Nak regulates Dlg basal localization in Drosophila salivary gland cells Yu-Huei Peng a,b , Wei-Kan Yang b , Wei-Hsiang Lin b , Tzu-Ting Lai b , Cheng-Ting Chien a,b, * a Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan b Institute of Molecular Biology, Academia Sinica, 128 Sec. 2, Academia Road, Nankang, Taipei 115, Taiwan article info Article history: Received 19 February 2009 Available online 1 March 2009 Keywords: Nak Dlg AP-1 AP-2 Protein trafficking Salivary gland Drosophila abstract Protein trafficking is highly regulated in polarized cells. During development, how the trafficking of cell junctional proteins is regulated for cell specialization is largely unknown. In the maturation of Drosophila larval salivary glands (SGs), the Dlg protein is essential for septate junction formation. We show that Dlg was enriched in the apical membrane domain of proximal cells and localized basolaterally in distal mature cells. The transition of Dlg distribution was disrupted in nak mutants. Nak associated with the AP-2 subunit a-Ada and the AP-1 subunit AP-1c. In SG cells disrupting AP-1 and AP-2 activities, Dlg was enriched in the apical membrane. Therefore, Nak regulates the transition of Dlg distribution likely through endocytosis of Dlg from the apical membrane domain and transcytosis of Dlg to the basolateral membrane domain during the maturation of SGs development. Ó 2009 Elsevier Inc. All rights reserved. Introduction Most cells exhibit subdivisions in plasma membranes and underlying subcortical regions with specialized compositions and structures. Typical examples include the apical and basolateral do- mains of polarized epithelial cells and the axonal and somatoden- dritic domains of neurons that bear distinct and vital functions to the cells. During the establishment of the epithelial cell polarity, different cell junctions that function in cell–cell contact and tissue integrity are organized. Many junctional components also play essential roles in the establishment and the maintenance processes [1–3]. Depositions of junctional components and organization of specialized cellular domains rely on endocytosis, protein sorting, endosomal recycling, and polarized transportation processes [4,5]. Little is known for the reorganization of junctional proteins and the underlying mechanism regulating this process. In clathrin-mediated trafficking processes, AP complexes recruit cargos, clathrin, and other accessory proteins to form clathrin- coated vesicles. The AP-2 complex is localized at the subcortical region of the plasma membrane and required for endocytosis. In mammalian cells, the AP-1A complex is ubiquitously expressed in all cell types and involved in trafficking between TGN and endo- somes. The AP-1B complex is only present in polarized cells and required for sorting from the recycling endosomes [6–9]. In Drosophila, the only AP-1 complex might play dual roles performed by AP-1A and AP-1B in mammalian cells. In addition, the Drosoph- ila AP-1 and AP-2 complexes share the b subunit, but differ in other subunits: the AP-1 complex includes c, l1, and r1 subunits; the AP-2 complex includes a, l2, and r2 subunits [10]. Activities of the AP complexes are regulated by the Ark/Prk fam- ily of protein kinases which share sequence homology in the serine/threonine kinase domains [11]. In endocytosis, yeast Ark1p and Prk1p negatively regulate the formation of protein complexes that include the Eps-15 homolog Pan1p, Sla1p, and End3p [12,13]. Mammalian AAK1 and GAK/auxilin2 have multiple and partially redundant roles in clathrin-mediated trafficking processes, such as endocytosis, endosomal recycling, or lysosomal sorting [14–18]. AAK1, GAK, or both regulate these processes through phosphorylations of the l subunits of AP-1 and AP-2 complexes by which facilitates the cargo binding efficiency [15,19–22]. Other mechanisms involve recruitment of adaptors through protein–pro- tein interactions, phosphorylation of accessory proteins, and uncoating the clathrin-coated vesicles [16,18,23]. We show that the Drosophila Ark/Prk homolog Nak is involved in the polarization of SG cells. In nak mutants, basolateral mem- brane distribution of Dlg and apical-to-basal transition in SG cells were disrupted, and the growth of SG cells was severely retarded in nak mutants. We identified the interaction between Nak and the a subunit of the AP-2 complex and the c subunit of AP-1 complex. Consistently, mutants for components of AP-1 or AP-2 complexes photocopied nak mutants, displayed similar Dlg distri- bution and cellular growth defects. Therefore, Nak regulates SGs 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.02.139 * Corresponding author. Address: Institute of Molecular Biology, Academia Sinica, 128 Sec. 2, Academia Road, Nankang, Taipei 115, Taiwan. Fax: +886 2 27826085. E-mail address: ctchien@gate.sinica.edu.tw (C.-T. Chien). Biochemical and Biophysical Research Communications 382 (2009) 108–113 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc