Volume 17, Issue 1, Pages 23-35 (January 2005) An FF Domain-Dependent Protein Interaction Mediates a Signaling Pathway for Growth Factor-Induced Gene Expression  Wei Jiang, Raffaella Sordella, Guang-Chao Chen, Shweta Hakre, Ananda L. Roy, Jeffrey Settleman  Molecular Cell  Volume 17, Issue 1, Pages 23-35 (January 2005) DOI: 10.1016/j.molcel.2004.11.024

Figure 1 Identification of TFII-I as a Candidate Interactor of the p190A FF Domains (A) Schematic representation of the GST-p190A fusion constructs used in (B). (B) Coomassie blue-stained protein gel indicating the purified GST-fusion proteins corresponding to various p190A constructs after expression in bacteria and isolation on glutathione beads. Note that the GST-GTPase fusion was used as a negative control in binding studies. (C) Silver-stained protein gel corresponding to “pull-downs” from neuroblastoma (N2A) cell lysates (or buffer-only control samples) with the indicated GST fusion proteins after a high-salt elution. Specific p190A-FF-interacting protein bands were analyzed by mass spectrometry, and the identified p130 peptide sequences, which were found to correspond to the TFII-I transcriptional regulator, are indicated. Molecular Cell 2005 17, 23-35DOI: (10.1016/j.molcel.2004.11.024)

Figure 2 p190A Interacts with TFII-I In Vitro and In Vivo (A) The FF domains of p190A interact with TFII-I in vitro. GST pull-down assays were performed with either GST beads (lanes 1 and 2) or GST-p190A-FF beads (lanes 3, 4, and 5) loaded with either buffer alone (lanes 1 and 3) or N2A cell lysates (lanes 2, 4, and 5). Phosphatase inhibitors (10 mM NaF and 1 mM Na3VO4) were included in the cell lysis buffer in lane 5 but omitted in lane 4. N2A lysate input for lanes 4 and 5 is shown in lanes 6 and 7. TFII-I was detected by immunoblotting. (B) Full-length p190A interacts directly with TFII-I in vitro. GST (lanes 2 and 3) or GST-TFII-I (lanes 4 and 5) proteins purified from Cos7 cells were used in a pull-down assay with either buffer alone (lanes 2 and 4) or with baculovirus-purified full-length p190A (lanes 3 and 5). Lane 1 is input of p190A for lanes 2 and 4. p190A was detected by immunoblotting. (C) p190A and TFII-I interact in vivo when both proteins are coexpressed. pEBG (lanes 1 and 3) or pEBG-TFII-I (lanes 2 and 4) were cotransfected with RcHAp190A into Cos7 cells. Cell lysates were prepared 2 days posttransfection and used for a glutathione-mediated pull-down assay. p190A was detected by immunoblotting with anti-HA epitope antibody. (D) Coimmuoprecipitation of endogenous TFII-I with p190A from serum-starved mouse fibroblasts. Lysates from p190A+/+ cells (lane 1) or p190A−/− cells (lane 2) were subjected to immunoprecipitation with a p190A-specific antibody and followed by immunoblotting with an anti-p190A antibody (top) or an anti-TFII-I antibody (bottom). (E) Coimmuoprecipitation of endogenous p190A with TFII-I from serum-starved mouse fibroblasts. Lysates from p190A+/+ cells (lane 1) or p190A−/− cells (lane 2) were subjected to immunoprecipitation with a TFII-I-specific antibody and then immunoblotted with anti-TFII-I antibody (bottom) or an anti-p190A antibody (top). (F) All four FF domains of p190A can bind weakly to TFII-I. Individual FF domains, expressed as GST-fusions, or the four tandem FF domains (GST-FF) were used in pull-down assays with N2A cell lysates to examine TFII-I binding by immunoblotting. (G) p190A binds the N-terminal region of TFII-I. Previously generated deletion/truncation mutants of TFII-I were prepared as purified GST-fusions and used in pull-down assays of cell lysates to detect associated p190A by immunoblotting. (H) Schematic of the truncation and deletion mutants used in panel (G). Molecular Cell 2005 17, 23-35DOI: (10.1016/j.molcel.2004.11.024)

Figure 3 p190A Antagonizes TFII-I's Transcriptional Activity on the Vβ Promoter (A) Cos-7 cells were transiently transfected with 250 ng of a Vβ-luciferase promoter construct and a β-galactosidase-expressing plasmid (internal control for transfection) together with p190A and/or TFII-I constructs, as indicated. 400 ng of pEBG-TFII-I, RcHAp190A, or 12-2 were included where indicated. “Empty” pEBG or RcHA vectors were used to equalize the DNA used in each transfection. Luciferase activity was measured 2 days posttransfection from cell extracts as described in Experimental Procedures. (B) Immunoblots of cell extracts indicating the expression of pEBG-TFII-I, RcHAp190A, and 12-2 in the transient expression experiments in (A). Expression of endogenous TFII-I is also detected. Molecular Cell 2005 17, 23-35DOI: (10.1016/j.molcel.2004.11.024)

