The checkpoint protein Chfr is a ligase that ubiquitinates Plk1 and inhibits Cdc2 at the G2 to M transition.
The checkpoint protein Chfr delays entry into mitosis, in the presence of mitotic stress (Scolnick, D.M., and T.D. Halazonetis. 2000. Nature. 406:430-435). We show here that Chfr is a ubiquitin ligase, both in vitro and in vivo. When transfected into HEK293T cells, Myc-Chfr promotes the formation of high molecular weight ubiquitin conjugates. The ring finger domain in Chfr is required for the ligase activity; this domain auto-ubiquitinates, and mutations of conserved residues in this domain abolish the ligase activity. Using Xenopus cell-free extracts, we demonstrated that Chfr delays the entry into mitosis by negatively regulating the activation of the Cdc2 kinase at the G2-M transition. Specifically, the Chfr pathway prolongs the phosphorylated state of tyrosine 15 in Cdc2. The Chfr-mediated cell cycle delay requires ubiquitin-dependent protein degradation, because inactivating mutations in Chfr, interference with poly-ubiquitination, and inhibition of proteasomes all abolish this delay in mitotic entry. The direct target of the Chfr pathway is Polo-like kinase 1 (Plk1). Ubiquitination of Plk1 by Chfr delays the activation of the Cdc25C phosphatase and the inactivation of the Wee1 kinase, leading to a delay in Cdc2 activation. Thus, the Chfr pathway represents a novel checkpoint pathway that regulates the entry into mitosis by ubiquitin-dependent proteolysis.
Kang
Dongmin
D
Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020, USA.
Chen
James
J
Wong
Jim
J
Fang
Guowei
G
D000818:Animals; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D002474:Cell-Free System; D004789:Enzyme Activation; D016195:G2 Phase; D006801:Humans; D008025:Ligases / Q000737:chemistry / Q000378:metabolism*; D016200:Maturation-Promoting Factor / Q000378:metabolism; D008938:Mitosis; D009363:Neoplasm Proteins; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D000075223:Poly-ADP-Ribose Binding Proteins; D011494:Protein Kinases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases; D014981:Xenopus; D029867:Xenopus Proteins; D000097869:Polo-Like Kinase 1
D016203:CDC2 Protein Kinase; D018797:Cell Cycle Proteins; D008025:Ligases; D016200:Maturation-Promoting Factor; D009363:Neoplasm Proteins; D019000:Phosphotyrosine; D000075223:Poly-ADP-Ribose Binding Proteins; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D029867:Xenopus Proteins; D000097869:Polo-Like Kinase 1; C412002:CHFR protein, human; C101085:Plk1 protein, Xenopus