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

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.

Fig. 1

T6BP and NDP52 are myosin VI binding partners. (A) T6BP and NDP52 interact with the myosin VI C-terminal tail in the mammalian two-hybrid assay. Plasmids encoding myosin VI tail constructs were in the bait vector and T6BP or NDP52 were in the prey vector. (B) T6BP and NDP52 interact with myosin VI in an F-actin pelleting assay. Each binding partner was mixed with full-length myosin VI and then with F-actin (input). After centrifugation, to remove the unbound fraction (unbound), the actomyosin pellet was resuspended in salt-ATP solution to dissociate myosin VI from F-actin. A second centrifugation step partitioned myosin VI and T6BP/NDP52 into the supernatant (SN) and F-actin into the pellet (P). Control samples without myosin VI show that T6BP and NDP52 do not bind to F-actin. (C) NDP52 and full-length T6BP bind the myosin VI tail in a pull-down assay using GST-myosin VI tail and in-vitro-translated T6BP and NDP52. GST was used as a control. (D) HeLa cell lysates probed with our affinity-purified rabbit polyclonal antibodies raised against T6BP or NDP52. (E) T6BP or NDP52 co-immunoprecipitate with myosin VI. HeLa cells either untransfected (U) or overexpressing GFP-NDP52 (T) were lysed and immunoprecipitation was performed using no antibody (Blank) or anti-NDP52. Immunoblots were tested using anti-myosin VI or anti-NDP52 antibodies. A431 cells were used for the T6BP-myosin VI co-immunoprecipitation with antibodies shown. Immunoblots were stained with anti-T6BP antibodies.

Fig. 2

Mapping the binding sites on T6BP, NDP52 and myosin VI. (A) The zinc fingers of T6BP bind to myosin VI. Expression plasmids encoding myosin VI tail constructs were used as bait and either T6BP truncation constructs or site-directed mutagenesis constructs as prey. (B) The LIM-like domain of NDP52 binds to myosin VI. Expression plasmids used were as in A but as prey either NDP52 truncation constructs or site-directed mutagenesis constructs were used. (C) T6BP and NDP52 cartoons showing the N-terminal SKICH domains, shaded boxes predicting regions of α-helical coiled coil and the zinc fingers (Z). (D) T6BP and NDP52 interact with the RRL motif in the myosin VI tail. Expression plasmids used were as in A except that those encoding myosin VI wild type (WT) and mutant (RRL to AAA) tail constructs were the bait and T6BP or NDP52 constructs were the prey.

Fig. 3

Intracellular localisation of T6BP and NDP52 in HeLa cells. (A) T6BP is localised at the Golgi complex and in the perinuclear region of the cell using anti-T6BP antibody (a and b) or by GFP-T6BP expression (c). Cells in d-f were pre-permeabilised with saponin and double labelled with antibody against TGN46 (e and e′), the box area in panel f is shown enlarged in panels d′-f′. Cells in panels g-i were double labelled with antibodies against T6BP and vinculin. (B) NDP52 is localised at the Golgi complex and in the perinuclear region of the cell by anti-NDP52 antibodies (a and b) or by GFP-NDP52 expression (c). To localise NDP52 around the Golgi complex, cells in panels d-f were pre-permeabilised with saponin and double labelled with TGN46 antibodies (e and e′). The boxed area in panel f is shown enlarged in panels d′-f′. Bars, 10 μm.

Fig. 4

T6BP and NDP52 colocalise with myosin VI in vesicles in the perinuclear region around the Golgi complex in HeLa cells. (A) Cells transiently overexpressing GFP-tagged myosin VI tail (a-d, e-h) were pre-permeabilised with saponin, fixed, double labelled with the antibodies against T6BP (a-d) and NDP52 (e-h), and analysed by immunofluorescence. The boxed areas in panels a and e are shown enlarged in panels panels b-d and f-h, respectively. Arrows indicate examples of colocalisation. Bars, 10 μm. (B) Immuno-electron microscopy shows endogenous T6BP (a,b) and endogenous NDP52 (c,d). Myosin VI and T6BP (b) or NDP52 (d) colocalise in vesicles near the Golgi complex. The number of gold particles observed in these sections reflects the expected levels of these endogenous proteins. Enlarged images highlight T6BP and NDP52 associated with vesicles. Bar, 450 nm.

