Dimerization is required for SH3PX1 tyrosine phosphorylation in response to epidermal growth factor signalling and interaction with ACK2

Weis Center for Research, Geisinger Clinic, 100 N. Academy Avenue, Danville, PA 17822, USA.
Biochemical Journal (Impact Factor: 4.4). 04/2006; 394(Pt 3):693-8. DOI: 10.1042/BJ20050576
Source: PubMed


SH3PX1 [SNX9 (sorting nexin 9)] is a member of SNX super-family that is recognized by sharing a PX (phox homology) domain. We have previously shown that SH3PX1, phosphorylated by ACK2 (activated Cdc42-associated tyrosine kinase 2), regulates the degradation of EGF (epidermal growth factor) receptor. In mapping the tyrosine phosphorylation region, we found that the C-terminus of SH3PX1 is required for its tyrosine phosphorylation. Further analysis indicates that this region, known as the coiled-coil domain or the BAR (Bin-amphiphysin-Rvs homology) domain, is the dimerization domain of SH3PX1. Truncation of as little as 13 amino acid residues at the very C-terminus in the coiled-coil/BAR domain of SH3PX1 resulted in no dimerization, no ACK2-catalysed and EGF-stimulated tyrosine phosphorylation and no interaction with ACK2. The intracellular localization of SH3PX1 became dysfunctional upon truncation in the BAR domain. Taken together, our results indicate that the dimerization, which is mediated by the BAR domain, is essential for the intracellular function of SH3PX1.

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    • "Ack1 is recruited to EGFR following EGF stimulation [21]. EGFR stability and recycling are regulated by interactions between Ack1 and multiple protein partners including ubiquitin [21], clathrin heavy chain [20] [42], and SH3PX1 [43] [44]. In HeLa cells, overexpression of Ack1 inhibits EGFR endocytosis and causes the accumulation of EGF in internal structures of endocytic origin, suggesting that Ack1 is involved in the intracellular sorting of activated EGFR [45]. "
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    ABSTRACT: Ack family non-receptor tyrosine kinases are unique with regard to their domain composition and regulatory properties. Human Ack1 (activated Cdc42-associated kinase) is ubiquitously expressed and is activated by signals that include growth factors and integrin-mediated cell adhesion. Stimulation leads to Ack1 autophosphorylation and to phosphorylation of additional residues in the C-terminus. The N-terminal SAM domain is required for full activation. Ack1 exerts some of its effects via protein-protein interactions that are independent of its kinase activity. In the basal state, Ack1 activity is suppressed by an intramolecular interaction between the catalytic domain and the C-terminal region. Inappropriate Ack1 activation and signaling has been implicated in the development, progression, and metastasis of several forms of cancer. Thus, there is increasing interest in Ack1 as a drug target, and studies of the regulatory properties of the enzyme may reveal features that can be exploited in inhibitor design.
    02/2011; 2011(12):742372. DOI:10.1155/2011/742372
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    • "It is possible that the PX-BAR unit of SNX9 cooperates with dynamin for the tubulation and scission reaction to release the coated vesicle. In addition to its proposed direct role in clathrin-mediated endocytosis, SNX9 has been suggested to be involved in a number of plasma membrane-related events such as receptor downregulation, clathrin-independent endocytosis , and actin dynamics (Howard et al, 1999; Lin et al, 2002; MaCaulay et al, 2003; Yeow-Fong et al, 2005; Childress et al, 2006; Badour et al, 2007; Yarar et al, 2007). "
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    ABSTRACT: Sorting nexins (SNXs) form a family of proteins known to interact with components in the endosomal system and to regulate various steps of vesicle transport. Sorting nexin 9 (SNX9) is involved in the late stages of clathrin-mediated endocytosis in non-neuronal cells, where together with the GTPase dynamin, it participates in the formation and scission of the vesicle neck. We report here crystal structures of the functional membrane-remodeling unit of SNX9 and show that it efficiently tubulates lipid membranes in vivo and in vitro. Elucidation of the protein superdomain structure, together with mutational analysis and biochemical and cell biological experiments, demonstrated how the SNX9 PX and BAR domains work in concert in targeting and tubulation of phosphoinositide-containing membranes. The study provides insights into the SNX9-induced membrane modulation mechanism.
    The EMBO Journal 12/2007; 26(22):4788-800. DOI:10.1038/sj.emboj.7601889 · 10.43 Impact Factor
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    ABSTRACT: The Wiskott-Aldrich syndrome protein (WASp) plays a major role in coupling T cell antigen receptor (TCR) stimulation to induction of actin cytoskeletal changes required for T cell activation. Here, we report that WASp inducibly binds the sorting nexin 9 (SNX9) in T cells and that WASp, SNX9, p85, and CD28 colocalize within clathrin-containing endocytic vesicles after TCR/CD28 costimulation. SNX9, implicated in clathrin-mediated endocytosis, binds WASp via its SH3 domain and uses its PX domain to interact with the phosphoinositol 3-kinase regulatory subunit p85 and product, phosphoinositol (3,4,5)P3. The data reveal ligation-induced CD28 endocytosis to be clathrin- and phosphoinositol 3-kinase-dependent and TCR/CD28-evoked CD28 internalization and NFAT activation to be markedly enhanced by SNX9 overexpression, but severely impaired by expression of an SNX9 mutant (SNX9DeltaPX) lacking p85-binding capacity. CD28 endocytosis and CD28-evoked actin polymerization also are impaired in WASp-deficient T cells. These findings suggest that SNX9 couples WASp to p85 and CD28 so as to link CD28 engagement to its internalization and to WASp-mediated actin remodeling required for CD28 cosignaling. Thus, the WASp/SNX9/p85/CD28 complex enables a unique interface of endocytic, actin polymerizing, and signal transduction pathways required for CD28-mediated T cell costimulation.
    Proceedings of the National Academy of Sciences 02/2007; 104(5):1593-8. DOI:10.1073/pnas.0610543104 · 9.67 Impact Factor
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