RACK1 works through Src to suppress Vav2 and Myc activation. (A) Effect of RACK1 overexpression on Src-mediated Vav2 tyrosine phosphorylation. Cells were transfected as indicated. Top panel, proteins were immunoprecipitated with antiphosphotyrosine from lysates containing 500 μg of cellular protein and subjected to immunoblot analysis with anti-Vav2. Bottom three panels, proteins from lysate containing 20 μg of total cellular protein were subjected to immunoblot analysis with anti-Vav2, Src, or RACK1. Data are representative of three independent experiments. (B) Stimulation of myc promoter activity in cells cotransfected with pMyc-Luc and expression vectors containing Vav2 (1 μg), Src (0.5 μg), RACK1 (1 μg), and mutant RACK1 (1 μg), as indicated. Luciferase activity was measured as described in the legend to Fig. 1. Data represent mean values ± standard errors from triplicate samples and are representative of three independent experiments. (C) Effect of Src, RACK1, and Vav2 overexpression on Myc protein expression. Cells were transfected as indicated, and lysate proteins containing 20 μg of total cellular protein were subjected to immunoblot analysis with anti-Myc, Vav2, Src, or RACK1. Data are representative of three independent experiments.

RACK1 works through Src to suppress Stat3 and Myc activation. Shown are the effects of Stat3 on Rac1-stimulated myc promoter activity (A) and Myc protein expression (B). Dominant-negative Stat3β is a naturally occurring splice variant with a deletion in the C terminus that harbors the transcriptional activation domain and the Ser 727 residue, phosphorylation of which is required for efficient transcriptional activation. As a result of this deletion, dimers formed with Stat3β lack transcriptional activity. (A) Stimulation of myc promoter activity in cells cotransfected with pMyc-Luc and expression vectors containing Rac1 (1 μg), Stat3 (1 μg), and Stat3β (1 μg), as indicated. Luciferase activity was measured as described in Materials and Methods. Data represent mean values ± standard errors from triplicate plates and are representative of three independent experiments. (B) Cells were transfected as indicated, and lysate proteins containing 20 μg of total cellular protein were subjected to immunoblot analysis with anti-Myc, Rac1, or Stat3. (C) Effect of Src and RACK1 overexpression on Stat3 tyrosine phosphorylation. Top panel, lysate proteins were immunoprecipitated with anti-Stat3 and immunoblotted with an antibody that recognizes phosphorylated Tyr 705, the Src-mediated phosphorylation site on Stat3. Bottom panels, proteins from lysate containing 20 μg of total cellular protein were subjected to immunoblot analysis with anti-Stat3, Myc, Src, or RACK1. (D) Effect of Src and RACK1 overexpression on ERK signaling. Lysate proteins were subjected to immunoblot analysis with an antibody that recognizes phosphorylated ERK1 and ERK2 (Thr 202/Tyr 204), ERK1 and ERK2, or RACK1 as indicated. Lysate proteins were immunoprecipitated with anti-ERK and assayed for in vitro protein kinase activity with MBP as a substrate. Data are representative of two or three independent experiments. (E) Effect of RACK1 inhibition on downstream effectors of Src. HEK 293 cells were transfected with RACK1 siRNAs as described in the legend to Fig. 1. Tyrosine phosphorylation of Vav2 and Stat3 was assayed as described in the legends to Fig. 2A and panel C, respectively. Lysate proteins were subjected to immunoblot analysis with anti-Vav2, Stat3, cyclin D1, phospho-ERK, or ERK as indicated. ERK in vitro kinase activity was assayed as described for panel D.

RACK1 regulates G₁/S progression via its interaction with Src. (A) Effect of RACK1 overexpression on G₁/S transition. NIH 3T3 cells were transiently transfected with vector, Src, RACK1, and RACK1 mutant (as indicated) and synchronized and held in G₀ by serum starvation for 48 h. Cells were released into G₁ with the addition of 10% FBS, harvested 6 h later, and analyzed for cellular DNA content by flow cytometry. (B) Effect of inhibiting RACK1 expression on G₁/S transition. HEK 293 cells were transfected with vector or RACK1 siRNA-A or siRNA-B, synchronized in G₀ by serum withdrawal for 24 h, released into G₁, and analyzed for cellular DNA content as described for panel A.

Model for RACK1 function in Src-mediated mitogenic signaling and cell cycle progression. (A) Engagement of PDGF with its cell surface tyrosine kinase receptor induces dimerization and autophosphorylation of the receptor; Src binding and activation; tyrosine phosphorylation of Vav2; activation of Rho GTPases; tyrosine phosphorylation of Stat3; dimerization and translocation of Stat3 to the nucleus; activation of Myc, cyclin D1, and other cell cycle regulators; cell cycle progression through the G₁/S checkpoint; and cell proliferation. (B) RACK1 works by inhibiting Src activity and thereby the tyrosine phosphorylation of Vav2; activation of Rho GTPases; tyrosine phosphorylation of Stat3; and activation of Myc, cyclin D1, and other cell cycle regulators. Consequently, G₁/S transition is delayed.

