Based on our previous work and the

Based on our previous work [13] and the observation that SNX9 is a partner for ACK, we have investigated interactions involving the SH3 domain of SNX9, and identified for the first time synaptojanin-1 as an alternate partner. This SH3 domain can bind a single site in ACK1, but multiple sites in the proline-rich domain of synaptojanin; one synaptojanin site only overlaps previously characterized synaptojanin partners amphiphysin and endophilin. When SNX9 binding to ACK1 was investigated in the context of full-length proteins, SNX9 was found to interact preferentially with inactive ACK1. This is consistent with a previous proposal [7] that phosphorylation of SNX9 by ACK (or Src kinases) downregulates SH3 binding to its target sequences. On this basis we suggest that ACK1 both binds to and regulates interaction of SNX9 with other proteins involved with endocytosis, including synaptojanin-1.
Materials and methods
Results
Discussion This study describes a system for quantative in vivo biotinylation of GST expressed fusion proteins in E. coli. Our 18 residue acceptor sequence performed well in terms of biotinylation, but underwent significant proteolysis in vivo probably by basic directed bacterial proteases. Shortening the acceptor sequence to 10 residues appeared to completely resolve this problem while maintaining the stoichiometry of modification (Fig. 1C). Mammalian activated Cdc42 associated kinase (ACK1) was the first effector to be described for the Rho family and is a cytoplasmic tyrosine kinases that binds active Cdc42 but not Rac1 or RhoA via a PAK-like CRIB motif [21]. The association of ACK1 with Cdc42 and adaptors such as Grb2 is likely to provide localization cues for the kinase (6). That kinase-dead ACK1 can interact with Grb2 but not with Nck (Fig. 4B) is consistent with the peptide overlay data (Fig. 3B): thus Nck binding would requires interaction of the SH2 domain with phospho-tyrosine residues on ACK1. The domain structure of ACK kinases consists of an N-terminal tyrosine kinase catalytic domain followed by an SH3 domain, a Cdc42 binding domain, clathrin binding domain and a proline-rich C-terminal region terminating in two UBA boxes (Fig. 4). Here we have identified different mitotic inhibitors in the extensive proline-rich region of ACK1 for both Grb2 and SNX9. This paves the way for future studies to address the issue of how these proteins might modulate ACK1 localization and activity. A connection between Nck and ACK1 was suggested by studies of the Drosophila orthologue of Nck (Dock): using an epitope-tagged SH2 domain of Dock five proteins were identified (molecular masses of 270, 145, 74, 69, and 63kDa) in immuno-precipitates [22]. The 145kDa protein represented DAck1 and the 63kDa protein identified as DSNX9. Although the SH3 domain of DSNX9 can bind the orthologue of Wiskott-Aldrich Syndrome protein (WASp), we did not identify this interaction in our analysis of SNX9 binding proteins (Fig. 2). DSNX9 is a substrate for DAck in vivo and in vitro [7] and a major site of DSNX9 phosphorylation is the conserved tyrosine residue (Tyr-56) present in the N-terminal SH3 domain that appears to block SH3 function. However we were able to detect tyrosine-phosphorylated SNX9 co-precipitating with ACK1. Sorting nexins (SNXs) are 400–700 amino-acid hydrophilic proteins that are characterized by the presence of a phospholipid-binding domain, the PX domain. In addition to the PX domain, SNXs have various protein–protein interaction motifs that are thought to determine their sub cellular localization and their ability to form specific complexes (as reviewed by Worby and Dixon [9]). Mammalian SNXs are thought to be important for the sorting of proteins in the endosomal pathway. Both over-expressed ACK2 or epidermal growth factor (EGF) can stimulate the tyrosine phosphorylation of SNX9 [4]. Both clathrin and dynamin-2, two other essential molecules in the endocytic process, interact with SNX9. CIN85 SH3 domains have recently been shown to pull-down synaptojanin-1 [23]: the authors identified four proteins in SH3 pull-downs but could only verify synaptojanin-1 and PAK2 as direct binders by overlay [23]: all three CIN85 SH3 domains bound synaptojanin-1. Other potential synaptojanin-1 binders are ArgBP2 and FISH [20]. What would be the significance of the interaction between SNX9 and synaptojanin? Synaptojanin-1 is a polyphospho-inositide phosphatase implicated in synaptic vesicle recycling and thus membrane trafficking. Cultured cortical neurons from synaptojanin-1 knockout mice show relatively normal endocytosis after a moderate synaptic stimulus but during prolonged stimulation the regeneration of fusion-competent synaptic vesicles is severely impaired [24]. These findings implicate synaptojanin-1 in progression and maturation of recycling vesicles: the participation of SNX9 which contains an inositol lipid binding domain (PX) would not be unexpected. It is thought endophilin is required to localize and stabilize synaptojanin-1 at membranes during synaptic vesicle recycling [25] however the potential participation of SNX9 deserves further study. The endophilin binding site in the synaptojanin C-terminal [17] does not overlap with sites we identify here for SNX9 (see Fig. 3A). Distinct sites for another SNX9 partner synaptophysin 1 are also marked in Fig. 3 (A1 and A2).