Abstract
The DNA replication-licensing factor Cdt1 is present during the G1 phase of the cell cycle. When cells initiate S phase or are UV-irradiated, Cdt1 is recruited to chromatin-bound PCNA and ubiquitinated by CRL4(Cdt2) for degradation. In both situations, the substrate-recognizing subunit Cdt2 is detected as a highly phosphorylated form. Here, we show that both caffeine-sensitive kinase and MAP kinases are responsible for Cdt2 phosphorylation following UV irradiation. We found that Cdt1 degradation was attenuated in the presence of caffeine. This attenuation was also observed in cells depleted of ATR, but not ATM. Following UV irradiation, Cdt2 was phosphorylated at the S/TQ sites. ATR phosphorylated Cdt2 in vitro, mostly in the C-terminal region. Cdt1 degradation was also induced by DNA damaging chemicals such as methyl methanesulfonate (MMS) or zeocin, depending on PCNA and CRL4-Cdt2, though it was less caffeine-sensitive. These findings suggest that ATR, activated after DNA damage, phosphorylates Cdt2 and promotes the rapid degradation of Cdt1 after UV irradiation in the G1 phase of the cell cycle.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Ataxia Telangiectasia Mutated Proteins
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Caffeine / pharmacology
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Cell Cycle Proteins / antagonists & inhibitors
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism*
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Cullin Proteins / metabolism
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DNA Damage
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / metabolism
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Gene Knockdown Techniques
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HEK293 Cells
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HeLa Cells
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Humans
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Mitogen-Activated Protein Kinases / antagonists & inhibitors
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Nuclear Proteins / metabolism*
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Phosphorylation
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Protein Processing, Post-Translational / radiation effects*
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Protein Serine-Threonine Kinases / antagonists & inhibitors
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Protein Serine-Threonine Kinases / genetics
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Protein Serine-Threonine Kinases / metabolism*
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Proteolysis / radiation effects*
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RNA Interference
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Tumor Suppressor Proteins / genetics
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Tumor Suppressor Proteins / metabolism
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Ubiquitin-Protein Ligases / metabolism*
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Ultraviolet Rays
Substances
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CDT1 protein, human
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CUL4A protein, human
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Cell Cycle Proteins
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Cullin Proteins
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DNA-Binding Proteins
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DTL protein, human
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Nuclear Proteins
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Tumor Suppressor Proteins
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Caffeine
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Ubiquitin-Protein Ligases
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ATM protein, human
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ATR protein, human
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Ataxia Telangiectasia Mutated Proteins
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Protein Serine-Threonine Kinases
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Mitogen-Activated Protein Kinases
Grants and funding
This work was supported by Grants in Aid for Scientific Research, Scientific Research in a Priority Area, and the Global Center of Excellence Program from the Ministry of Education, Culture, Sport, Science, and Technology of Japan; and the Naito Foundation; Hyogo Science and Technology Association; and The Uehara Memorial Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.