RACK1 inhibits Src-mediated Myc activation. (A) Effect of RACK1 overexpression on Src in vitro kinase activity and Myc expression. NIH 3T3 cells were transfected with vector (lane 1), Src (lane 2), Src and wild-type HA-RACK1 (lane 3), Src and a mutant RACK1 (Y228F/Y246F HA-RACK1) containing a phenylalanine substitution for tyrosine at positions 228 and 246 (lane 4), mutant HA-RACK1 (lane 5), or wild-type HA-RACK1 (lane 6). Top panel, proteins were immunoprecipitated with excess anti-Src (MAb 327) from lysate containing 500 μg of cellular protein and incubated with [γ-³²P]ATP, MnCl₂, and enolase for 10 min at 30°C in an in vitro protein kinase assay. Bottom three panels, proteins from lysate containing 20 μg of total cellular protein were subjected to immunoblot analysis with anti-Myc, Src, or RACK1. Data are representative of three independent experiments. (B) Stimulation of myc promoter activity in cells cotransfected with pMyc-Luc and expression vectors containing Src (0.5 or 1 μg), RACK1 (0.5, 1, or 1.5 μg), mutant RACK1 (0.5, 1, or 1.5 μg), and dl155 Src (1 μg), as indicated. +, the highest concentration of Src or RACK1 was transfected. Results represent luciferase activity in each sample normalized to efficiency of transfection. Luciferase activity was normalized to a simultaneously transfected internal control plasmid (pRL-TK, Renilla luciferase). Data represent mean values ± standard errors from triplicate samples and are representative of three independent experiments. (C) Effect of introducing RACK1 siRNAs on RACK1 and Myc expression and Src in vitro kinase activity. HEK 293 cells were transfected with 5 μg of vector (lane 1), RACK1 siRNA-A (lane 2), or RACK1 siRNA-B (lane 3), and lysate proteins were subjected to immunoblot analysis with anti-RACK1 (top panel) or anti-Myc (bottom panel) or were immunoprecipitated with anti-Src and assayed for in vitro protein kinase activity or subjected to immunoblot analysis with anti-Src (middle panels) as described for panel A. Data are representative of three independent experiments.

RACK1 works through Src to suppress Rho GTPases and Myc activation. Shown are the effects of Src and RACK1 overexpression on Rac1-stimulated myc promoter activity (A) and Myc protein expression (B and C). Dominant-negative Rac1N17 contains a threonine-to-asparagine change and cannot bind GTP. (A) Stimulation of myc promoter activity in cells cotransfected with pMyc-Luc and expression vectors containing Rac1 (1.5 μg), Src (0.5 μg), RACK1 (1 μg), mutant RACK1 (1 μg), and Rac1N17 (1.5 μg) as indicated. Luciferase activity was measured as described. Data represent mean values ± standard errors from triplicate plates and are representative of three independent experiments. (B and C) Cells were transfected as indicated, and lysate proteins containing 20 μg of total cellular protein were subjected to immunoblot analysis with anti-Myc, Rac1, Rac1N17, Src, or RACK1. Data are representative of three independent experiments.

RACK1 inhibits cell cycle regulators in G₁ by suppressing Src activity. NIH 3T3 cells were transiently transfected with vector, Src, and wild-type or mutant HA-RACK1 or GFP-RACK1, as indicated. (A) Effect of RACK1 overexpression on cell cycle regulators in G₁. Transfectants were synchronized and held in G₀ by serum starvation for 48 h and then released into G₁ with the addition of 10% FBS for 6 h. First, third, and fourth panels from the top, proteins were precipitated with antibody to Src, CDK4, or CDK2, respectively, from lysates containing 500 μg of cellular protein and incubated with [γ-³²P]ATP together with MnCl₂ and enolase (Src immunoprecipitates), MgCl₂ and Rb peptide (CDK4 immunoprecipitates), or MgCl₂ and histone H1 (CDK2 immunoprecipitates) for 10 min at 30°C and analyzed for in vitro protein kinase activity. Other panels, proteins from lysates containing 20 μg of total cellular protein were subjected to immunoblot analysis with antibodies to cyclin D1, p27, p21, pRb, E2F1, or tubulin. (B) Effect of RACK1 overexpression on nuclear cyclin D1 expression in G₁. Transfectants were serum starved for 24 h, incubated with 10% FBS for 3 h, replated onto fibronectin-coated chamber slides in 10% FBS, and fixed 4 h later. Cells were analyzed for cyclin D1 expression by immunofluorescence with an Alexa Fluor 594-conjugated secondary antibody (red), for GFP by fluorescence with a GFP filter (green), and for nuclei with DAPI (blue).

RACK1 inhibits Src- but not MAPK-dependent PDGF signaling. NIH 3T3 cells were transiently transfected with vector, RACK1, or mutant RACK1 as indicated; serum starved to quiescence; and treated with PDGF (30 ng ml⁻¹) for 30 min prior to lysis, as indicated (+). (A) Effect of RACK1 overexpression on Src-mediated PDGF signaling. Tyrosine phosphorylation of Vav2 and Stat3 was assayed as described in the text. Lysate proteins were subjected to immunoblot analysis with anti-Vav2, Stat3, Myc, cyclin D1, or RACK1 as indicated. (B) Effect of RACK1 overexpression on MAPK-mediated PDGF signaling. Lysate proteins were subjected to immunoblot analysis with antibody that recognizes phospho-ERK (top panel) or ERK (middle panel). ERK in vitro kinase activity was assayed as described in the text (bottom panel).