Figure 4 p190A Regulates the Nuclear Translocation of TFII-I (A and B) p190A+/+ 3T3 cells (A) and p190A−/− 3T3 cells (B) were serum starved for 2 days before treatment with PDGF at 25 ng/ml for 15 min. Cells were then fixed and costained with anti-TFII-I antibody (red) and DAPI (blue) to indicate nuclei. Note that in p190A-deficient cells, TFII-I is largely localized within nuclei even in the absence of PDGF treatment. (C) p190A+/+ and p190A−/− 3T3 cells were serum starved for 2 days, and cells were then subjected to a nuclear-cytoplasmic fractionation in the presence or absence of PDGF stimulation. Cytoplasmic (C) and nuclear (N) fractions were immunoblotted for TFII-I, CREB (a nuclear marker), and paxillin (a cytoplasmic marker). (D) p190A−/− 3T3 cells were transfected with RcHAp190A and GFP (marker for transfected cells), starved for 2 days, and then fixed and stained with anti-TFII-I antibody and DAPI. Note that the p190A−/− cell into which p190A was reintroduced exhibits a diffuse TFII-I distribution relative to the untransfected cell. (E) Immunodepletion of p190A from the postnuclear fraction of serum-starved wild-type fibroblasts depletes the majority of TFII-I, as revealed by immunoblotting. As a negative control, the same analysis was performed in parallel in p190A−/− mutant fibroblasts. mDia immunoblotting was used as a normalization control. Molecular Cell 2005 17, 23-35DOI: (10.1016/j.molcel.2004.11.024)

Figure 5 p190A Regulates Serum-Induced c-fos Promoter Activity and Cell Cycle Reentry (A) p190A+/+ and p190A−/− 3T3 cells were transiently transfected with a c-fos luciferase reporter plasmid and a RNL-TK plasmid, with or without RcHA-C3, and then serum starved for 2 days and stimulated with 15% FBS for 4 hr. The bar graph indicates the relative increase in c-fos luciferase activity upon serum treatment. All experiments were performed in triplicate and standard deviations are indicated. (B) p190A+/+ and p190A−/− 3T3 cells with a stably integrated c-fos luciferase reporter plasmid were serum starved for 2 days and stimulated with either 20 ng/ml PDGF or 15% FBS. c-fos luciferase activity is shown as a bar graph reflecting the average of three independent experiments and indicating standard deviations. (C) p190A−/− cells were transiently transfected with a c-fos luciferase plasmid and the RNL-TK plasmid together with one of the following p190A constructs: RcHA vector, RcHAp190A, F2, 12-2, 30-1, or F4. The cells were serum starved for 2 days after transfection and then either maintained in serum-free medium or treated with 15% FBS for 4 hr before assaying for c-fos luciferase activity. All experiments were performed in triplicate. (D) A schematic representation of the p190A constructs used in (C). (E) Northern analysis of serum-induced c-fos mRNA in p190A−/− cells stably transfected with various p190A constructs. Cells were serum starved and then stimulated with 15% FBS for 30 min. p190A−/− (+ RcHA vector) exhibit a 2.5-fold increase in c-fos mRNA relative to p190A+/+ (+ RcHA vector), and the increase was abolished when wild-type p190A (RcHA-p190A), Rho-GAP-deficient p190A (30-1), or C-terminally-truncated p190A (12-2) were integrated in the genome of p190A−/− cells. (F) p190A+/+ and p190A−/− 3T3 cells were grown to confluency, serum starved for 2 days, and reseeded into 10% serum-containing medium at subconfluency (70%–80%). Cells were pulsed with 10 μM BrdU for 30 min and then collected and fixed in 75% ethanol at the indicated time points. FACS analysis was used to quantify the percentage of cells in S phase (BrdU incorporation). Molecular Cell 2005 17, 23-35DOI: (10.1016/j.molcel.2004.11.024)

