Two nuclear export signals of Cdc6 are differentially associated with CDK-mediated phosphorylation residues for cytoplasmic translocation.
Cdc6 is cleaved at residues 442 and 290 by caspase-3 during apoptosis producing p49-tCdc6 and p32-tCdc6, respectively. While p32-tCdc6 is unable to translocate into the cytoplasm, p49-tCdc6 retains cytoplasmic translocation activity, but it has a lower efficiency than wild-type Cdc6. We hypothesized that a novel nuclear export signal (NES) sequence exists between amino acids 290 and 442. Cdc6 contains a novel NES in the region of amino acids 300-315 (NES2) that shares sequence similarity with NES1 at residues 462-476. In mutant versions of Cdc6, we replaced leucine with alanine in NES1 and NES2 and co-expressed the mutant constructs with cyclin A. We observed that the cytoplasmic translocation of these mutants was reduced in comparison to wild-type Cdc6. Moreover, the cytoplasmic translocation of a mutant in which all four leucine residues were mutated to alanine was significantly inhibited in comparison to the translocation of wild-type Cdc6. The Crm1 binding activities of Cdc6 NES mutants were consistent with the efficiency of its cytoplasmic translocation. Further studies have revealed that L468 and L470 of NES1 are required for cytoplasmic translocation of Cdc6 phosphorylated at S74, while L311 and L313 of NES2 accelerate the cytoplasmic translocation of Cdc6 phosphorylated at S54. These results suggest that the two NESs of Cdc6 work cooperatively and distinctly for the cytoplasmic translocation of Cdc6 phosphorylated at S74 and S54 by cyclin A/Cdk2.
Division of Pharmaceutical Biosciences, Research Institute for Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea.
In Sun
IS
Hwang
Department of Pharmacy, College of Pharmacy, Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan, Gyeonggi-do 426-791, Republic of Korea.
Sang Uk
SU
Woo
Division of Pharmaceutical Biosciences, Research Institute for Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea.
Ji-Woong
JW
Park
Division of Pharmaceutical Biosciences, Research Institute for Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea.
Seung Ki
SK
Lee
Department of Pharmacy, College of Pharmacy, Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan, Gyeonggi-do 426-791, Republic of Korea. Electronic address: hsyim@hanyang.ac.kr.
Hyungshin
H
Yim
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism*; D019925:Cyclin A / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D028884:Karyopherins / Q000378:metabolism; D008969:Molecular Sequence Data; D049790:Nuclear Export Signals; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism; D021381:Protein Transport; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D016196:S Phase; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D000097606:Exportin 1 Protein
D000596:Amino Acids; D018797:Cell Cycle Proteins; D019925:Cyclin A; D051357:Cyclin-Dependent Kinase 2; D028884:Karyopherins; D049790:Nuclear Export Signals; D009687:Nuclear Proteins; D020033:Protein Isoforms; D018160:Receptors, Cytoplasmic and Nuclear; D000097606:Exportin 1 Protein; C103706:CDC6 protein, human
ASA-Cdc6; Cdc6; Crm1; Localization; NES; NMT-Cdc6; Nuclear export signal; Phosphorylation; SAA-Cdc6; a mutant version of Cdc6 with Lys to Ala in NES sequences; a mutant version of Cdc6 with S54A and S106A; a mutant version of Cdc6 with S74A and S106A; a truncated Cdc6 at D290, producing a 32kDa fragment; a truncated Cdc6 at D442, producing a 49kDa fragment; nuclear export signal; p32-tCdc6; p49-tCdc6