Fig. 5

T6BP and NDP52 colocalise and form a complex in vivo. (A) T6BP and NDP52 colocalise in vesicles around the Golgi complex in HeLa cells. Cells transiently overexpressing GFP-T6BP (a-d) or GFP-NDP52 (e-g) were double labelled with antibodies to NDP52 (a-d) and T6BP (e-h). The boxed areas in panels a and e are shown enlarged in adjacent panels b-d and f-h, respectively. Arrows indicate colocalisation. Bar, 10 μm. (B) Immuno-electron microscopy shows that endogenous T6BP and NDP52 colocalise on vesicles on the trans-side of the Golgi complex. Bar, 370 nm. (C) T6BP co-immunoprecipitates with NDP52. HeLa cells either untransfected (U) or overexpressing GFP-NDP52 (T) were lysed and immunoprecipitations were performed using no antibody (Blank) or anti-NDP52. Immunoprecipitates were immunoblotted using anti-T6BP antibodies. (D) T6BP and NDP52 interact in the mammalian two-hybrid assay. T6BP constructs were used as the bait and NDP52 constructs as the prey.

Fig. 6

T6BP and NDP52 are not recruited into membrane ruffles upon EGF stimulation. A431 cells were stimulated with EGF and labelled with antibodies against myosin VI (MVI), T6BP or NDP52 after 0, 5 and 10 minutes. After 5-minute stimulation myosin VI is recruited into membrane ruffles (see arrows) whereas T6BP and NDP52 are not recruited into these membrane ruffles. Bar, 10 μm.

Fig. 7

Loss of T6BP and NDP52 leads to dramatic changes in actin filament organisation and focal adhesion formation. HeLa cells were depleted of T6BP, NDP52 or both proteins by transfection with siRNA and stained for immunofluorescence microscopy using Rhodamine-phalloidin and antibodies against vinculin. Whereas control cells show membrane ruffles at the plasma membrane but few focal adhesions and stress fibres, the KD cells have lost membrane ruffles and display a substantial increase in size and number of focal adhesions and stress fibres, respectively.

Fig. 8

Depleting T6BP or NDP52 by RNAi KD increases the rate of constitutive secretion in HeLa cells. (A) HeLa cells expressing SEAP were either mock-transfected or transfected with siRNAs specifically targeting myosin VI, T6BP or NDP52 and were analysed by immunoblotting using anti-actin antibodies as a loading control. Myosin VI, T6BP and NDP52 expression was <10% of normal levels. (B) In HeLa cells depletion of T6BP or NDP52 or both proteins by single siRNA (P1) or SMARTpool (SP) primers causes a significant increase in SEAP secretion (30-90% increase), whereas depletion of myosin VI leads to a 80% decrease in secretion. The amount of SEAP activity in the growth medium was analysed by chemiluminescence and normalised relative to cell density.

Fig. 9

T6BP is an inhibitor of TRAF6-mediated NF-κB activation. (A) Overexpression of T6BP inhibits NF-κB activation induced by TRAF6. HEK293 ET cells were transfected with the plasmids encoding either GFP or wild-type or mutant T6BP, NDP52 or myosin VI (400 ng per well) and plasmids encoding TRAF6 (200 ng per well). An empty pEGFP vector was included as a control. At 24 hours after transfection the luciferase activities in cell lysates were measured. T6BP inhibits TRAF6-mediated NF-κB activation, whereas myosin VI and NDP52 have no significant effect. (B) Overexpression of T6BP inhibits NF-κB activation downstream of a TRAF6-dependent pathway. HEK293 ET cells were transfected with the plasmids encoding either GFP or wild-type or mutant T6BP (600 ng per well) and plasmids encoding TLR2 (100 ng per well). At 24 hours after transfection, cells were stimulated for 5 hours with 10 μg/ml of peptidoglycan. The luciferase activities in cell lysates were then measured. (C) NF-κB activation via a TRAF6-independent pathway is unaffected by T6BP overexpression. HEK293 ET cells were transfected with the GFP plasmid or increasing amounts (200, 400 and 800 ng per well) of T6BP plasmid. At 24 hours after transfection cells were stimulated for 5 hours with 10 ng/ml of TNF-α and then the luciferase activities in the cell lysates were measured.