T6BP and NDP52 are myosin VI binding partners with potential roles in cytokine signalling and cell adhesion

Abstract

Summary Myosin VI has been implicated in many cellular processes including endocytosis, secretion, membrane ruffling and cell motility. We carried out a yeast two-hybrid screen and identified TRAF6-binding protein (T6BP) and nuclear dot protein 52 (NDP52) as myosin VI binding partners. Myosin VI interaction with T6BP and NDP52 was confirmed in vitro and in vivo and the binding sites on each protein were accurately mapped. Immunofluorescence and electron microscopy showed that T6BP, NDP52 and myosin VI are present at the trans side of the Golgi complex, and on vesicles in the perinuclear region. Although the SKICH domain in T6BP and NDP52 does not mediate recruitment into membrane ruffles, loss of T6BP and NDP52 in RNAi knockdown cells results in reduced membrane ruffling activity and increased stress fibre and focal adhesion formation. Furthermore, we observed in these knockdown cells an upregulation of constitutive secretion of alkaline phosphatase, implying that both proteins act as negative regulators of secretory traffic at the Golgi complex. T6BP was also found to inhibit NF-κB activation, implicating it in the regulation of TRAF6-mediated cytokine signalling. Thus myosin VI-T6BP interactions may link membrane trafficking pathways with cell adhesion and cytokine-dependent cell signalling.

Myosin VI and its cargo adaptors – linking endocytosis and autophagy

“…For example, the region containing the RRL motif interacts with nuclear dot protein 52 (NDP52), Traf6-binding protein (T6BP), optineurin and GAIP-interacting protein C-terminus (GIPC), and the region encompassing the WWY motif is required for binding to Tom1, Dab2 and lemur tyrosine kinase-2 (LMTK2) (Bunn et al, 1999;Chibalina et al, 2007;Morris et al, 2002;Morriswood et al, 2007;Sahlender et al, 2005;Spudich et al, 2007;Tumbarello et al, 2012). For other myosin VI adaptor molecules, such as SAP97 (Wu et al, 2002), otoferlin (Heidrych et al, 2009), phospholipase Cd3 (Sakurai et al, 2011) and Dock7 (Majewski et al, 2012), the exact binding domain has not yet been determined.…”

“…The third autophagy receptor that specifically binds to myosin VI is the NDP52-related protein T6BP, which also localises to autophagosomes and is required for autophagosome biogenesis (Tumbarello et al, 2012). Structurally, T6BP and NDP52 are most similar and consist of an N-terminal SKICH domain, followed by a non-canonical LIR, a coiled-coil domain and C-terminal zinc finger domains that mediate ubiquitin-binding (Morriswood et al, 2007;von Muhlinen et al, 2012). Optineurin also consists of multiple coiled-coil regions, a leucine zipper domain and a C-terminal zinc finger domain (Fig.…”

Genetic Characterization of the Drosophila jaguar322 Mutant Reveals That Complete Myosin VI Loss of Function Is Not Lethal

“…Myosin VI mutations in mice, fish, and humans result in deafness due to degeneration of hair cell stereocilia (Avraham et al 1995;Self et al 1999;Ahmed et al 2003;Kappler et al 2004). In addition, vertebrate myosin VI has been implicated in processes similar to those affected in Drosophila, such as endocytosis, cell adhesion, and basolateral sorting (Dance et al 2004;Ameen and Apodaca 2007;Au et al 2007;Maddugoda et al 2007;Morriswood et al 2007). Since myosin VI is involved in similar processes in vertebrates as Drosophila, the lethality of myosin VI in flies but not in vertebrates seems surprising.…”

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

“…The functional and phenotypic diversity associated with MYO6 arises from interactions with multiple cargo adaptors including disabled‐2 (DAB2), GAIP‐interacting protein C‐terminus (GIPC1), target of Myb 1 (TOM1), lemur tyrosine kinase 2 (LMTK2), optineurin (OPTN), TAX1 binding protein 1 (TAX1BP1) and nuclear dot protein 52 (NDP52) 8, 9, 10, 11, 12, 13. 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.…”