Role of the N-terminal Forkhead-associated Domain in the Cell Cycle Checkpoint Function of the Rad53 Kinase

Brietta L Pike,Andrew Hammet,Jörg Heierhorst

doi:10.1074/jbc.m009558200

Brietta L Pike, Andrew Hammet + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m009558200

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Abstract

Forkhead-associated (FHA) domains are multifunctional phosphopeptide-binding modules and are the hallmark of the conserved family of Rad53-like checkpoint protein kinases. Rad53-like kinases, including the human tumor suppressor protein Chk2, play crucial roles in cell cycle arrest and activation of repair processes following DNA damage and replication blocks. Here we show that ectopic expression of the N-terminal FHA domain (FHA1) of the yeast Rad53 kinase causes a growth defect by arresting the cell cycle in G(1). This phenotype was highly specific for the Rad53-FHA1 domain and not observed with the similar Rad53-FHA2, Dun1-FHA, and Chk2-FHA domains, and it was abrogated by mutations that abolished binding to a phosphothreonine-containing peptide in vitro. Furthermore, replacement of the RAD53 gene with alleles containing amino acid substitutions in the FHA1 domain resulted in an increased DNA damage sensitivity in vivo. Taken together, these data demonstrate that the FHA1 domain contributes to the checkpoint function of Rad53, possibly by associating with a phosphorylated target protein in response to DNA damage in G(1).

Highlights

Checkpoint signaling pathways are crucial for maintaining genome integrity by delaying the cell cycle in response to DNA damage, replicational stress or mitotic spindle assembly defects, and are highly conserved throughout evolution [1, 2]
Strains overexpressing the entire Rad53 N terminus or expressing a construct containing only the biochemically defined FHA1 domain had a dramatic growth defect and failed to form visible colonies on solid media containing galactose (Fig. 1A). This growth defect was highly specific for the Rad53-FHA1 domain as it was not observed with the related Rad53-FHA2, Dun1-FHA, and Chk2FHA domains (Fig. 1A) that were expressed at similar protein levels (Fig. 1B)
The Rad53-FHA2 and Dun1-FHA domains are fully functional in this system, and the fact that only the FHA1 domain causes a growth defect underscores the specificity of this phenotype

Summary

Introduction

Checkpoint signaling pathways are crucial for maintaining genome integrity by delaying the cell cycle in response to DNA damage, replicational stress or mitotic spindle assembly defects, and are highly conserved throughout evolution [1, 2]. We show that ectopic expression of the N-terminal FHA domain (FHA1) of the yeast Rad53 kinase causes a growth defect by arresting the cell cycle in G1.

Results

Conclusion