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. 2006 Mar;26(6):2202-14.
doi: 10.1128/MCB.26.6.2202-2214.2006.

The molecular scaffold kinase suppressor of Ras 1 is a modifier of RasV12-induced and replicative senescence

Robert L Kortum  1 , Heidi J Johnson, Diane L Costanzo, Deanna J Volle, Gina L Razidlo, Angela M Fusello, Andrey S Shaw, Robert E Lewis
Affiliations

The molecular scaffold kinase suppressor of Ras 1 is a modifier of RasV12-induced and replicative senescence

Robert L Kortum et al. Mol Cell Biol. 2006 Mar.

Abstract

In primary mouse embryo fibroblasts (MEFs), oncogenic Ras induces growth arrest via Raf/MEK/extracellular signal-regulated kinase (ERK)-mediated activation of the p19ARF/p53 and INK4/Rb tumor suppressor pathways. Ablation of these same pathways causes spontaneous immortalization in MEFs, and oncogenic transformation by Ras requires ablation of one or both of these pathways. We show that Kinase Suppressor of Ras 1 (KSR1), a molecular scaffold for the Raf/MEK/ERK cascade, is necessary for RasV12-induced senescence, and its disruption enhances primary MEF immortalization. RasV12 failed to induce p53, p19ARF, p16INK4a, and p15INK4b expression in KSR1-/- MEFs and increased proliferation instead of causing growth arrest. Reintroduction of wild-type KSR1, but not a mutated KSR1 construct unable to bind activated ERK, rescued RasV12-induced senescence. On continuous culture, deletion of KSR1 accelerated the establishment of spontaneously immortalized cultures and increased the proportion of cultures escaping replicative crisis. Despite enhancing escape from both RasV12-induced and replicative senescence, however, both primary and immortalized KSR1-/- MEFs are completely resistant to RasV12-induced transformation. These data show that escape from senescence is not necessarily a precursor for oncogenic transformation. Furthermore, these data indicate that KSR1 is a member of a unique class of proteins whose deletion blocks both senescence and transformation.

