br Experimental Procedures br Acknowledgments br

Experimental Procedures
Acknowledgments
Introduction Hematopoietic stem ARRY-142886 (HSCs) are capable of both self-renewal and production of mature blood lineages. Players in this balanced regulation include transcription factors, cell-cycle regulators, signaling molecules, surface receptors, and cytokines (Rossi et al., 2012). The type III receptor tyrosine kinases (RTKs), which include FMS-like tyrosine kinase-3 (FLT3), c-KIT (referred to hereafter as KIT), cFMS, and PDGFR play a key role in normal and malignant hematopoiesis. Importantly, FLT3 and KIT are highly expressed on hematopoietic stem and progenitor cells (HSPCs) (Adolfsson et al., 2005; Boyer et al., 2011; Buza-Vidas et al., 2009) as well as on the surface of leukemic blasts in most patients with acute myeloid leukemia (Sargin et al., 2007; Toffalini and Demoulin, 2010). Extracellular binding of a specific ligand to its respective RTK induces dimerization and autophosphorylation on specific tyrosine residues, followed by activation of intracellular signaling cascades. The amplitude and duration of RTK signaling is tightly controlled by receptor ubiquitination, internalization, and degradation, resulting in signal termination (Verstraete and Savvides, 2012). In this context, E3 ligases mediate ubiquitination, thereby initiating internalization and endocytosis (Ryan et al., 2006; Toffalini and Demoulin, 2010; Verstraete and Savvides, 2012). Dephosphorylation of RTKs by phosphatases has been less studied so far and appears to be a transient and fine-tuned negative regulation of RTK signaling (Dikic and Giordano, 2003; Sastry and Elferink, 2011). We have reported previously that the E3 ligase CBL binds to autophosphorylated FLT3 and KIT and leads to FLT3 and KIT ubiquitination via its E3 ligase activity (Bandi et al., 2009; Sargin et al., 2007). It has been shown that FLT3 signaling is greatly amplified in FLT3+ multipotent progenitors (MPPs) in a genetic mouse model expressing a RING finger mutant of CBL, leading to a myeloid proliferative disease. This pheonotype was reversible by treatment with the FLT3 kinase inhibitor AC220 (Rathinam et al., 2010; Taylor et al., 2012). Here we analyze the function of two known binding partners of CBL and evaluate their potential phosphatase activity toward FLT3 and KIT: STS1 and STS2 (suppressor of T cell receptor signaling 1 and 2, also known as TULA2 and TULA and UBASH3B and UBASH3A, respectively). STS1 and STS2 proteins share a 75% amino acid homology and are characterized by a ubiquitin binding domain (UBA), a SH3 domain, and a phosphoglycerate mutase-like domain (PGM) (Carpino et al., 2002, 2004). Both proteins bind CBL through their SH3 domain and regulate interactions between trafficking receptors and the ubiquitin sorting machinery in the endosome through their UBA domain (Kowanetz et al., 2004; Mikhailik et al., 2007; Raguz et al., 2007). STS1/STS2 have been shown to constitutively interact with CBL and inhibit CBL-mediated degradation of the epidermal growth factor (EGF) receptor (Feshchenko et al., 2004; Raguz et al., 2007). Importantly, STS1 has been shown to be a tyrosine phosphatase for the EGF and PDGF receptors, with the PGM domain encoding the phosphatase activity (Hoeller et al., 2006; Mikhailik et al., 2007). Interestingly, the phosphatase activity of STS2 is much weaker, although the PGM domains of STS1 and STS2 are highly homologous (Carpino et al., 2009; Chen et al., 2009a, 2009b). Single STS1 or STS2 knockout mice are viable, develop normally, and do not display any obvious abnormalities, and no differences were detected concerning bone marrow cellularity, B and T cell development, or proliferative capacity. Mice lacking both STS proteins are shown to be hyper-responsive to T cell receptor stimulation, resulting in an increase in both cytokine production and susceptibility to autoimmunity (Carpino et al., 2002, 2004).