Figure 6 Phosphorylation of Y308 in the p190A FF Domain Regulates the p190A-TFII-I Interaction (A) p190A+/+ and p190A−/− 3T3 cells were grown to confluency, serum starved for 2 days, and treated with either 20 ng/ml PDGF or 15% FBS. Cells were then lysed and immunoprecipitated with anti-p190A antibody, and immunoprecipitates were immunoblotted with RC20 (anti-phosphotyrosine) antibody, anti-p190A antibody, or anti-TFII-I antibody, as indicated. The numbers below blot images indicate the relative signal intensity. Note that serum/PDGF-induced tyrosine phosphorylation of p190A inversely correlates with TFII-I binding. (B) p190A−/− cells were transiently transfected with RcHA vector (lanes 1 and 2), RcHAp190A (lanes 3 and 4), or RcHAp190AY308F (lanes 5 and 6). Cells were grown to confluency and serum starved for 36 hr before treatment with PDGF for 15 min. Cells were then lysed, and lysates were subjected to immunoprecipitation with the 12CA5 anti-HA antibody. Immunoprecipitates were immunoblotted with the RC20 (anti-phosphotyrosine) antibody or the 12CA5 anti-HA antibody. (C) In vitro kinase assay with purified PDGF receptor to phosphorylate immobilized GST-FF or the GST-FF-Y308F mutant of p190A. Phosphorylation of PDGFR as well as the fusion protein was visualized by SDS-PAGE and autoradiography (top). The Coomassie blue-stained protein components of the reaction are shown in the bottom. (D) Glutathione bead-mediated pull-down assays with GST (lane 1), GST-p190A-FF (lane 2), the GST-p190A-FF-Y308F mutant (lane 3), and the GST-p190A-FF-Y308D mutant (lane 4) to detect associated TFII-I from N2A cell lysates. TFII-I was detected by immunoblotting with an anti-TFII-I antibody. Coomassie blue staining was used to visualize GST fusion proteins. (E) The proper folding of the Y308F and Y308D mutants of full-length p190A was verified by transfecting Cos7 cells with each construct and immunoprecipitating the HA-tagged forms of p190A and immunoblotting with either anti-HA antibody or an antibody directed against the major p190A binding partner, p120 RasGAP, as indicated. Cells were pretreated with pervanadate to promote phospho-tyrosine mediated p190A-p120 RasGAP association. Protein in total lysates was also detected by immunoblotting. Both mutants of p190A bind p120 RasGAP as efficiently as wild-type p190A does. (F) pEBG (lanes 1, 3, and 5) or pEBG-TFII-I (lanes 2, 4, and 6) was cotransfected with RcHAp190A (lanes 1 and 2), RcHAp190AY308F (lanes 3 and 4), RcHAp190AY308D (lanes 5 and 6), or RcHA vector (lane 7) into Cos7 cells. Two days after transfection, cells were lysed, and the lysates were incubated with glutathione beads to detect associated p190A by immunoblotting with anti-HA epitope antibody. Lysates were also immunoblotted with anti-HA antibody to confirm expression of the p190A proteins. (G) Purified GST, GST-FF, and GST-FF-Y308F were subjected to in vitro phosphorylation with PDGFR, and a GST pull-down assay followed by immunoblotting was performed with N2A cell lysates to examine the effect of phosphorylation on TFII-I binding. Molecular Cell 2005 17, 23-35DOI: (10.1016/j.molcel.2004.11.024)

Figure 7 Phosphorylation of Y308 in p190A Regulates Nuclear Translocation and Transcriptional Activity of TFII-I (A) Cos-7 cells were cotransfected with GFP (to mark transfected cells) and either RcHA-FF-Y308, RcHA-FF-Y308D, or RcHA-FF-Y308F, as indicated. Two days after transfection, cells were fixed and stained with anti-TFII-I antibody (red) and DAPI (blue). Note that in cells transfected with the wild-type FF domains or with the phospho-defective Y308F mutant, TFII-I exhibits a diffuse localization, whereas in the cell transfected with the phospho-mimicking Y308D mutant, TFII-I is largely nuclear. (B) Wild-type fibroblasts were starved and then stimulated with PDGF, and lysates were prepared at indicated time points to determine the relative kinetics of p190A tyrosine phosphorylation and the nuclear translocation of TFII-I. p190A phosphorylation peaks ∼5 min after PDGF exposure, whereas nuclear translocation of TFII-I peaks ∼12 min after PDGF treatment. (C) p190A−/− 3T3 cells were transiently transfected with a c-fos luciferase reporter plasmid and a RNL-TK plasmid with either RcHA, RcHAF2, RcHAF2-Y308F, or RcHAF2-Y308D. Cells were serum starved for 2 days and stimulated with 15% FBS for 4 hr. The bar graph indicates the relative increase in c-fos luciferase activity upon serum treatment. All experiments were performed in triplicate, and standard deviations are indicated. Molecular Cell 2005 17, 23-35DOI: (10.1016/j.molcel.2004.11.024)