Abstract
Rad53 is an essential protein kinase governing DNA damage and replication stress checkpoints in budding yeast. It also appears to be involved in cellular morphogenesis processes. Mass spectrometry analyses revealed that Rad53 is phosphorylated at multiple SQ/TQ and at SP/TP residues, which are typical consensus sites for phosphatidylinositol 3-kinase-related kinases and CDKs, respectively. Here we show that Clb-CDK1 phosphorylates Rad53 at Ser(774) in metaphase. This phosphorylation event does not influence the DNA damage and replication checkpoint roles of Rad53, and it is independent of the spindle assembly checkpoint network. Moreover, the Ser-to-Asp mutation, mimicking a constitutive phosphorylation state at site 774, causes sensitivity to calcofluor, supporting a functional linkage between Rad53 and cellular morphogenesis.
Highlights
Saccharomyces cerevisiae Rad[53] is a serine/threonine/tyrosine kinase, and it is well established that Rad[53] family members, including the Chk[2] protein kinase in human cells, play a central role in the signal transduction pathway activated in response to DNA lesions and help prevent genome rearrangements and cancer (1, 2)
It is unlikely that Rad[53] kinase activity plays a role in response to spindle damage because Rad[53], prepared from cells treated with nocodazole, does not exhibit autophosphorylation activity, as determined by in situ kinase assay (8); rad53⌬ cells are not sensitive to spindle-damaging agents
These findings suggested that the CDK1 cell cycle kinase may directly phosphorylate Rad[53] and influence its activity, as supported by recent observations implicating CDK1 in DNA damage checkpoint activation (12, 13)
Summary
Saccharomyces cerevisiae Rad[53] is a serine/threonine/tyrosine kinase, and it is well established that Rad[53] family members, including the Chk[2] protein kinase in human cells, play a central role in the signal transduction pathway activated in response to DNA lesions and help prevent genome rearrangements and cancer (1, 2). None of the CDK1-dependent phosphorylation events occurring on Rad[53] at metaphase appear to have any role in promoting Rad[53] activation in response to DNA damage or replication stress.
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