Clathrin-coated vesicles (CCVs) are involved in fundamental intracellular transport pathways such as receptor-mediated endocytosis at the plasma membrane and the sorting of lysosomal enzymes at the trans-Golgi network (TGN). The formation of CCVs is a dynamic and complex process that is mediated and controlled by the coat protein clathrin and a battery of accessory proteins. Recently, a novel CCV-associated protein with a serine/threonine kinase homology domain was identified in a tandem MS

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... ysis of rat brain CCVs and termed CVAK104 (coated-vesicle-associated kinase of 104 kDa) (Blondeau et al., 2004; Conner and Schmid, 2005) . In this study CVAK104 was characterized by biochemical and cell biological methods. Although CVAK104 is capable of binding to ATP, sequence analysis of the kinase domain and data from in vitro kinase assays indicated that CVAK is catalytically inactive and can therefore be classified as a pseudokinase. Instead, it was demonstrated that a C-terminal segment of CVAK104 interacts directly with the N-terminal domain of clathrin and with the α-appendage domain of the adaptor protein AP2. Fluorescence microscopy examinations revealed that CVAK104 localizes predominantly to the perinuclear region of HeLa and COS-7 cells but it is also present on peripheral vesicular structures that are accessible to internalized transferrin. The distribution of CVAK104 overlaps extensively with that of AP1, AP3, the mannose 6-phosphate receptor and perinuclear clathrin but not at all with that of its in vitro binding partner AP2. RNAi-mediated clathrin knockdown reduced the membrane association of CVAK104. The recruitment of CVAK104 to perinuclear membranes of permeabilized cells is enhanced by GTPγS. Moreover, upon treatment of cells with Brefeldin A, CVAK104 redistributes into the cytosol. Both observations suggest a direct or indirect requirement for GTP-binding proteins in the membrane association of CVAK104. Live-cell imaging showed colocalization of GFP-CVAK104 with endocytosed transferrin and with RFP-clathrin on rapidly moving endosomes. Like AP1-depleted COS-7 cells, CVAK104depleted cells missort the lysosomal hydrolase cathepsin D. Taken together, the present data suggest a function for CVAK104 in clathrin-dependent pathways between the TGN and the endosomal system.