L2DTL/CDT2 interacts with the CUL4/DDB1 complex and PCNA and regulates CDT1 proteolysis in response to DNA damage.
The CUL4 (cullin 4) proteins are the core components of a new class of ubiquitin E3 ligases that regulate cell cycle, DNA replication and DNA damage response. To determine the composition of CUL4 ubiquitin E3 ligase complex, we used anti-CUL4 antibody affinity chromatography to isolate the proteins that associated with human CUL4 complexes and identified them by mass-spectrometry. A novel and conserved WD40 domain-containing protein, the human homologue of Drosophila lethal(2) denticleless protein (L2DTL) or fission yeast CDT2, was found to associate with CUL4 and DDB1. L2DTL also interacts with replication licensing protein CDT1 in vivo. Loss of L2DTL in Drosophila S2 and human cells suppressed proteolysis of CDT1 in response to DNA damage. We further isolated the human L2DTL complexes by anti-L2DTL immuno-affinity chromatography from HeLa cells and found it associates with DDB1, components of the COP9-signalosome complex (CSN) and PCNA. We found that PCNA interacts with CDT1 and loss of PCNA suppressed CDT1 proteolysis after DNA damage. Our data also revealed that in vivo, inactivation of L2DTL causes the dissociation of DDB1 from the CUL4 complex. Our studies suggest that L2DTL and PCNA interact with CUL4/DDB1 complexes and are involved in CDT1 degradation after DNA damage.
Higa
Leigh Ann
LA
Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Banks
Damon
D
Wu
Min
M
Kobayashi
Ryuji
R
Sun
Hong
H
Zhang
Hui
H
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000172:deficiency / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D018797:Cell Cycle Proteins / Q000378:metabolism*; D044842:Cullin Proteins / Q000378:metabolism*; D004249:DNA Damage / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D029721:Drosophila Proteins / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000378:metabolism; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000172:deficiency / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D044767:Ubiquitin-Protein Ligases
D048868:Adaptor Proteins, Signal Transducing; C467199:CDT1 protein, human; C114234:CUL4A protein, human; D018797:Cell Cycle Proteins; D044842:Cullin Proteins; C091435:DDB1 protein, human; D004268:DNA-Binding Proteins; C429530:DTL protein, human; D029721:Drosophila Proteins; D006360:Heat-Shock Proteins; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; C099924:l(2)dtl protein, Drosophila; D044767:Ubiquitin-Protein Ligases