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First published online 9 December 2008
doi: 10.1242/jcs.036251

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DLC1 interacts with 14-3-3 proteins to inhibit RhoGAP activity and block nucleocytoplasmic shuttling

Rolf-Peter Scholz^*, Jennifer Regner^*,, Anke Theil, Patrik Erlmann, Gerlinde Holeiter, Ruth Jähne, Simone Schmid, Angelika Hausser and Monilola A. Olayioye

University of Stuttgart, Institute of Cell Biology and Immunology, Allmandring 31, 70569 Stuttgart, Germany

View larger version (31K): [in this window] [in a new window]   Fig. 1. Interaction of DLC1 with recombinant GST-14-3-3 proteins. (A) HEK293T cells were transiently transfected with a plasmid encoding Myc-tagged DLC1. Whole-cell extracts (WCE) were incubated with glutathione beads coupled to the indicated GST–14-3-3 isoforms, or GST alone, and bound proteins were separated by SDS-PAGE. DLC1 was detected by western blotting with a Myc-specific antibody (top panel). The integrity of GST (marked with an arrowhead) and GST-14-3-3 isoforms (marked with an arrow) was verified by probing the membrane with GST-specific antibody. (B) Whole-cell extracts of HEK293T cells transiently expressing Myc-DLC1 were subjected to a pull-down (PD) with wild-type (WT) or R56/60A GST-14-3-3, and bound proteins were analyzed as described in A. (C) Schematic representation of DLC1 truncation mutants and putative PKD phosphorylation sites. (D) Whole-cell extracts of HEK293T cells transiently expressing the indicated Flag-tagged DLC1 mutants were subjected to a pull-down with GST-14-3-3β and bound proteins were analyzed with Flag-specific antibody as described in A. Expression of the different DLC1 variants was verified by immunoblotting of whole-cell extracts with Flag-specific antibody (right panel).

View larger version (27K): [in this window] [in a new window]   Fig. 2. Coimmunoprecipitation of DLC1 with 14-3-3 proteins. (A) HEK293T cells were transiently transfected with Myc-tagged DLC1 and empty vector (–), EE-tagged 14-3-3 or 14-3-3 or HA-tagged 14-3-3 expression vectors. 14-3-3 isoforms were immunoprecipitated from whole-cell extracts with EE- and HA-specific antibodies, respectively, and immune complexes were separated by SDS-PAGE. Coprecipitated DLC1 was detected by western blotting with Myc-specific antibody (top panels). Immunoprecipitation of 14-3-3 isoforms was verified by probing the membrane with EE- and HA-specific antibodies, respectively (middle panels), and expression of DLC1 was verified by immunoblotting of whole-cell extracts with Myc-specific antibody (bottom panels). (B) HEK293T cells were transiently transfected with expression vectors encoding EE-tagged 14-3-3 or 14-3-3, or HA-tagged 14-3-3, and empty vector or Myc-tagged DLC1. DLC1 was immunoprecipitated from whole-cell extracts with Myc-specific antibody and immune complexes were separated by SDS-PAGE. Coprecipitated 14-3-3 isoforms were detected by western blotting with EE- and HA-specific antibodies, respectively (middle panels, indicated with arrowheads). IgG chains are indicated with asterisks. Immunoprecipitation of DLC1 was verified with Myc-specific antibody (top panels), and expression of 14-3-3 isoforms was verified by immunoblotting of whole-cell extracts with EE- and HA-specific antibodies (bottom panels). (C) HEK293T cells transiently expressing Myc-tagged DLC1 were lysed and 14-3-3 proteins were immunoprecipitated from whole-cell extracts with 14-3-3-specific rabbit pAb. An unrelated rabbit pAb was used as a control (con IgG). Immune complexes were separated by SDS-PAGE. Coprecipitated DLC1 was detected by western blotting with Myc-specific antibody (top panel). Immunoprecipitation of 14-3-3 proteins were verified by probing the membrane with 14-3-3-specific mouse mAb (middle panel). Expression of DLC1 was verified by immunoblotting of whole-cell extracts with Myc-specific antibody (bottom panel).

View larger version (28K): [in this window] [in a new window]   Fig. 3. Phosphorylation-dependent interaction of DLC1 with 14-3-3 proteins. HEK293T cells were transiently transfected with a Myc-DLC1 expression vector and treated with (A) 100 nM okadaic acid or solvent (DMSO), or (B) 100 nM PDBu for the indicated times prior to lysis. (C) Cells were incubated with 1 µM staurosporine (ST), 5 µM Gö6983, 5 µM Gö6976 or solvent (DMSO) for 90 minutes before stimulation with 1 µM PDBu for 15 minutes. (D) MDAMB231 cells were left untreated or treated with 1 µM PDBu for 15 minutes prior to lysis. Whole-cell extracts were subjected to a pull-down with GST-14-3-3 beads and bound proteins were separated by SDS-PAGE. DLC1 was detected with Myc-specific antibody in A-C and DLC1-specific antibody in D (top panels). The integrity of recombinant GST-14-3-3 was verified by probing the membrane with GST-specific antibody (middle panels), and expression of DLC1 was verified by immunoblotting of whole-cell extracts with Myc-specific antibody in A-C and DLC1-specific antibody in D (bottom panels).

