Proposed model for RhoA signaling to the c-jun promoter. Activated RhoA can initiate a signaling pathway inducing ERK6, which in turn activates MEF2A and ATF2 transcription factors bound to the jAP1 and MEF2 sites on the c-jun promoter, thereby enhancing c-jun expression. Available data suggest that Rho A, PKN, MKK3/MKK6, and ERK6 (p38γ) are components of a novel signal transduction pathway involved in the regulation of gene expression and cellular transformation. Parallel pathways are depicted and described in the text. Unknown molecules are indicated by question marks.

The transforming activity of Rho is inhibited by dominant-negative c-Jun and kinases upstream of ERK6. (A) NIH-3T3 cells were transfected by the calcium phosphate technique with pCDNAIII β-Gal pCEFL–AU5-RhoA QL, or pCEV–MEK1 EE (1μg each), alone or in combination with c-Jun Tam67 (1 μg). Cells were cultured for 3 wk in 5% calf serum, and then fixed and stained. Representative plates for each transfection are shown. (B) Cells were transfected as above with with pCDNAIII β-Gal (1μg), pCEFL–AU5–RhoA QL (1 μg) or pCEFL–AU5 RasV12 (0.5 μg), alone or in combination with MKK3 AA, MKK6 KR, or MEK1 AA (1 μg each). The data represent percentage of foci developed with respect to those developed by RhoA QL or Ras V12 alone, whose values were taken as 100%. Results are the average ± s.e. of three experiments.

Activated mutants of small GTP-binding proteins stimulate the c-jun promoter. NIH-3T3 cells were cotransfected with (A) pJLuc or (B) pSRE together with pRL-null plasmid DNAs (0.1 and 0.01 μg per plate each, respectively) using the Lipofectamine Plus reagent. Empty expression vector (control) or expression vectors for Ras V12, RhoA QL, Rac1 QL, and Cdc42 (1 μg each) were included in the transfection mixtures, as indicated. Twenty-four hours after transfection, cells were collected and the lysates were assayed for dual luciferase activities. The data represents firefly luciferase activity normalized by Renilla luciferase activity present in each sample expressed as fold induction relative to control. Values are the average ± s.e. of triplicate samples from a typical experiment. Nearly identical results were obtained in four additional experiments.

RhoA stimulates the phosphorylation and kinase activity of ERK6 (p38γ). NIH-3T3 cells were transfected with expression vectors containing (A) HA-tagged ERK2, JNK, ERK5, or (B) HA-tagged p38α or ERK6 along with Ras V12, RhoA QL, Rac1 QL, or Cdc42 QL. After serum starvation, lysates were immunoprecipitated with anti-HA antibody and used for kinase reactions. ³²P-labeled substrates are indicated. Histograms represent the kinase activity relative to that in cells transfected with empty expression vector (control), whose value was taken as 1. Data represent the means ± s.e. of triplicate samples from a typical experiment. Similar results were obtained in three additional experiments. (Bottom) Expression of HA-tagged kinases in lysates from control and each indicated transfectant upon analysis by Western blot using a specific anti-HA antibody. (C) NIH-3T3 cells were cotransfected with HA-tagged ERK6 together with RhoA QL, RhoA N19, or MKK6. Twenty-four hours later, lysates were immunoprecipitated with anti-HA antibody and used for Western blot. Gels show phosphorylated ERK6 (p38γ) (top) and expression of HA-tagged ERK6 (bottom) from the same samples recognized by anti-phospho ERK6 and anti-HA specific antibodies, respectively. (D) Cells were seeded on coverslips and transfected as above with HA-tagged ERK6, together with AU5-tagged-RhoA QL, RhoA N19, Ras V12, or GST-tagged MKK6, or HA-tagged ERK2 along with AU5-tagged Ras V12, as indicated. Twenty-four hours after transfection, cells were transferred to serum-free medium for an additional 24 h, fixed, and analyzed by immunofluorescence for nuclear staining (DAPI, top) as well as for ERK6, ERK2, AU5, GST, and Ras with specific antibodies, as indicated (middle and bottom).

