Loss of oncogenic H-ras-induced cell cycle arrest and p38 mitogen-activated protein kinase activation by disruption of Gadd45a

Abstract

The activation of p53 is a guardian mechanism to protect primary cells from malignant transformation; however, the details of the activation of p53 by oncogenic stress are still incomplete. In this report we show that in Gadd45a(-/-) mouse embryo fibroblasts (MEF), overexpression of H-ras activates extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) but not p38 kinase, and this correlates with the loss of H-ras-induced cell cycle arrest (premature senescence). Inhibition of p38 mitogen-activated protein kinase (MAPK) activation correlated with the deregulation of p53 activation, and both a p38 MAPK chemical inhibitor and the expression of a dominant-negative p38alpha inhibited p53 activation in the presence of H-ras in wild-type MEF. p38, but not ERK or JNK, was found in a complex with Gadd45 proteins. The region of interaction was mapped to amino acids 71 to 96, and the central portion (amino acids 71 to 124) of Gadd45a was required for p38 MAPK activation in the presence of H-ras. Our results indicate that this Gadd45/p38 pathway plays an important role in preventing oncogene-induced growth at least in part by regulating the p53 tumor suppressor.

FIG. 1.

H-ras-induced permanent cell cycle arrest is abolished in Gadd45a^−/− MEF. wt, Gadd45a^−/−, and Trp53^−/− MEF were infected with retrovirus expressing oncogenic H-ras and selected for 4 days with 2 μg of puromycin (puro)/ml. The numbers of BrdU-positive cells (S-phase cells [S]) (left panel) and growth rates (right panel) after selection in puromycin are shown.

FIG. 2.

Disruption of Gadd45a abolishes p38 activation after H-ras overexpression. (A) wt and Gadd45a^−/− MEF were infected with H-ras-expressing retrovirus, and activities of ERK, JNK, and p38 were analyzed, with MBP, GST-Jun, and GST-ATF2 as substrates, respectively. puro, puromycin. (B) At the same time that the protein extracts were obtained, the levels of p16/Ink4a, p53, and p21/Waf1 were determined. (C) MEF were infected with either puromycin- or H-ras-expressing retroviruses, and 5 days after selection with puromycin, mRNA was purified and the levels of p53-inducible genes (those encoding p21/Waf1, XPC, and ATF2) were analyzed by using a quantitative filter hybridization procedure (29). The relative induction ratio was obtained after dividing the relative level of mRNA after infection with H-ras retrovirus by the level of mRNA after infection with puromycin vector alone. (D) The levels of Gadd45a and p19/ARF mRNA in wt and Gadd45a^−/− MEF on day 5 after retroviral infection were measured by Northern blotting. GAPD was included as a loading control.

FIG. 3.

Gadd45a and p38 are required for p53 activation after H-ras overexpression. (A) wt and Gadd45a^−/− MEF were incubated in the presence of a MEK1 (50 μM PD98059) or p38 (10 μM SB202190) inhibitor, and protein extracts were obtained on day 5 after selection (see Materials and Methods). The levels of p16/Ink4a, p21/Waf1, and p53 proteins were analyzed. DMSO, dimethyl sulfoxide; puro, puromycin. (B) wt and Gadd45a^−/− MEF were cotransfected with p53RE-CAT reporter plasmid and expression vectors containing either puromycin or H-ras. Some cells were additionally transfected with a dominant-negative p38α vector (p38DN). Four days later, cells were treated with either a MEK1 (PD90859) or p38 (SB202190) inhibitor, and CAT assays were carried out 12 h later. (C) wt and Gadd45a^−/− MEF were cotransfected with p53RE-CAT reporter plasmid and expression vectors containing either puromycin or MKK6(E). Four days later, CAT activity was analyzed, and representative results are shown. Relative induction, as measured by increased CAT activity, was consistently twofold or greater in wt MEF compared to that in Gadd45a^−/− MEF.

FIG. 4.

