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formation of procentrioles in S phase is dependent on the activity of CDK2. The
Sluder laboratory used Xenopus egg extracts and sperm nuclei to demonstrate that
cells incubated with control egg extracts could form multiple centrosomes fol-
lowing S phase arrest with the DNA polymerase inhibitor aphidicolin. However,
cells incubated with egg extracts containing recombinant Xic-1 p27 , a CDK2-cyclin
E inhibitor, did not undergo repeated rounds of centrosome replication (Hinchcliffe
et al. 1999 ). The Stearns lab used S phase-arrested Xenopus embryos in which
individual blastomeres were microinjected with p21 or p27. Compared to control
non-injected blastomeres that underwent multiple rounds of centrosome doubling,
repeated centrosome doubling was not observed in blastomeres from the same
embryo that had been microinjected with the CDK2 inhibitors (Lacey et al. 1999 ).
Similar observations were made in mammalian cells. Nishida and colleagues
demonstrated that Chinese hamster ovary (CHO) cells that were arrested in S
phase by hydroxyurea treatment had high levels of CDK2 and could undergo
repeated centrosome replication, whilst cells arrested in G1 phase by mimosine
treatment had low levels of CDK2 and could not undergo multiple rounds of
centrosome replication (Matsumoto et al. 1999 ). In addition, blocking CDK2
activity in S phase-arrested cells by treatment with roscovitine or butyrolactone or
expression of p21 significantly inhibited the formation of extra centrioles (Mat-
sumoto et al. 1999 ). The Nigg laboratory used hydroxyurea-arrested CHO cells to
demonstrate a role for the Retinoblastoma (Rb)-E2F transcription factor pathway
in centrosome reduplication (Meraldi et al. 1999 ). They found that CDK2 in
complex with cyclin A, rather than cyclin E, was necessary for centrosome
reduplication in this system (Meraldi et al. 1999 ).
More recent evidence for a requirement for CDK2 in centrosome replication
came from a study on p53-/- CHO cells, which formed multiple centrin foci
following G1/S arrest with hydroxyurea. These centrin foci were found to repre-
sent procentrioles that could mature into functional centrosomes and were
dependent on the presence of active CDK2 (Prosser et al. 2009 ). We have also
found that overexpression of the CDK-cyclin activator CDC25B in G1/S-arrested
U2OS cells results in multiple centrosomes formed within a single S phase
(Boutros et al. 2007 ). We found that centrosome re-replication in these cells could
be blocked by specific inhibition of CDK2 but not CDK1 activity (R Boutros,
unpublished data).
CDK2 in complex with cyclin E and/or cyclin A therefore appears to be
required for the initiation of centrosome replication. However, the exact mecha-
nism for this is unclear. And the findings that CDK2 knockout mice are viable
suggest that other kinases, most likely CDK1, can take on the role of CDK2 in both
centrosome replication and DNA replication (Berthet et al. 2003 ; Ortega et al.
2003 ). Nonetheless, a number of kinase targets for CDK2-cyclins E/A in cen-
trosome replication have been identified to date, which suggest that CDK2 exerts
its effects on centrosome replication through the timely phosphorylation of its
substrates. The polo kinase 4 (PLK4), also essential for centrosome replication,
functions in co-operation with CDK2 (Habedanck et al. 2005 ).
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