Raf-MEK-Erk cascade in anoikis is controlled by Rac1 and Cdc42 via Akt - PubMed (original) (raw)

Raf-MEK-Erk cascade in anoikis is controlled by Rac1 and Cdc42 via Akt

O Zugasti et al. Mol Cell Biol. 2001 Oct.

Abstract

Signals from the extracellular matrix are essential for the survival of many cell types. Dominant-negative mutants of two members of Rho family GTPases, Rac1 and Cdc42, mimic the loss of anchorage in primary mouse fibroblasts and are potent inducers of apoptosis. This pathway of cell death requires the activation of both the p53 tumor suppressor and the extracellular signal-regulated mitogen-activated protein kinases (Erks). Here we characterize the proapoptotic Erk signal and show that it differs from the classically observed survival-promoting one by the intensity of the kinase activation. The disappearance of the GTP-bound forms of Rac1 and Cdc42 gives rise to proapoptotic, moderate activation of the Raf-MEK-Erk cascade via a signaling pathway involving the kinases phosphatidlyinositol 3-kinase and Akt. Moreover, concomitant activation of p53 and inhibition of Akt are both necessary and sufficient to signal anoikis in primary fibroblasts. Our data demonstrate that the GTPases of the Rho family control three major components of cellular signal transduction, namely, p53, Akt, and Erks, which collaborate in the induction of apoptosis due to the loss of anchorage.

PubMed Disclaimer

Figures

FIG. 1

Loss of anchorage leads to inactivation of Ras family GTPases and apoptosis. Mouse primary fibroblasts were kept in suspension over a layer of 1% agar in culture medium supplemented with 10% FCS. At the indicated times, cells were collected and lysed, and the GTP-bound form of the GTPases was precipitated as described in Materials and Methods. (A) Rac1-GTP precipitated with GST-PAK1 and total Rac1 present in the lysates were analyzed by immunoblotting with an anti-Rac1 antibody. The histogram represents the relative proportions of GTP-loaded Rac1, with the 0-h time point being arbitrarily set as 100% loading. (B) Cdc42 was analyzed as described in panel A with an anti-Cdc42 antibody. (C) Ras-GTP loading was assayed as described in panels A and B using GST-RalGDSRBD to bind the GTPase. (D) At the indicated times, cells were collected, and their viability was assayed by trypan blue exclusion. The results represent means and SDs from three experiments.

FIG. 2

Erk phosphorylation status in anchorage-deprived MEFs. Cells were kept in suspension over a layer of 1% agar in culture medium supplemented with 10% FCS. (A) At the indicated times, cells were collected and lysed, and proteins were analyzed by immunoblotting. Activated Erk was detected by its phosphorylation status using an antibody specific for Erk1 and Erk2 doubly phosphorylated on Thr 202 and Tyr 204 (ERK-P). (B) At the indicated times, cells were collected, lysed, and electrophoresed in MBP-containing SDS-polyacrylamide gels. After renaturation, kinase activity was revealed by MBP phosphorylation in situ. Band intensities were quantified by scanning with a PhosphorImager and are represented as the ratio of activated Erk to total Erk, with the ratio found in exponentially grown adherent cells set as 100%. For the Western analysis, the results are presented as means and SDs from three experiments.

FIG. 3

Inhibition of Rac1 or Cdc42 signaling in adherent MEFs activates Erk. (A) Exponentially growing cells were cotransfected with vectors coding for an HA-tagged Erk2 construct and one of the following: dominant-negative form of Rac1 (Rac1N17), dominant-negative form of Cdc42 (Cdc42N17), N-WASP DN, constitutively active Ras (RasV12), or pcDNA3 as a negative control. At 24 h after transfection, cells were serum starved by culturing in medium supplemented with 1% FCS for 16 h and lysed, and HA-Erk2 was immunoprecipitated with anti-HA monoclonal antibody 12CA5. Kinase activity was assayed by in vitro phosphorylation of GST-MBP (upper panel). Erk2 activation was normalized (lower panel) relative to the total amount of transfected HA-Erk2, as quantitated by immunoblotting with antibody 12CA5 (middle panel). (B) Analysis like that in panel A, except that the MEK1 inhibitor PD98059 (20 μM) was included in the culture medium.