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Figures

FIG. 1.
FIG. 1.
KSR1 is necessary for RasV12-induced senescence in primary MEFs. (A) Passage 4 to 6 KSR1−/− (squares) or KSR1+/+ (triangles) MEFs expressing RasV12 (closed) or control (open) vectors were seeded at 5 × 104 cells per 35-mm dish. Separate duplicate dishes were assessed for cell number every 48 h on a Beckman Coulter Counter. Data are expressed as means ± standard deviations (SD) of three independent experiments. (B) Photomicrographs (10×) of cells analyzed in panel A. (C) Photomicrographs (10×) of RasV12-expressing cells analyzed in panel A and stained to visualize SA β-galactosidase activity as described in Materials and Methods. (D) Western blot analysis of whole-cell extracts prepared from passage 4 to 6 KSR1−/− and KSR1+/+ MEFs expressing RasV12 or control vector. Lysates were probed with the indicated antibodies to detect induction and activation of each protein by RasV12. Actin and tubulin were used to demonstrate equal loading of each sample, and Ras and KSR1 expression results are shown as controls. Data are representative of four independent experiments.
FIG. 2.
FIG. 2.
Induction of p53 activity by γ irradiation is independent of KSR1. Passage 4 KSR1−/− and KSR1+/+ MEFs were irradiated with 8 Gy, and whole-cell extracts were prepared at the indicated times after radiation exposure. Lysates were probed with the indicated antibodies to assess the induction of p53 activity. Actin was used to demonstrate equal loading of each sample. Data are representative of two independent experiments.
FIG. 3.
FIG. 3.
The KSR1-ERK interaction is required, but the KSR1-MEK interaction is dispensable, for RasV12-induced senescence in KSR1−/− MEFs. Passage 5 KSR1−/− MEFs were infected with recombinant retrovirus encoding KSR1, a KSR1 construct unable to bind activated ERK (KSR1.FSFP/AAAP) or a KSR1 construct unable to bind MEK (KSR1.C809Y), and RasV12 or a control virus. Low levels of KSR1 expression were selected by FACS analysis for levels of GFP expression previously shown to correlate with 1× to 5× KSR1+/+ levels. (A) Proliferation in passage 5 KSR1−/− MEFs expressing the indicated KSR1 construct and RasV12 (closed squares) or control (open diamonds) vectors. Separate duplicate dishes were assessed for cell number every 48 h on a Beckman Coulter Counter. Data are expressed as means ± SD of three independent experiments. (B) Photomicrographs (10×) of cells analyzed in panel A and stained to visualize SA β-galactosidase activity as described in Materials and Methods. (C) Quantification of cells analyzed in panel A and stained to visualize SA β-galactosidase (β-Gal) activity as described in Materials and Methods. Separate triplicate dishes were quantified for each cell line, with a minimum of 50 cells quantified per well. Data are expressed as mean percentages of cells  ± SD from three independent experiments. (D) Western blot analysis of whole-cell extracts prepared from passage 5 KSR1−/− MEFs expressing the indicated KSR1 constructs and RasV12 or control vector. Lysates were probed with the indicated antibodies to detect induction and activation of each protein by RasV12. Actin and tubulin were used to demonstrate equal loading of each sample, and Ras and KSR1 expression results are shown as controls. Data are representative of four independent experiments.
FIG. 4.
FIG. 4.
The KSR1-ERK interaction is required, but the KSR1-MEK interaction is dispensable, for RasV12-induced transformation and PDGF-induced ERK activation in immortal KSR1−/− MEFs. Immortal KSR1−/− MEFs were infected with recombinant retrovirus encoding KSR1, KSR1.FSFP/AAAP, or KSR1.C809Y and RasV12 or a control virus. Low levels of KSR1 expression were selected by FACS analysis for levels of GFP expression previously shown to correlate with 1× to 5× KSR1+/+ levels. (A) Cells were assessed for transformation by growth on soft agar as described in Materials and Methods. Data are expressed as means ± SD from two independent experiments. (B) Triplicate wells expressing the indicated KSR1 construct were treated with 25 ng of PDGF/ml for the indicated times, and ERK1/2 phosphorylation levels were determined by in-cell Western blotting for ERK1 and pERK1/2 with a Li-Cor Odyssey system. Data are expressed as ratios of pERK1/2 to ERK1. Data are expressed as means ± SD.
FIG. 5.
FIG. 5.
KSR1 is not required for, but enhances, MEKEE-induced transformation in immortal KSR1−/− MEFs. Immortal KSR1−/− and KSR1+/+ MEFs were infected with recombinant retrovirus encoding MEKEE, RasV12, or a control virus. (A) Western blot analysis of whole-cell extracts prepared from immortal KSR1−/− and KSR1+/+ MEFs expressing MEKEE, RasV12, or control vector. Lysates were probed with antibodies against pERK1/2 and ERK1/2 to detect activation of ERK by MEKEE or RasV12. Actin was used to demonstrate equal loading of each sample, and expression blots of Ras, MEK1, and KSR1 are shown as controls. For MEK1 blots, low exposures are shown to indicate cells that have received the MEKEE transgene and not expression of endogenous MEK1. Data are representative of four independent experiments. (B and C) Cells were assessed for transformation by growth on soft agar (B) and focus formation (C) as described in Materials and Methods. Data are expressed as means ± SD from two independent experiments.
FIG. 6.
FIG. 6.
KSR1 is necessary for MEKEE-induced senescence in primary MEFs. (A) Western blot analysis of whole-cell extracts prepared from passage 4 to 6 KSR1−/− and KSR1+/+ MEFs expressing MEKEE, RasV12, or control vector. Lysates were probed with the indicated antibodies to detect induction and activation of each protein by MEKEE or RasV12. Actin was used to demonstrate equal loading of each sample, and expression blots of Ras, MEK1, and KSR1 are shown as controls. For MEK1 blots, low exposures are shown to indicate cells that have received the MEKEE transgene and not expression of endogenous MEK1. (B) Passage 4 to 6 KSR1−/− (squares) or KSR1+/+ (triangles) MEFs expressing RasV12 (closed), MEKEE (gray), or control (open) vectors were seeded at 1 × 104 cells per well in a 24-well plate. Separate duplicate dishes were assessed for cell number every 72 h by trypan blue exclusion. Data are expressed as means ± SD from two independent experiments. (C) Photomicrographs (10×) of cells analyzed in panel A and stained to visualize SA β-galactosidase activity as described in Materials and Methods. (D) Quantification of cells analyzed in panel A and stained to visualize SA β-galactosidase activity as described in Materials and Methods. Separate triplicate wells were quantified for each cell line, with a minimum of 100 cells quantified per well. Data are expressed as means ± SD from four independent experiments.
FIG. 7.
FIG. 7.
Loss of KSR1 allows for early immortalization of primary mouse embryo fibroblasts. (A) Passage 3 KSR1−/− and KSR1+/+ MEFs were passaged by a 3T9 protocol until immortalized populations were obtained. Data are means ± SD of four embryos of each genotype. (B) At passages 5, 10, 15, and 18, 2 × 104 MEFs were plated in 35-mm dishes and counted either daily or every 72 h to assess low-density growth. Data are means ± SD of three embryos of each genotype. (C) KSR1+/+, KSR1+/−, and KSR1−/− MEFs at passage 9 were seeded at 2 × 104 cells/60-mm dish and fed twice weekly for 4 weeks. Colonies consisting of at least 16 cells that arose from MEFs of each genotype were counted. Data are expressed as means ± SD from two embryos of each genotype.
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