Geldanamycin-induced degradation of Chk1 is mediated by proteasome.
Checkpoint kinase 1 (Chk1) is a cell cycle regulator and a heat shock protein 90 (Hsp90) client. It is essential for cell proliferation and survival. In this report, we analyzed the mechanisms of Chk1 regulation in U87MG glioblastoma cells using Geldanamycin (GA), which interferes with the function of Hsp90. GA reduced Chk1 protein level but not its mRNA level in glioblastoma cells. Co-treatment with GA and cycloheximide (CHX), a protein synthesis inhibitor, induced a decrease of half-life of the Chk1 protein to 3h and resulted in Chk1 down-regulation. CHX alone induced only 32% reduction of Chk1 protein even after 24h. These findings indicated that reduction of Chk1 by GA was due to destabilization and degradation of the protein. In addition, GA-induced down-regulation of Chk1 was reversed by MG132, a specific proteasome inhibitor. And it was revealed that Chk1 was ubiquitinated by GA. These results have indicated that degradation of Chk1 by GA was mediated by the ubiquitin-proteasome pathway in U87MG glioblastoma cells.
Nomura
M
M
Department of Neurosurgery, Yokohama Sakae Kyosai Hospital, Yokohama, Japan. nomura413jp@yahoo.co.jp
Nomura
N
N
Yamashita
J
J
D001483:Base Sequence; D016227:Benzoquinones; D045744:Cell Line, Tumor; D000071877:Checkpoint Kinase 1; D003513:Cycloheximide / Q000494:pharmacology; D017931:DNA Primers; D006207:Half-Life; D006801:Humans; D006868:Hydrolysis; D047029:Lactams, Macrocyclic; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D011809:Quinones / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D025801:Ubiquitin / Q000378:metabolism
D016227:Benzoquinones; D017931:DNA Primers; D047029:Lactams, Macrocyclic; D011809:Quinones; D012333:RNA, Messenger; D025801:Ubiquitin; D003513:Cycloheximide; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D046988:Proteasome Endopeptidase Complex; C001277:geldanamycin