14-3-3 proteins, FHA domains and BRCT domains in the DNA damage response

Abstract

The DNA damage response depends on the concerted activity of protein serine/threonine kinases and modular phosphoserine/threonine-binding domains to relay the damage signal and recruit repair proteins. The PIKK family of protein kinases, which includes ATM/ATR/DNA-PK, preferentially phosphorylate Ser-Gln sites, while their basophilic downstream effecter kinases, Chk1/Chk2/MK2 preferentially phosphorylate hydrophobic-X-Arg-X-X-Ser/Thr-hydrophobic sites. A subset of tandem BRCT domains act as phosphopeptide binding modules that bind to ATM/ATR/DNA-PK substrates after DNA damage. Conversely, 14-3-3 proteins interact with substrates of Chk1/Chk2/MK2. FHA domains have been shown to interact with substrates of ATM/ATR/DNA-PK and CK2. In this review we consider how substrate phosphorylation together with BRCT domains, FHA domains and 14-3-3 proteins function to regulate ionizing radiation-induced nuclear foci and help to establish the G(2)/M checkpoint. We discuss the role of MDC1 a molecular scaffold that recruits early proteins to foci, such as NBS1 and RNF8, through distinct phosphodependent interactions. In addition, we consider the role of 14-3-3 proteins and the Chk2 FHA domain in initiating and maintaining cell cycle arrest.

Figure 1. Kinase class-specific phosphopeptide-binding domains

The DNA damage-responsive phosphopeptide binding domains can be segregated by kinase motif. ATM/ATR/DNA-PK are gluamine-directed kinases that recruit a subset BRCT domains and FHA domains. Significant accumulation of ATM/ATR/DNA-PK substrates is observed at Ionizing Radiation Induced Foci (IRIF). Conversely, checkpoint kinases preferentially phosphorylate ΦxRxxSΦ sites, where Φ denotes hydrophobic amino acids sites, and a subset of their substrates result in the generation of 14-3-3 motifs. Selectivity in interactions are a result of the positive selection for binding to the substrates of the proper kinase family and negative selection to prevent binding to substrates of the incorrect kinase family.

Figure 2. Protein domain architecture and phospho-motif selectivity for DNA damage-response proteins

A. The domain structure of the proteins that will be discussed in this review. BRCT domains in gray have empirically determined phosphopeptide binding activity. B. Table summarizing the domains, selected binding motifs and example ligands. “X” stands for any amino acid, “pS” stands for phosphoserine, “pT” for phosphothreonine and ψ, for aromatic residues.

Figure 3. Overview of protein-protein interactions in IRIF formation

ATM is activated rapidly after IR treatment, resulting in the phosphorylation of H2AX at Ser-139. Phosphorylation of H2AX generates an optimal phosphobinding epitope that is recognized by the BRCT domains of MDC1 [42]. MDC1 recruits RNF8 and NBS1 through phospho-dependent interactions. NBS1 regulates the localization of the MRN complex and amplifies ATM activation [61-64], while RNF8 recruitment is required for BRCA1 and 53BP1 localization to IRIF [56, 57].