Subcellular Proteomics
Reveals a Role for Nucleo-cytoplasmic Trafficking at the DNA Replication
Origin Activation Checkpoint
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Abstract
Depletion of DNA replication initiation factors such
as CDC7 kinase triggers the origin activation checkpoint in healthy
cells and leads to a protective cell cycle arrest at the G1 phase
of the mitotic cell division cycle. This protective mechanism is thought
to be defective in cancer cells. To investigate how this checkpoint
is activated and maintained in healthy cells, we conducted a quantitative
SILAC analysis of the nuclear- and cytoplasmic-enriched compartments
of CDC7-depleted fibroblasts and compared them to a total cell lysate
preparation. Substantial changes in total abundance and/or subcellular
location were detected for 124 proteins, including many essential
proteins associated with DNA replication/cell cycle. Similar changes
in protein abundance and subcellular distribution were observed for
various metabolic processes, including oxidative stress, iron metabolism,
protein translation and the tricarboxylic acid cycle. This is accompanied
by reduced abundance of two karyopherin proteins, suggestive of reduced
nuclear import. We propose that altered nucleo-cytoplasmic trafficking
plays a key role in the regulation of cell cycle arrest. The results
increase understanding of the mechanisms underlying maintenance of
the DNA replication origin activation checkpoint and are consistent
with our proposal that cell cycle arrest is an actively maintained
process that appears to be distributed over various subcellular locations