UC San Diego

While the prevailing dogma states that cell cycle progression is associated with the activation of the cyclin-dependent kinases (CDKs), and cell cycle arrest and apoptosis with the inhibition of CDKs, there is growing evidence that regulation of CDK activity is not that straight forward and the balance between proliferation and checkpoint arrest is intricately weaved. This suggests that specific mechanisms of regulation will exist to accomplish this apparent paradoxical regulation. The Speedy/RINGO family of CDK regulators appears to play a role in this specialized regulation. Spy1 is the originally identified member of the Speedy/RINGO family, and has been shown to atypically activate CDKs, even in the face of inhibition. Here I report a role for Spy1- regulation of CDK2 in apoptosis and checkpoint activation in response to Ultraviolet (UV) irradiation. Using an inducible system allowing for regulated expression of Spy1, I show that Spy1 expression suppresses apoptosis in a p53 and p21 dependent fashion. Spy1 expression also allows for UV irradiation resistant DNA synthesis (UVDS) and inhibits the S- and G2/M- checkpoints through the inhibition of checkpoint response proteins. This leads to DNA damage tolerance and prevention of repair of UV-induced cyclobutane pyrimidine dimers through suppression of nucleotide excision repair. Furthermore, knockdown of Spy1 activates intrinsic damage responses indicating that Spy1 is required to promote tolerance of damage that may occur endogenously or exogenously. p27Kip1 binds to cyclin E/ CDK2 complexes inhibiting its kinase activity, yet certain critical events trigger CDK2 to phosphorylate its own inhibitor leading to p27 degradation and cell cycle progression. Utilizing recombinant proteins, we demonstrate that Spy1 activates CDK2 to phosphorylate p27 at T187 in vitro. Spy1 expression in vivo leads to enhanced T187 phosphorylation and degradation of endogenous p27 in late G1 and throughout S-phase. The mechanisms of action conferred by the Speedy/RINGO family represent novel modes by which CDKs are regulated. This type of regulation may be important at cell cycle transitions, in the tolerance of normal intrinsic damage, or in response to exogenous DNA damage