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

Brooke Morriswood,Claudia Puri,Calliope Dendrou,Folma Buss,John Kendrick-Jones,Susan D Arden,Rhys Roberts,Grigory Ryzhakov

doi:10.1242/jcs.007005

Brooke Morriswood, Claudia Puri + Show 6 more

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https://doi.org/10.1242/jcs.007005

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Abstract

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-kappaB 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.

Highlights

Myosins are a superfamily of motor proteins found virtually throughout the eukaryota (Foth et al, 2006) and, harnessing the energy liberated by ATP hydrolysis, they move along the actin filament cytoskeleton and instigate a plethora of cellular processes ranging from cell migration to membrane traffic (Coureux et al, 2004; De La Cruz and Ostap, 2004; Tuxworth and Titus, 2000)
To confirm that the two proteins interacted with myosin VI in a mammalian cellular environment, we carried out a series of two-hybrid assays in Chinese hamster ovary (CHO) cells (Fig. 1A)
The interactions were shown to be specific to the globular domain of the myosin VI tail, because a truncated construct of the myosin VI tail did not bind to either TNF␣-receptor-associated factor 6 (TRAF6)-binding protein (T6BP) or nuclear dot protein 52 (NDP52)

Summary

Introduction

Myosins are a superfamily of motor proteins found virtually throughout the eukaryota (Foth et al, 2006) and, harnessing the energy liberated by ATP hydrolysis, they move along the actin filament cytoskeleton and instigate a plethora of cellular processes ranging from cell migration to membrane traffic (Coureux et al, 2004; De La Cruz and Ostap, 2004; Tuxworth and Titus, 2000). Their biophysical and kinetic properties have been intensively studied, they remain less well characterised at a cellular level and the molecular details of many of their various tasks remain to be elucidated. Optineurin and myosin VI are found at the Golgi complex and the recycling endosome, where they play a role in the delivery of newly synthesised proteins to the plasma membrane (Sahlender et al, 2005; Au et al, 2007)

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