Myosin VI Binds to and Localises with Dab2, Potentially Linking Receptor‐Mediated Endocytosis and the Actin Cytoskeleton

Abstract

Myosin VI, an actin-based motor protein, and Disabled 2 (Dab2), a molecule involved in endocytosis and cell signalling, have been found to bind together using yeast and mammalian two-hybrid screens. In polarised epithelial cells, myosin VI is known to be associated with apical clathrin-coated vesicles and is believed to move them towards the minus end of actin filaments, away from the plasma membrane and into the cell. Dab2 belongs to a group of signal transduction proteins that bind in vitro to the FXNPXY sequence found in the cytosolic tails of members of the low-density lipoprotein receptor family. The central region of Dab2, containing two DPF motifs, binds to the clathrin adaptor protein AP-2, whereas a C-terminal region contains the binding site for myosin VI. This site is conserved in Dab1, the neuronal counterpart of Dab2. The interaction between Dab2 and myosin VI was confirmed by in vitro binding assays and coimmunoprecipitation and by their colocalisation in clathrin-coated pits/vesicles concentrated at the apical domain of polarised cells. These results suggest that the myosin VI-Dab2 interaction may be one link between the actin cytoskeleton and receptors undergoing endocytosis.

The MYO6 interactome reveals adaptor complexes coordinating early endosome and cytoskeletal dynamics

“…To verify the novel MYO6 interaction network, we introduced mutations into the known cargo‐binding and lipid‐binding sites, the WWY, RRL and PIP 2 binding motifs 10, 11, 14 and assessed differences in the relative abundance of BirA*‐MYO6 binding partners using a SILAC‐based approach (Fig 1F). Analysis of these cells by confocal microscopy also revealed that the ΔRRL and ΔPIP2, but not the ΔWWY mutation, perturbed MYO6 targeting to APPL1 endosomes (Fig EV1A).…”

“…These interactions occur at two major protein binding motifs, the RRL and WWY (named after their amino acid composition), which are located within two distinct subdomains of a unique C‐terminal cargo‐binding tail 10, 11. The tail also contains a phosphatidylinositol 4,5‐bisphosphate (PIP 2 ) binding motif, which aids recruitment of the motor to membranes along with its binding partners 14.…”

The inhibitory effects of Disabled-2 (Dab2) on Wnt signaling are mediated through Axin

“…Dab2 contains a conserved N-terminal phosphotyrosine-binding domain (Yun et al, 2003) that binds to members of the low-density lipoprotein receptor (LDLR) family and with phosphoinositides (Morris and Cooper, 2001). Its linker and C-terminal regions bind clathrin, the clathrin adaptor protein AP2 and myosin VI, facilitating clathrincoated pit assembly and receptor-mediated endocytosis (Morris and Cooper, 2001;Mishra et al, 2002;Morris et al, 2002). The endocytic and vesicular trafficking functions of Dab2 are postulated to mediate its effects on cellular signaling (Morris et al, 2002).…”

“…Its linker and C-terminal regions bind clathrin, the clathrin adaptor protein AP2 and myosin VI, facilitating clathrincoated pit assembly and receptor-mediated endocytosis (Morris and Cooper, 2001;Mishra et al, 2002;Morris et al, 2002). The endocytic and vesicular trafficking functions of Dab2 are postulated to mediate its effects on cellular signaling (Morris et al, 2002).…”

Src and FAK Kinases Cooperate to Phosphorylate Paxillin Kinase Linker, Stimulate Its Focal Adhesion Localization, and Regulate Cell Spreading and Protrusiveness

“…Glutathione S-transferase (GST)-fusion proteins were expressed and purified onto glutathione-Sepharose 4B beads (Amersham Biosciences) as described previously (Morris et al, 2002) with pull-down assays performed using 250 g Triton lysis buffer cell lysates and 5 g of GST or GST-fusion. After 2 h of sample incubation end-over-end at 4°C, proteins specifically bound to the GST-fusion coupled to the Sepharose beads were washed extensively with lysis buffer and processed as described above.…”