Regulation of ERK6 (p38γ) by Rho-dependent pathways. (A) NIH-3T3 cells were transfected with HA-tagged ERK6. After serum starvation, they were treated with 10 μM LPA for the indicated times. Lysates were immunoprecipitated with anti-HA antibody and used for kinase reactions. Histograms represent the kinase activity relative to that of untreated cells (control), whose value was taken as 1. Data represent the means ± s.e. of triplicate samples from a typical experiment. Similar results were obtained in three additional experiments. (B) NIH-3T3 cells were transfected with HA-tagged ERK6 or HA-tagged MAPK and C3 toxin, as indicated. After serum starvation, they were treated with 10 μM LPA for 5 min. Cellular lysates were immunoprecipitated with anti-HA antibody and used for kinase reactions. In each case, the top gel shows ³²P-labeled substrates and the bottom gel shows expression of HA-tagged ERK6 or MAPK in lysates from the same samples upon analysis by Western blot by use of a specific anti-HA antibody. Autoradiograms are representative of three independent experiments. (C) Total lysates from NIH-3T3 cells (control) and HEK-293T cells transfected with empty vector (control) or with HA-ERK6 (p38γ) and HA-p38α, as indicated, were analyzed by Western blotting by use of two different anti-ERK6 antibodies (a and b) and an anti-p38α antibody as described in Materials and Methods. Autoradiograms are representative of three similar blots. ERK6 (p38γ) transcripts were also detected in both cell lines (data not shown). (D) NIH-3T3 cells were treated with 10 μM LPA or transfected with RhoA QL and MKK3 EE as above. Endogenous ERK6 (p38γ) kinase activity was measured as above after immunoprecipitating ERK6 with the anti-ERK6a goat polyclonal antibody. Autoradiograms are representative of three independent experiments.

Regulation of ERK6 (p38γ) by molecules acting downstream from Rho. (A) NIH-3T3 (top) and HEK-293T cells (bottom) were transfected with expression vectors containing HA-tagged ERK6 along with RhoA QL, PKN wt, PKN Δ ROKα wt, ROKα Δ, or MEK3 EE (1 μg each), as indicated. Kinase and Western blotting assays were performed as described above. Histograms represent the kinase activity relative to that in cells transfected with empty expression vector (control), whose value was taken as 1. Gels are representative of three independent experiments. (B) Lysates from 293T cells transfected with the indicated expression vectors were analyzed by Western blotting by use of anti-PKN and anti-tag (myc) antibodies. (C) NIH-3T3 cells were transfected as above with pSREmutL (0.1 μg per plate) or pJLuc together with pRL-null reporter plasmid DNAs (0.01 μg per plate) and treated with 10 μM LPA for 5 h or cotransfected along with PKN Δ alone or in combination with MKK3 AA (1 μg), as indicated. Transfection with MKK3 EE served as a control. Twenty-four hours after transfection, cells were collected and the lysates were assayed for dual luciferase activities. The data represent firefly luciferase activity normalized by Renilla luciferase activity present in each sample expressed as fold induction relative to control, and are the average ± s.e. of triplicate samples from a typical experiment. Similar results were obtained in three independent experiments.

The MAPK kinases MKK6 and MKK3 mediate the stimulation of ERK6 (p38γ) and the c-jun promoter by RhoA in NIH-3T3 cells. (A) NIH-3T3 cells were cotransfected with HA-tagged ERK6 together with RhoA QL alone or in combination with MKK3 AA (1 μg) and MKK6 KR (1 μg) as indicated. Twenty-four hours later, lysates were immunoprecipitated with anti-HA antibody and used for kinase reactions. ³²P-labeled substrates are indicated. Autoradiograms correspond to a representative experiment. Data represent the means ± s.e. of triplicate samples from a typical experiment expressed as fold induction with respect to cells transfected with empty expression vector, whose value was taken as 1. Similar results were obtained in three additional experiments. The bottom gel shows expression of HA-tagged ERK6 in lysates from the indicated samples upon analysis by Western blot by use of a specific anti-HA antibody. (B) The expression levels of MKK3 AA and GST-tagged MKK6 KR in extracts from transfected cells were determined by Western blotting by use of anti-MKK3 and anti-GST antibodies. (MW) Molecular mass in kilodaltons. (C) NIH-3T3 cells were transfected as above with pSREmutL (0.1 μg per plate) or pJLuc together with pRL-null reporter plasmid DNAs (0.01 μg per plate) along with RhoA QL alone or in combination with MKK3 AA, MKK6 KR, MEK1 AA, MKK7 KR, MKK4 KR, and MEK5 AA (1μg), as indicated. Transfection with MKK3 EE served as a control. Twenty-four hours after transfection, cells were collected and the lysates were assayed for dual luciferase activities. The data represent firefly luciferase activity normalized by Renilla luciferase activity present in each sample expressed as fold induction relative to control, and are the average ± s.e. of triplicate samples from a typical experiment. Similar results were obtained in four independent experiments.