View larger version (61K): [in this window] [in a new window]   Fig. 4. DLC1 interaction with 14-3-3 proteins requires phosphorylation of Ser327 and Ser431. HEK293T cells were transiently transfected with GFP-tagged DLC1 WT, S327A, S431A, or S327/431A expression vectors. Whole-cell extracts were subjected to a pull-down with GST-14-3-3 beads and bound proteins were separated by SDS-PAGE. DLC1 variants were detected with a GFP-specific antibody (top panel). The integrity of recombinant GST-14-3-3 was verified by probing the membrane with GST-specific antibody (middle panel), and expression of DLC1 variants was verified by immunoblotting of whole-cell extracts with GFP-specific antibody (bottom panel). (B) An HA-tagged 14-3-3 expression vector was transiently transfected into HEK293T cells along with DLC1 WT, S327/431A or empty vector (con). DLC1 variants were immunoprecipitated from whole-cell extracts with GFP-specific antibody and immune complexes were separated by SDS-PAGE. Coprecipitated 14-3-3 was detected by western blotting with HA-specific antibody (middle panel). Immunoprecipitation of DLC1 variants was verified by probing the membrane with GFP-specific antibody (top panel). Equal expression of 14-3-3 was verified by immunoblotting of whole-cell extracts with HA-specific antibody (bottom panel). (C) HEK293T cells were transiently transfected with expression plasmids encoding Flag-tagged DLC1 WT or S327/431 and empty vector (–) or GFP-tagged PKD1 (+), respectively. Whole-cell extracts were subjected to GST-14-3-3 pull-downs and analyzed as described in A. Expression of PKD1 and DLC1 variants was verified by immunoblotting of whole-cell extracts with GFP- and Flag-specific antibodies, respectively (bottom panels). (D) HEK293T transiently expressing GFP-DLC1 WT or S327/431A were treated left untreated or treated with 100 nM okadaic acid for 2 hours or 1 µM PDBu for 15 minutes. Whole-cell extracts were subjected to GST-14-3-3 pull-downs and analyzed as described in A. (E) HEK293T cells transiently expressing GFP-tagged DLC1 WT, S327/431A or S327/431D were left untreated (–) or treated with 1 µM PDBu (+) for 15 minutes. Whole-cell extracts were subjected to GST-14-3-3 pull-downs and analyzed as described in A. (F) HEK293T cells transiently expressing GFP-tagged DLC1 WT, S327/431A, S327/419/431A, or S236/327/419/431A were left untreated (–) or treated with 1 µM PDBu (+) for 15 minutes. Whole-cell extracts were subjected to GST-14-3-3 pull-downs and analyzed as described in A. (G) Recombinant GST-DLC1(aa242-569) proteins were incubated in kinase buffer containing [-32P]ATP in the absence (left lanes) or presence of purified Myc-tagged PKD1 (right lanes). Proteins were separated by SDS-PAGE and transferred to membrane. Incorporation of radioactive phosphate was analyzed using a PhosphoImager (top panel), followed by immunoblotting with GST- and Myc-specific antibodies to verify equal loading (bottom panels). The GST-DLC1 fusion proteins and Myc-PKD1 are indicated with arrows.

View larger version (40K): [in this window] [in a new window]   Fig. 5. Binding of 14-3-3 proteins inhibits DLC1 RhoGAP activity. (A) HEK293T cells were transiently transfected with expression vectors encoding Raichu-RhoA (con) along with Flag-DLC1 WT or S327/431A, and HA-14-3-3, where indicated. The emission ratio of Raichu-RhoA was determined by measuring YFP (FRET) and CFP fluorescence (excitation 433 nm) in cell lysates. Equal expression of 14-3-3 and DLC1 proteins was verified by immunoblotting of lysates with HA- and Flag-specific antibodies, respectively (not shown). The mean of three independent experiments performed with triplicate samples is shown; error bars represent s.e.m. Results were statistically significant for the wild-type protein (two-tailed unpaired t-test, P=0.0006); no significant difference was observed for the control and the S327/431A mutant protein (NS, P>0.05). (B) HEK293 Flp-In-DLC1 cells were left untreated (–) or treated with 10 ng/ml doxycycline (+) overnight to induce GFP-DLC1 expression. Whole-cell extracts were separated by SDS-PAGE and analyzed by western blotting with GFP-specific (top panel) and RhoA-specific antibodies (bottom panel). For SRF reporter assays, Flp-In-DLC1 cells were transiently transfected with the 3DA.Luc reporter, pTK-Renilla and HA-14-3-3 or empty vector. Cells were left untreated (–DOX) or treated with 10 ng/ml doxycycline (+DOX) for 4 hours, followed by PDBu stimulation (100 nM) for additional 4 hours. Firefly luciferase activity in cell lysates was determined and normalized by Renilla luciferase activity. Fold induction after PDBu stimulation was calculated and values for uninduced cells (–DOX) were set to 100%. Expression of DLC1 and 14-3-3 proteins was verified by immunoblotting of lysates with GFP- and HA-specific antibodies, respectively (not shown). The mean of three independent experiments performed with triplicate samples is shown, error bars represent s.e.m. (C) Flp-In-DLC1 cells were left untreated or treated with 10 ng/ml doxycycline over night and photographed with a Leitz DM IRB microscope equipped with a NPLAN 10/0.25 PH1 objective (Leica) and an AxioCam MRc camera (Zeiss) (left panels). Flp-In-DLC1 cells were transiently transfected with empty vector or EE-14-3-3 expression plasmid, followed by induction of GFP-DLC1 expression with 10 ng/ml doxycycline overnight. Cells were fixed and stained with EE-specific antibody (red) (right panels). Stacks of several confocal sections are shown. Scale bars: 20 µm. (D) Flp-In-DLC1 cells were treated with 10 ng/ml doxycycline overnight, trypsinized and kept in suspension for 1 hour. Cells were then plated onto collagen-coated dishes for the indicated times and lysed. Whole-cell extracts were subjected to a pull-down with GST-14-3-3 beads and bound proteins were separated by SDS-PAGE. DLC1 was detected with GFP-specific antibody (top panel). The integrity of recombinant GST-14-3-3 was verified by probing the membrane with GST-specific antibody (middle panel), and expression of DLC1 was verified by immunoblotting of whole-cell extracts with GFP-specific antibody (bottom panel).

