Abstract
Spy1 is the originally identified member of the Speedy/Ringo family of vertebrate cell cycle regulators, which can control cell proliferation and survival through the atypical activation of cyclin-dependent kinases. Here we report a role for Spy1 in apoptosis and checkpoint activation in response to UV irradiation. Using an inducible system allowing for regulated expression of Spy1, we show that Spy1 expression prevents activation of caspase-3 and suppresses apoptosis in response to UV irradiation. Spy1 expression also allows for UV irradiation-resistant DNA synthesis and permits cells to progress into mitosis, as demonstrated by phosphorylation on histone H3, indicating that Spy1 expression can inhibit the S-phase/replication and G2/M checkpoints. We demonstrate that Spy1 expression inhibits phosphorylation of Chk1, RPA, and histone H2A.X, which may directly contribute to the decrease in apoptosis and checkpoint bypass. Furthermore, mutation of the conserved Speedy/Ringo box, known to mediate interaction with CDK2, abrogates the ability of Spy1 to inhibit apoptosis and the phosphorylation of Chk1 and RPA. The data presented indicate that Spy1 expression allows cells to evade checkpoints and apoptosis and suggest that Spy1 regulation of CDK2 is important for the response to DNA damage.
Highlights
Spy1-expressing U2OS cells (Spy1) is the originally identified member of the Speedy/Ringo family of vertebrate cell cycle regulators, which can control cell proliferation and survival through the atypical activation of cyclin-dependent kinases
To examine whether Spy1 requires CDK2 binding to mediate responses to UV irradiation, we created a construct based on mutations previously shown to prevent CDK2 binding and activation [6, 7], mutating the five glutamate residues and the one aspartate residue within the Speedy/Ringo box to glutamine and asparagine, respectively (Spy1S/R box)
We report here a role for Spy1 expression in checkpoint activation and apoptosis
Summary
S/R, Speedy/Ringo; UVDS, UV irradiation-resistant DNA synthesis; RXR, retinoid X receptor; Spy1:U2OS, Spy1-expressing U2OS cells; PBS, phosphate-buffered saline; CDK, cyclin-dependent kinase. Spy expression prevents the activation of checkpoint proteins, such as Chk and the histone variant H2A.X, in response to UV irradiation and prevents other ATR-mediated signaling events, such as the phosphorylation of RPA32 on its N terminus. Mutations within the S/R box of Spy, known to mediate the interaction with and activation of CDK2 [6, 7], prevent these effects of Spy. Mutations within the S/R box of Spy, known to mediate the interaction with and activation of CDK2 [6, 7], prevent these effects of Spy1 Expression of this mutant does not suppress the phosphorylation of Chk or RPA32 in response to UV-induced DNA damage, indicating a specific role for Spy and Spy1-associated CDK2 activity in the regulation of the DNA damage response. The expression of Spy facilitates the evasion of checkpoints and apoptotic pathways that are activated in response to DNA damage
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