RhoA stimulates the transcriptional activity of MEF2A and ATF2 through the MKK3/6-ERK6 (p38γ) pathway. (A) Cells were transfected with pcDNA3.1/GS-MEF2A (V5-tagged) or pCGN-ATF2 (HA-tagged) alone or in combination with RhoA QL, MKK3 (0.5 μg), MKK3 EE + ERK6, MKK3 EE + p38α, and MEK5  + ERK5 (0.5 μg each). Lysates were immunoprecipitated with mouse monoclonal anti-V5 and anti-HA antibodies and used in Western blotting experiments. Phospho-MEF2A and phospho-ATF2 were detected by a mouse monoclonal anti-P-Thr-Pro antibody (1:4000) and a rabbit polyclonal P-ATF2 antibody (1:1000). V5-MEF2 and HA-ATF2 in total lysates were detected by anti V5 and HA antibodies (1:1000 each). (B) The expression of the Gal4-fusion proteins containing the transactivation domains of MEF2A (amino acids 266–360) and ATF2 (amino acids 1–96) was determined by Western blotting by use of an anti-Gal4 (DBD) monoclonal antibody. (MW) Molecular mass in kilodaltons. (C) NIH-3T3 cells were cotransfected with the pcDNAIII–Gal4–MEF2A and pcDNAIII–Gal4–ATF2 chimeric molecules (0.05 μg) along with pGal4–Luc and pRL-null (0.1 and 0.01 μg per plate each). Expression vectors for RhoA QL (1 μg), MEKK (0.5 μg), and MEK5 DD + ERK5 (0.5 μg each) were included in the transfection mixtures, as indicated. Twenty-four hours after transfections, cells were collected and the lysates were assayed for dual luciferase activities. Total amount of plasmid DNAs was adjusted with empty vector. The data represents firefly luciferase activity normalized by Renilla luciferase activity present in each sample expressed as fold induction relative to their respective controls (pcDNAIII–Gal4–MEF2A or ATF2 alone) whose values were taken as 1. Values are the average ± s.e. of triplicate samples from a typical experiment. Nearly identical results were obtained in three additional experiments. (D) Cells were transfected with pDNAIII–Gal4–MEF2A and pDNAIII–Gal4–ATF2 pGal4–Luc and pRL-null reporter plasmid DNAs as described above along with RhoA QL, MEK5 DD + ERK5 or MEKK alone or in combination with MKK3 AA or MKK6 KR as indicated. Twenty-four hours later, cells were lysed and assayed for dual luciferase activities. The data represent firefly luciferase activity normalized by Renilla activity present in each sample expressed as fold induction relative to their corresponding controls (pcDNAIII–Gal4–MEF2A or pcDNAIII–Gal4–ATF2 alone), whose values were taken as 1. Results are the average ± s.e. of triplicate samples from a typical experiment. Similar results were obtained in three additional experiments.

Rho induces c-jun expression and the activation of the c-jun promoter through distinct regulatory elements. (A) NIH-3T3 cells were transfected with pcDNAIII-β-gal and RhoA QL as indicated 24 h after serum starvation, left untreated or treated with 10 μM LPA for 30 min, and total RNA extracted. Samples containing 20 μg of total RNA were fractionated and analyzed by Northern blotting by use of ³²P-labeled murine c-jun cDNA as a probe. Total RNA present in each lane was assessed to be equivalent by ethidium bromide staining of ribosomal RNAs. The autoradiogram corresponds to a representative experiment. (B) Schematic representation of the c-jun promoter depicting the relative position of response elements. (C) NIH-3T3 cells were cotransfected with the reporter plasmids pcDNAIII-β-gal (0.5 μg) together with pJC6, pJTX, pJSX, or pJSTX (0.1 μg per plate), as indicated. Crosses indicate the sites of point mutations in the jAP1 (pJTX) and the MEF2 (pJSX) binding sites. The plasmid pJSTX contains mutations for both the jAP1 and MEF2 sites. MEKK (0.5 μg), MEK5 + ERK5 (0.5 μg each), and RhoA QL (1 μg) were included in the transfection mixtures, as indicated. Twenty-four hours later, cells were collected and the lysates were assayed for CAT and β-galactosidase activities. The data represent CAT activity normalized by the β-galactosidase activity present in each sample, expressed as the percentage of the pJC6 induction elicited by the activating molecules. (D) Cells were transfected as above with the reporter plasmids pjAP1–MEF2, pjAP1m–MEF2, pjAP1–MEF2m, or pjAP1m–MEF2m (0.05 μg each) along with pRL-null (0.01 μg per plate). Crosses indicate mutations on the jAP1 or MEF2 sites. Twenty-four hours after transfection, cells were collected and the lysates were assayed for dual luciferase activities. The data represent firefly activity normalized by Renilla luciferase activity present in each sample, expressed as the percentage of the pjAP1–MEF2 induction elicited by RhoA QL. (E) Cells were transfected with the reporter plasmids pjAP1–MEF2, pMEF2, or pjAP1 (0.05 μg each) and pRL-null (0.01 μg per plate). Empty expression vector (control) or expressing RhoA QL (1 μg) was added to the transfection mix for each reporter. Dual luciferase activities were assayed as indicated above. The data represent firefly luciferase activity normalized by Renilla luciferase activity present in each sample expressed as fold induction relative to controls for each reporter, whose values were taken as 1. All values are the average ± s.e. of triplicate samples from a typical experiment. In each case, similar results were obtained in three additional experiments.