FIG. 4

Cytoplasmic retention of activated Erk inhibits RacN17- and Cdc42N17-induced apoptosis. (A) Adherent MEFs were transfected with myc epitope-tagged MKP3C/S. After 24 h, the cells were starved by culturing in medium containing 1% FCS for 24 h (panels a, c, e, and g) and then were stimulated with 20% serum for 10 min (panels f and h) or 2 h (panels b and d). Cells were stained with anti-ERK antibody (panels c and d) or anti-phospho-ERK antibody (panels g and h). The transfected cells (arrowheads) were identified by immunofluorescence detection of myc-MKP3C/S (panels a, b, e, and f). Bar, 10 μm. The results shown are representative of four independent experiments. (B) Adherent cells were cotransfected with the indicated constructs and truncated rat CD2 as a marker of transfection. Apoptosis in the transfected-cell population was assayed by the Annexin V binding assay. The results represent the means and SDs from two independent experiments performed in triplicate. (C) Western blot analysis of the levels of expression of the myc epitope-tagged GTPases with the different cotransfection combinations.

FIG. 5

Inhibition of Rac1 or Cdc42 signaling in adherent MEFs activates Erk via the MAPK kinase kinase Raf. (A to C and E) Exponentially growing cells were cotransfected with a vector coding for an HA-tagged Erk2 construct and vector pcDNA3 (control) or a plasmid encoding either a constitutively active form of Ras (RasV12) (A) or a dominant-negative form of Cdc42 (CdcN17) (B), Rac1 (RacN17) (C), or Ras (RasN17) (E). Dominant-negative mutants of mKsr-1 (CA5) and B-Raf (NB-Raf), which prevent MEK activation by Raf proteins, were included as indicated. At 24 h after transfection, the cells were serum starved by culturing in medium containing 1% FCS for 16 h, lysed, and immunoprecipitated with anti-HA monoclonal antibody 12CA5. Erk2 kinase activity was assayed by in vitro phosphorylation of GST-MBP and normalized relative to the total amount of transfected HA-Erk, quantitated by Western blotting with antibody 12CA5. (D) Cells were transfected with vectors encoding the indicated GTPases or empty vector pcDNA3 as a negative control and a construct coding for myc epitope-tagged B-Raf. After 24 h, cells were starved for serum for 16 h, lysed, and incubated with anti-myc monoclonal antibody 9E10. Raf activity in the immunoprecipitate was measured by an indirect kinase assay based on serial additions of purified GST-MEK1, GST-Erk2, and GST-MBP. The total amount of myc epitope-tagged B-Raf protein was estimated by immunoblotting with antibody 9E10 and was used to normalize the kinase activity. (F) Western blot analysis indicating the level of expression of the GTPases in the different cotransfections. Rac1N17 and Cdc42N17 are tagged with a myc epitope, RasV12 is tagged with HA, and RasN17 is not tagged.

FIG. 6

Akt activity is regulated by anchorage. (A) Exponentially growing MEFs were deprived of anchorage by culturing in complete medium over a layer of 1% agar. At the indicated time points, cells were collected, lysed, and analyzed by immunoblotting with polyclonal antibodies directed against Akt phosphorylated on serine 473 (P-Akt) or Akt (total Akt). A Western blot and a histogram from a representative experiment are shown. (B) Exponentially growing MEFs were cotransfected with an expression vector encoding a myristoylated form of Akt (Akt-myr) or an empty vector as a control. After 24 h, the cells were trypsinized and placed in suspension over a layer of 1% agar for 18 h. Cell viability was assayed by Annexin V-fluorescein isothiocyanate labeling in the transfected population identified by anti-CD2 staining. Results represent the mean and range for two independent experiments.

