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First published online 28 March 2006
doi: 10.1242/jcs.02889

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TRAF6 activation of PI 3-kinase-dependent cytoskeletal changes is cooperative with Ras and is mediated by an interaction with cytoplasmic Src

¹ Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA
² Department of Periodontology, Faculty of Dentistry, Khon Kaen University, Khon Kaen, 40002, Thailand
³ Department of Medicine, Harvard Medical School and Beth Israel Deaconess Medical Center, Boston, MA 02115, USA
⁴ Department of Immunology, University of Occupational and Environmental Health, Kitakyushu, 807, Japan
⁵ Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University School of Pharmacy, West Lafayette, IN 47907, USA
⁶ Center for Bone Biology, Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA

View larger version (45K): [in a new window]   Fig. 1. IL-1ß induces PI 3-kinase-dependent filopodia growth and translocation of PH-Akt-GFP. Hep3B (A) and HEK293R (B,C) cells were transfected with 15 ng of PH-Akt-GFP expression vector in a 96-well plate for 24 hours. IL-1ß was then added, or not (24h-No IL-1), into each well at a final concentration of 10 ng/ml for the indicated time. (C) Following 24 hours of IL-1 treatment, cells transfected with PH-Akt-GFP were exposed to a final concentration of 25 µM LY294002 (Ly). (D) HEK293R cells were fixed and stained with phalloidin-TRITC following incubation for 24 hours either in the absence or presence of IL-1ß, as indicated. Arrows locate long, branched filopodia-like structures.

View larger version (62K): [in a new window]   Fig. 2. TRAF6 induces translocation of PH-Akt-GFP to cell membranes and de novo protrusions. (A) A PH-Akt-GFP expression vector reporter was co-transfected with expression vectors encoding a second activator protein, as indicated above each panel, into either HEK293 (a-c) or RAW264.7 (d-f) cells for 24 hours. Transfection of only the activator protein, followed by fixation and phalloidin-TRITC staining in order to visualize polymerized actin (g-i). (B) PH-Akt-GFP expression vector reporter co-transfected with a TRAF6 expression vector into HEK293 cells in either the absence or presence of 25 µM of Ly294002 (LY) and 10 mM methyl-ß-cyclodextrin (MbCD) inhibitors for the indicated treatment time (a-i). Two different sets of cells are shown for MbCD. In one (d-f), a long exposure time reveals the details of the filopodial network. In another (g-i), a shorter exposure reveals that the PH-Akt-GFP is relocalized from cell membranes to the cytoplasm, following treatment with MbCD. A control set shows the co-transfected vectors evaluated in the presence of the DMSO vehicle for an equivalent treatment time (j-l) with a long exposure time to allow visualization of filopodia.

View larger version (50K): [in a new window]   Fig. 3. TRAF6 induces the localization of PH-Akt-GFP to actin branch points. (A) HEK293 cells were co-transfected with a PH-Akt-GFP expression vector reporter and a TRAF6 expression vector for 24 hours, followed by fixation and phalloidin-TRITC staining. GFP (green), FITC (red) and merged images were used to determine signal co-localization (yellow). (B) HEK293 cells were co-transfected with PH-Akt-GFP and TRAF6 expression vectors for 24 hours, then treated for the indicated times with 5 µM cytochalasin D.

View larger version (32K): [in a new window]   Fig. 4. TRAF6 and Src proteins used in these studies, showing the relative location of structural-functional domains. (A) Schematic of wild-type (wt) TRAF6 trimer structure and the location of point mutations. (B) Wild-type human Src and the location of point and deletion mutations.

View larger version (30K): [in a new window]   Fig. 5. Prolines within the TRAF6 MATH domain are required for morphological changes and activation of both PI 3-kinase and NF-B. (A) HEK293 cells were co-transfected with a PH-Akt-GFP expression vector reporter and various TRAF6 constructs (as described in Fig. 4A) for 24 hours. (B) An NF-B-dependent luciferase reporter containing four tandem B-binding sites adjacent to a minimal fos promoter was assayed 24 hours following cell transfection with either 150 ng of various TRAF6 expression vectors or control vector into HEK293 cells as described in the Materials and Methods. The PI 3-kinase inhibitor LY294002 (Ly) was added at a final concentration of 25 µM into cells after 2 hours of transfection and maintained until analyzed for luciferase activity. Numbers in parentheses indicate fold activity over control vector.

