T6BP and NDP52 are myosin VI binding partners with potential roles in cytokine signalling and cell adhesion.
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.
Morriswood
Brooke
B
MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK.
Ryzhakov
Grigory
G
Puri
Claudia
C
Arden
Susan D
SD
Roberts
Rhys
R
Dendrou
Calliope
C
Kendrick-Jones
John
J
Buss
Folma
F
D000199:Actins / Q000302:isolation & purification / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D001665:Binding Sites; D002448:Cell Adhesion; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000302:isolation & purification / Q000378:metabolism*; D053858:Metabolic Networks and Pathways; D046529:Microscopy, Electron, Transmission; D018995:Myosin Heavy Chains / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism*; D034622:RNA Interference; D015398:Signal Transduction; D048029:TNF Receptor-Associated Factor 6 / Q000378:metabolism*
D000199:Actins; C094297:CALCOCO2 protein, human; D016207:Cytokines; D047908:Intracellular Signaling Peptides and Proteins; D016328:NF-kappa B; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; C120635:TAX1BP1 protein, human; D048029:TNF Receptor-Associated Factor 6; C089785:myosin VI; D000469:Alkaline Phosphatase; D018995:Myosin Heavy Chains