FIG. 7

Akt acts downstream of Rac1 and Cdc42. (A) HA-tagged Akt was expressed in exponentially growing MEFs together with a constitutively active form of Rac1 or Cdc42 or empty vector pcDNA3 as a control. Where indicated (+ LY), LY294002 (50 μM), a pharmacological inhibitor of PI(3)K, was added to the culture medium. After 24 h, HA-Akt was immunoprecipitated, and its activity was assayed in vitro by phosphorylation of histone H2B. (B) Adherent MEFs were cultured in complete medium containing 0.1 ng of C. difficile toxin B/ml. At the indicated times, the cells were lysed and analyzed by immunoblotting for the presence of Akt phosphorylated on serine 473 (P-Akt) and total Akt. The blots were scanned, and the relative level of phosphorlyated Akt was plotted as a percentage of the control (no toxin B treatment), arbitrarily set as 100%.

FIG. 8

Dominant-negative Akt mutants lead to Erk activation. (A) MEFs were cotransfected with either of two Akt dominant-negative mutants, Akt K179M or Akt T308AS473A (AktAA), or an empty vector as a negative control. The cells were processed as described in the legend to Fig. 3A. (B) Cells were transfected with an HA-tagged Erk2 construct, a dominant-negative mutant of either Rac1 or Cdc42, or a myristoylated form of Akt (Akt-myr). At 24 h after transfection, cells were lysed, HA-Erk was immunoprecipitated, and its kinase activity was measured. (C) MEFs were cotransfected with a truncated rat CD2 antigen and either pcDNA3 as a negative control or a dominant-negative form of Rac1 or Cdc42 (RacN17 or CdcN17). A myristoylated form of Akt (Akt-myr) was also included where indicated. Cell survival was quantitated by Annexin V labeling of the transfected subpopulation identified by anti-CD2 staining. Results represent means and SDs for three experiments. (D) Cells were transfected with HA-tagged Erk2 and the HA-tagged Akt K179M mutant with or without m-Ksr1 (CA5). Erk2 kinase activity was assayed as described in the legend to Fig. 3A.

FIG. 9

Inhibition of Akt, in conjunction with p53 activation, mimics apoptosis induction by dominant-negative forms of Rac1 and Cdc42. Exponentially growing MEFs were cotransfected with truncated rat CD2 and a combination of the following constructs, as indicated below the bars: a dominant-negative Cdc42 mutant (CdcN17), a dominant-negative Rac1 mutant (RacN17), wild-type p53 (p53), a kinase-dead Akt mutant (Akt179M), and an inactive Akt mutant (AktAA). Empty vector pcDNA3 was used as a control. Where indicated, the MEK inhibitor PD98059 (20 μM) was included in the culture medium. At 48 h after transfection, apoptotic cells in the transfected subpopulations were scored by Annexin V labeling. The data represent the means and SDs from three experiments.

FIG. 10

High-intensity Erk signaling protects cells from apoptosis induced by Rac1 or Cdc42 inhibition. (A) Exponentially growing MEFs were cotransfected with Raf-CAAX- and HA-tagged Erk2-encoding vectors. After 24 h, cells were collected, and Erk activity was measured by an in vitro kinase assay after immunoprecipitation with an anti-HA antibody. (B) Cells were transfected with the indicated constructs. After 48 h, apoptosis was measured by Annexin V labeling of the transfected subpopulation. Data represent means and SDs of six measurements from two independent experiments.

References

1. Alessi D R, Cohen P, Ashworth A, Cowley S, Leevers S J, Marshall C J. Assay and expression of mitogen-activated protein kinase, MAP kinase kinase, and Raf. Methods Enzymol. 1995;255:279–290. - PubMed
1. Aplin A E, Howe A K, Juliano R L. Cell adhesion molecules, signal transduction and cell growth. Curr Opin Cell Biol. 1999;11:737–744. - PubMed
1. Bar-Sagi D, Hall A. Ras and Rho GTPases: a family reunion. Cell. 2000;103:227–238. - PubMed
1. Brunet A, Roux D, Lenormand P, Dowd S, Keyse S, Pouyssegur J. Nuclear translocation of p42/p44 mitogen-activated protein kinase is required for growth factor-induced gene expression and cell cycle entry. EMBO J. 1999;18:664–674. - PMC - PubMed
1. Burack W R, Shaw A S. Signal transduction: hanging on a scaffold. Curr Opin Cell Biol. 2000;12:211–216. - PubMed

Raf-MEK-Erk cascade in anoikis is controlled by Rac1 and Cdc42 via Akt - PubMed (original) (raw)