View larger version (46K): [in a new window]   Fig. 6. Examination of subcellular localization and protein-protein interaction in living cells for de novo-expressed Src and TRAF6. Bimolecular fluorescence complementation (BiFC) involving split YFP tags was used to detect intracellular protein-protein interaction between TRAF6 and Src that could be compared with the expression pattern for these proteins tagged with full-length YFP in HEK293T cells transiently transfected for 24 hours. (A) Transfection of 200 ng of individual, uncomplemented BiFC vectors; –YN, N-terminal YFP fragment (aa 1-154), –YC, C-terminal YFP fragment (aa 155-238). (B) Transfection of 200 ng of each BiFC vector complementary pair (–YN + –YC). (C) Subcellular localization of Src-YFP and variants in cells transfected with 50 ng of expression vectors encoding each protein form, as indicated. (D) Subcellular localization of TRAF6-YFP and variants in cells transfected with 20 ng of expression vectors as indicated. (E) Image enhancement of Fig. 6B panels a and c, revealing distinct TRAF6 and Src morphologies.

View larger version (47K): [in a new window]   Fig. 7. Src and Ras mediate TRAF6 activation of PI 3-kinase. A PH-Akt-GFP expression vector reporter was co-transfected along with indicated plasmids into HEK293 cells. Src- and Ras-specific inhibitors were each added, as indicated above each panel, to a final concentration of 500 nM into individual wells at 6 hours. Images were taken 24 hours after transfection.

View larger version (29K): [in a new window]   Fig. 8. TRAF6 is relocalized when co-expressed with either an upstream or downstream activator. (A) Subcellular localization of TRAF6-YFP co-transfected with various expression vectors encoding non-labeled activator proteins. 70 ng total DNA (20 ng of TRAF6-YFP and 50 ng of expression vector, as indicated) were co-transfected into HEK293 cells. `Control' represents cells co-transfected with 20 ng TRAF6-YFP and 50 ng of empty expression vector. Src, MyD88 and IRAK2 are wild-type expression vectors. Src(1-142) and Src(1-82) expression vectors contain the regions indicated in Fig. 4. (B) NF-B activity resulting from co-transfection in HEK293 cells with expression vectors, as indicated, and the luciferase reporter described in Fig. 5. The amount of transfected vectors is the same as in Fig. 8A. A total of 70 ng of DNA was used for each transfection, including the empty `Vector' control. (C) PH-Akt-GFP expression vector reporter was co-transfected together with cDNA vectors encoding proteins, as indicated above each panel, into HEK293 cells.

View larger version (31K): [in a new window]   Fig. 9. Schematic representation of TRAF-dependent PI 3-kinase pathways. The data presented in this paper are consistent with a pathway involving both Ras and Src as key molecules in generating TRAF6 and PI 3-kinase-dependent activation of actin-mediated changes in cell morphology. The schematic incorporates relevant pathways derived from other reports as described in the text. Solid black arrows connect sequential pathway elements, red arrows designate enzymatic action, and broken arrows indicate protein-protein interactions resulting in a directional activation, as indicated by arrowheads. The activation of NF-B by TRAF6 is indicated by tandem arrows, representative of the multistep process involving IB kinase-activated phosphorylation and degradation of IB resulting in subsequent release of active p50p65. Abbreviations: IL1RIIL1RAcP, IL-1 ligand-binding transmembrane receptor complex comprising the type I and accessory receptors; TIR, Toll-IL-1/18 homology domain; DD, death domain; TIM, TRAF-interaction motif; WASP, Wiskott-Aldrich syndrome protein; PI, phosphatidylinositol; PIP, phosphatidylinositol 4-monophosphate; PIP2, phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2]; PIP3, phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3]; PIP5Kinase, phosphatidylinositol-4-phosphate 5-kinase.