Gadd45a associates with p38 both in vivo and in vitro. (A) Total lysates from 33-F cells stably expressing Flag-tagged Gadd45a and from control cell lines (approximately 10 mg of total protein) were immunoprecipitated (IP) with anti-Flag immunomatrix followed by immunoblot (IB) analysis of precipitates with anti-p38 (top panel), anti-JNK (second panel), anti-ERK2 (third panel), and anti-Gadd45a (bottom panel) polyclonal Abs. The lysates (approximately 100 μg per lane) were also analyzed directly by immunoblotting (two right lanes of each panel). (B) ³⁵S-labeled Flag-p38α, obtained by coupled transcription-translation, was pulled down by GST-Gadd45a or GST beads and visualized by autoradiography. (C) ³⁵S-labeled Flag-p38a and HA-Gadd45a obtained by coupled transcription-translation were mixed and precipitated with anti-Flag immunomatrix. Precipitates were analyzed either by using autoradiography (left panel) or by probing with anti-HA Ab (right panel). (D) Another experiment was carried out as described in the legend to Fig. 2C, left panel, except that anti-HA immunomatrix was used for precipitation. As controls for the experiments described in the legends to panels C and D, the ³⁵S-labeled protein was mixed with in vitro transcription-translation labeling mixtures containing only vector.

FIG. 5.

The central region of Gadd45a protein is involved in the interaction with p38. RKO cells were transiently transfected with Flag-p38α and Myc-tagged Gadd45a deletion proteins (1-71)Gadd45a, (1-96)Gadd45a, (1-124)Gadd45a, (48-132)Gadd45a, and (48-165)Gadd45a as well as full-size Myc-tagged Gadd45a. The total lysates of transfected cells were immunoprecipitated (IP) with either anti-Myc (A and C, top panels) or anti-Flag (B, top panel) immunomatrix, and the presence of deletion mutant proteins in precipitate was analyzed by immunoblotting (IB) with anti-Flag or anti-Myc Ab, respectively. The abundant expression of Flag-tagged p38α (A and C, middle panels) and Myc-tagged Gadd45a proteins (B, middle panel, and C, bottom panel) in total lysates was confirmed by immunoblotting with anti-Flag and anti-Myc Abs, respectively. The blots presented at the top of panels A and B were also reprobed with anti-Myc (bands that correspond to various forms of Gadd45a are marked with asterisks in lanes where there is more than one band) and anti-Flag Abs, respectively (A and B, bottom panels) to confirm comparable levels of protein in the primary immunoprecipitates. Cotransfection with Myc vector and Flag-p38α or Flag vector and full-size Myc-Gadd45a was used as for controls.

FIG. 5.

The central region of Gadd45a protein is involved in the interaction with p38. RKO cells were transiently transfected with Flag-p38α and Myc-tagged Gadd45a deletion proteins (1-71)Gadd45a, (1-96)Gadd45a, (1-124)Gadd45a, (48-132)Gadd45a, and (48-165)Gadd45a as well as full-size Myc-tagged Gadd45a. The total lysates of transfected cells were immunoprecipitated (IP) with either anti-Myc (A and C, top panels) or anti-Flag (B, top panel) immunomatrix, and the presence of deletion mutant proteins in precipitate was analyzed by immunoblotting (IB) with anti-Flag or anti-Myc Ab, respectively. The abundant expression of Flag-tagged p38α (A and C, middle panels) and Myc-tagged Gadd45a proteins (B, middle panel, and C, bottom panel) in total lysates was confirmed by immunoblotting with anti-Flag and anti-Myc Abs, respectively. The blots presented at the top of panels A and B were also reprobed with anti-Myc (bands that correspond to various forms of Gadd45a are marked with asterisks in lanes where there is more than one band) and anti-Flag Abs, respectively (A and B, bottom panels) to confirm comparable levels of protein in the primary immunoprecipitates. Cotransfection with Myc vector and Flag-p38α or Flag vector and full-size Myc-Gadd45a was used as for controls.

FIG. 6.

The central region of Gadd45a is required for p38 activation by H-ras. Gadd45a^−/− MEF were cotransfected with p38α-Flag, different forms of Myc-Gadd45a, and plasmids with either puromycin (puro) or H-ras (1:3:9 ratio). Two days later, p38 activity was analyzed after immunoprecipitation with anti-Flag Ab in an in vitro kinase reaction with GST-ATF2 as the substrate.

FIG. 7.

(A) Schematic diagram of regions involved in protein associations as determined by analysis of Gadd45a deletion proteins (see reference for more detail). Results for p38 binding are taken from the experiment described in the legend to Fig. 5 and those for p38 activation by H-ras (gray bar) are from the experiment described in the legend to Fig. 6. (B) Model for growth-inhibitory responses triggered by oncogenic Ras and role of Gadd45/p38 pathway in activation of p53. The positive-feedback loop is designated by the gray circle with arrowheads (see text for further explanation). Some other relevant components, including Wip1, which is not induced by H-ras (9), are shown. To simplify the diagram, multiple components, such as MTK1, which also interacts with Gadd45 proteins (37, 47), have been omitted.