The Sam domain of the lipid phosphatase Ship2 adopts a common model to interact with Arap3-Sam and EphA2-Sam

Here, we determine the NMR solution structure of Arap3-Sam and implement a multidisciplinary approach consisting of NMR spectroscopy, ITC (Isothermal Titration Calorimetry), mutagenesis and molecular modeling studies to analyze the interaction between Ship2-Sam and Arap3-Sam. This work reveals that Arap3-Sam may associate with Ship2-Sam by adopting a binding mode common to other Sam domains. This binding mode is identical to what we have very recently observed for the association between Ship2-Sam and the Sam domain from the Ephrin A2 receptor.Our studies further clarify the structural features that are relevant for Sam-Sam interactions involving Ship2 and give additional hints that could be used for the identification of new molecules able to selectively inhibit Sam-Sam associations.Arap3 is a protein involved in phospatidylinositol 3 kinase (PI3K) signaling pathways linked to regulation of the actin cytoskeleton, cell spreading and the formation of lamellipodia [1,2]. It works as a GTP-ase activator protein (GAP) for the small G-proteins Arf and Rho [2]. Previous studies have reported that Arap3 binds Ship2 (Src homology 2 domain-containing phosphoinositide-5-phosphatase 2) by forming a hetero-dimer via the sterile alpha motif domains (Sam) of both proteins [3]. The dissociation constant for this complex is about 100 nM as determined by previous Isothermal titration Calorimetry (ITC) studies [3].Although the consequences of the interaction between the Sam domains of Arap3 (Arap3-Sam) and Ship2 (Ship2-Sam) are not completely clear, it has been speculated that the heterotypic Sam-Sam association may be used by Arap3 to link a negative regulator of PI3K signaling (i.e., Ship2) to the effector complex [3]. In fact, while Arap3 needs to bind phosphatidylinositol(3,4,5)P3 (PtIns(3,4,5)P3) with one of its pleckstrin homology (PH) domains to migrate to the plasma membrane and be activated [4]; Ship2, by converting PtIns(3,4,5)P3 to phosphatidylinositol(3,4)P2 (PtIns(3,4)P