Diverse but overlapping functions of the two forkhead-associated (FHA) domains in Rad53 checkpoint kinase activation.
Forkhead-associated (FHA) domains are phosphothreonine-binding modules prevalent in proteins with important cell cycle and DNA damage response functions. The yeast checkpoint kinase Rad53 is unique in containing two FHA domains. We have generated novel recessive rad53 alleles with abolished FHA domain functions resulting from Ala substitution of the critical phosphothreonine-binding residues Arg70 and Arg605. In asynchronous cells, inactivation of the N-terminal FHA1 domain did not impair Rad53 activation and downstream functions, whereas inactivation of the C-terminal FHA2 domain led to reduced Rad53 activation and significantly increased DNA damage sensitivity. Simultaneous inactivation of both FHA domains abolished Rad53 activation and all downstream functions and dramatically increased the sensitivity to DNA damage and replication blocks similar to kinase-defective and rad53 null alleles, but did not compromise the essential viability function of Rad53. Interestingly, in G2/M synchronized cells, mutation of either FHA domain prevented Rad53 activation and impaired the cell cycle arrest checkpoint. Our data demonstrate that both FHA domains are required for normal Rad53 functions and indicate that the two FHA domains have differential but partially overlapping roles in Rad53 activation and downstream signaling.
Pike
Brietta L
BL
St. Vincent's Institute of Medical Research, and Department of Medicine, St. Vincent's Hospital, The University of Melbourne, 9 Princes Street, Fitzroy, Victoria 3065, Australia.
Yongkiettrakul
Suganya
S
Tsai
Ming-Daw
MD
Heierhorst
Jörg
J
D000483:Alleles; D019943:Amino Acid Substitution; D018797:Cell Cycle Proteins; D064447:Checkpoint Kinase 2; D004249:DNA Damage / Q000502:physiology*; D004789:Enzyme Activation / Q000502:physiology; D051858:Forkhead Transcription Factors; D016195:G2 Phase / Q000502:physiology; D005808:Genes, Recessive; D008938:Mitosis / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics; D010641:Phenotype; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction / Q000502:physiology; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics; D015003:Yeasts / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
D018797:Cell Cycle Proteins; D051858:Forkhead Transcription Factors; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae