Abstract
KSR1 (kinase suppressor of Ras 1) is a molecular scaffold and positive regulator of the Raf/MEK/ERK phosphorylation cascade. KSR1 is required for maximal ERK activation induced by growth factors and by some cytotoxic agents. We show here that KSR1 is also required for maximal ERK activation induced by UV light, ionizing radiation, or the DNA interstrand cross-linking agent mitomycin C (MMC). We further demonstrate a role for KSR1 in the reinitiation of the cell cycle and proliferation following cell cycle arrest induced by MMC. Cells lacking KSR1 underwent but did not recover from MMC-induced G(2)/M arrest. Expression of KSR1 allowed KSR1(-/-) cells to re-enter the cell cycle following MMC treatment. However, cells expressing a mutated form of KSR1 unable to bind ERK did not recover from MMC-induced cell cycle arrest, demonstrating the requirement for the KSR1-ERK interaction. In addition, constitutive activation of ERK was not sufficient to promote cell cycle reinitiation in MMC-treated KSR1(-/-) cells. Only cells expressing KSR1 recovered from MMC-induced cell cycle arrest. Importantly, MMC-induced DNA damage was repaired in KSR1(-/-) cells, as determined by resolution of gamma-H2AX-containing foci. These data indicate that cell cycle reinitiation is not actively signaled in the absence of KSR1, even when DNA damage has been resolved. These data reveal a specific role for the molecular scaffold KSR1 and KSR1-mediated ERK signaling in the cellular response to DNA interstrand cross-links.
Highlights
MARCH 13, 2009 VOLUME 284 NUMBER 11 types of DNA damage [1]
In addition to mitogenic stimulation, ERK is activated in response to multiple types of DNA damage including UV photoproducts induced by UV irradiation [12], DNA interstrand cross-links (ICLs) generated by cisplatin and mitomycin C (MMC) [13,14,15], and double strand breaks (DSBs) introduced by IR, hydroxyurea, and etoposide (16 –18)
KSR1 is required for maximal ERK activation induced by growth factor stimulation and cisplatin treatment [7, 13]
Summary
Cell Culture and Generation of Stable Cell Lines—Mouse embryo fibroblasts (MEFs) were generated from day 13.5 embryos from KSR1Ϫ/Ϫ and KSR1ϩ/ϩ mice on a DBA1/LacJ background and were immortalized by a 3T9 protocol [26]. MMC was removed by washing four times with PBS, and normal culture medium was replaced. MMC was removed by washing cells with PBS four times, and normal culture medium was replaced. The cells were analyzed using phase contrast and fluorescent microscopy (filter set UV-2E/C; excitation 330 – 380 nm, dichromatic mirror 400, barrier filter emission 435– 485 nm) and were scored as having one or two nuclei. Binucleated cells were those that had entered mitosis and were arrested prior to cytokinesis by cytochalasin B. MMC was removed by washing four times with PBS, and fresh medium was replaced. Statistical analyses were performed using a Student’s t test where p Ͻ 0.05 was considered significant
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