Activation of ATR and related PIKKs

Abstract

The DNA damage response kinase ATR is an essential regulator of genome integrity. TopBP1 functions as a general activator of ATR. We have recently shown that TopBP1 activates ATR through its regulatory subunit ATRIP and a PIKK regulatory domain (PRD) located adjacent to its kinase domain. This mechanism of ATR activation is conserved in the S. cerevisiae ortholog Mec1. ATR is a member of the PIKK family of protein kinases that includes ATM, DNA-PKcs, mTOR and SMG1. The PRD regulates the kinase activity of other PIKKs and may serve as a site of interaction between these kinase and their respective activators. Activation of ATR by TopBP1 is maximal at low substrate concentrations and declines exponentially as substrate concentration increases. These data are consistent with a model in which TopBP1 acts to alter the conformation of ATR-ATRIP to increase the ability of ATR to bind substrates. A further understanding of the mechanism of ATR activation will likely provide insights into the regulation of related PIKKs.

Figure 1. Functional Domains of ATRIP and ATR and Activation of ATR by TopBP1 as a function of substrate concentration

(A) ATRIP contains an N-terminal Checkpoint Recruitment Domain (CRD), a coiled-coil domain that mediates it homo-dimerization, a TopBP1 interacting region, and a C-terminal ATR binding domain., , , (B) ATR contains an N-terminal ATRIP binding domain. ATR contains a similar domain architecture to other PIKK family members. Its kinase domain is flanked by two regions of high sequence similarity named the FAT and FATC domains. The FAT domain is part of a long N-terminal region predicted to fold into an extended alpha-helical HEAT repeat structure. The small FATC domain of at least some of the kinases is interchangeable. The FATC domain is required for ATR kinase activity. The functions of the FAT and FATC domain are unknown. Between the kinase domain and the FATC domain is the PRD (PIKK regulatory domain), which interacts with TopBP1, and is necessary for ATR activation. Sequence alignment of the PRD among ATR orthologs and ATM is presented. (C) In vitro ATR kinase assays were performed as described. The amount of the substrate, MCM2, was varied as indicated. Quantification of substrate phosphorylation was performed using a phosphorimager. Apparent K_m (μM) and V_max were calculated by nonlinear regression. Velocity is presented in arbitrary units.