLT3: ITDoes matter in leukemia. Leukemia 17: 1738–1752.
[9]
Yamamoto Y, Kiyoi H, Nakano Y, Suzuki R, Kodera Y, et al. (2001) Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies. Blood 97: 2434–2439.
[10]
Peschard P, Park M (2003) Escape from Cbl-mediated downregulation: a recurrent theme for oncogenic deregulation of receptor tyrosine kinases. Cancer Cell 3: 519–523.
[11]
Razumovskaya E, Masson K, Khan R, Bengtsson S, Ronnstrand L (2009) Oncogenic Flt3 receptors display different specificity and kinetics of autophosphorylation. Exp Hematol 37: 979–989.
[12]
Lin DC, Yin T, Koren-Michowitz M, Ding LW, Gueller S, et al. (2012) Adaptor protein Lnk binds to and inhibits normal and leukemic FLT3. Blood 120: 3310–3317.
[13]
Kazi JU, Sun J, Phung B, Zadjali F, Flores-Morales A, et al. (2012) Suppressor of Cytokine Signaling 6 (SOCS6) Negatively Regulates Flt3 Signal Transduction through Direct Binding to Phosphorylated Tyrosines 591 and 919 of Flt3. J Biol Chem 287: 36509–36517.
[14]
Voytyuk O, Lennartsson J, Mogi A, Caruana G, Courtneidge S, et al. (2003) Src family kinases are involved in the differential signaling from two splice forms of c-Kit. J Biol Chem 278: 9159–9166.
[15]
Marasca R, Maffei R, Zucchini P, Castelli I, Saviola A, et al. (2006) Gene expression profiling of acute promyelocytic leukaemia identifies two subtypes mainly associated with flt3 mutational status. Leukemia 20: 103–114.
[16]
Dragone LL, Shaw LA, Myers MD, Weiss A (2009) SLAP, a regulator of immunoreceptor ubiquitination, signaling, and trafficking. Immunol Rev 232: 218–228.
[17]
Heiss E, Masson K, Sundberg C, Pedersen M, Sun J, et al. (2006) Identification of Y589 and Y599 in the juxtamembrane domain of Flt3 as ligand-induced autophosphorylation sites involved in binding of Src family kinases and the protein tyrosine phosphatase SHP2. Blood 108: 1542–1550.
[18]
Liontos LM, Dissanayake D, Ohashi PS, Weiss A, Dragone LL, et al. (2011) The Src-like adaptor protein regulates GM-CSFR signaling and monocytic dendritic cell maturation. J Immunol 186: 1923–1933.
[19]
Myers MD, Dragone LL, Weiss A (2005) Src-like adaptor protein down-regulates T cell receptor (TCR)-CD3 expression by targeting TCRzeta for degradation. J Cell Biol 170: 285–294.
[20]
Uhlen M, Oksvold P, Fagerberg L, Lundberg E, Jonasson K, et al. (2010) Towards a knowledge-based Human Protein Atlas. Nat Biotechnol 28: 1248–1250.
[21]
Lebigot I, Gardellin P, Lefebvre L, Beug H, Ghysdael J, et al. (2003) Up-regulation of SLAP in FLI-1-transformed erythroblasts interferes with EpoR signaling. Blood 102: 4555–4562.
[22]
Sirvent A, Leroy C, Boureux A, Simon V, Roche S (2008) The Src-like adaptor protein regulates PDGF-induced actin dorsal ruffles in a c-Cbl-dependent manner. Oncogene 27: 3494–3500.
[23]
Sargin B, Choudhary C, Crosetto N, Schmidt MH, Grundler R, et al. (2007) Flt3-dependent transformation by inactivating c-Cbl mutations in AML. Blood 110: 1004–1012.
[24]
Tang J, Sawasdikosol S, Chang JH, Burakoff SJ (1999) SLAP, a dimeric adapter protein, plays a functional role in T cell receptor signaling. Proc Natl Acad Sci U S A 96: 9775–9780.
[25]
Hiragun T, Peng Z, Beaven MA (2006) Cutting edge: dexamethasone negatively regulates Syk in mast cells by up-regulating SRC-like adaptor protein. J Immunol 177: 2047–2050.
[26]
Yu M, Lowell CA, Neel BG, Gu H (2006) Scaffolding adapter Grb2-associated binder 2 requires Syk to transmit signals from FcepsilonRI. J Immunol 176: 2421–2429.
[27]
Kottaridis PD, Gale RE, Frew ME, Harrison G, Langabeer SE, et al. (2001) The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy: analysis of 854 patients from the United Kingdom Medical Research Council AML 10 and 12 trials. Blood 98: 1752–1759.
[28]
Asyali MH, Alci M (2005) Reliability analysis of microarray data using fuzzy c-means and normal mixture modeling based classification methods. Bioinformatics 21: 644–649.