View larger version (33K): [in this window] [in a new window]   Fig. 6. Stable expression of DLC1 variants in MCF7 cells inhibits proliferation. (A) Stable expression of DLC1 variants in MCF7 cells was verified by immunoblotting of whole-cell extracts using a DLC1-specific antibody (top panel). Equal loading was confirmed by probing the membrane with tubulin-specific antibody (bottom panel). (B) Proliferation of MCF7 cells stably overexpressing DLC1 WT and S327/431A, respectively, and vector control cells was measured by MTT assay. Data are normalized to absorbance at day 0. (C) MCF7 DLC1 WT and S327/431A cells were left untreated or treated with 1 µM PDBu for 15 minutes prior to lysis. Whole-cell extracts were subjected to a pull-down with GST-14-3-3 beads and bound proteins were separated by SDS-PAGE. DLC1 was detected with DLC1-specific antibody (top panel). The integrity of recombinant GST-14-3-3 was verified by probing the membrane with GST-specific antibody (middle panel), and expression of DLC1 was verified by immunoblotting of whole-cell extracts with DLC1-specific antibody (bottom panel). (D) MCF7 DLC1 WT cells were left untreated or treated with 1 µM PDBu prior to lysis. 14-3-3 proteins were immunoprecipitated from whole-cell extracts with 14-3-3-specific rabbit pAb. Immune complexes were separated by SDS-PAGE. Coprecipitated DLC1 was detected by Western blotting with DLC1-specific antibody (top panel). Immunoprecipitation of 14-3-3 proteins were verified by probing the membrane with 14-3-3-specific rabbit pAb (middle panel; IgG chains are indicated with an asterisk). Expression of DLC1 was verified by immunoblotting of whole-cell extracts with DLC1-specific antibody (bottom panel).

View larger version (40K): [in this window] [in a new window]   Fig. 7. Binding of 14-3-3 proteins inhibits DLC1 nuclear transport mediated by an NLS spanning residues 423-429. (A) Schematic representation of DLC1 WT and Arg-Gly exchange mutants. The putative bipartite NLS is highlighted in bold, the pat7 NLS is underlined. (B,C) MCF7 cells were transiently transfected with GFP-tagged DLC1 WT or R428/429G and treated with LMB for 2 hours prior to fixation. (C) Cells were stimulated with 100 nM PDBu prior to LMB addition. (D) MCF7 cells transiently expressing the indicated GFP-tagged DLC1 variants were treated with LMB for 2 hours and fixed. The number of cells displaying mainly cytosolic, homogenous (nuclear + cytosolic) or mainly nuclear protein distribution was determined by counting 100 cells each in random microscopic fields. Values correspond to the mean of two independent experiments. (E) COS7 cells were transiently transfected with both GFP-tagged DLC1 and EE-14-3-3 expression vectors and treated with 10 ng/ml LMB for 1 hour prior to fixation and staining with EE-specific antibody. Cells expressing only DLC1 are indicated with asterisks, cells expressing DLC1 and 14-3-3 are marked with arrows. All images are single confocal sections. Scale bars: 20 µm.

View larger version (27K): [in this window] [in a new window]   Fig. 8. Regulation of DLC1 by 14-3-3 proteins. Activation of PKC/PKD kinases leads to DLC1 phosphorylation on sites that include Ser327 and Ser431. This creates binding sites for 14-3-3 adaptor proteins, whereby DLC1 RhoGAP activity is inhibited, most likely by cytosolic sequestration. 14-3-3 binding furthermore masks a nuclear localization signal spanning residues 423-429, thus preventing DLC1 nucleocytoplasmic shuttling.

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