Common motifs in ETAA1 and TOPBP1 required for ATR kinase activation

Abstract

DNA damage response Ser/Thr kinases, including ataxia telangiectasia-mutated (ATM) and Rad3-related (ATR), control cell cycle progression, DNA repair, and apoptosis. ATR is activated by ETAA1 activator of ATR kinase (ETAA1) or DNA topoisomerase II binding protein 1 (TOPBP1). Both ETAA1 and TOPBP1 contain experimentally defined ATR activation domains (AADs) that are mostly unstructured and have minimal sequence similarity. A tryptophan residue in both AADs is required for ATR activation, but the other features of these domains and the mechanism by which they activate ATR are unknown. In this study, using bioinformatic analyses, kinase assays, co-immunoprecipitation, and immunofluorescence measures of signaling, we more specifically defined the TOPBP1 and ETAA1 AADs and identified additional features of the AADs needed for ATR activation. We found that both ETAA1 and TOPBP1 contain a predicted coiled-coil motif that is required for ATR activation in vitro and in cells. Mutation of the predicted coiled coils does not alter AAD oligomerization but does impair binding of the AADs to ATR. These results suggest that TOPBP1 and ETAA1 activate ATR using similar motifs and mechanisms.

Keywords: ATR; ATR activation domain (AAD); DNA damage response; DNA replication; DNA topoisomerase II binding protein 1 (TOPBP1); ETAA1 activator of ATR kinase (ETAA1); PI3K-related protein kinase (PIKK); cancer; cell cycle; serine/threonine protein kinase; signaling.

Figure 1.

Identification of the ETAA1 ATR activation domain. A, schematic of ETAA1 with the AAD and RPA interaction motifs indicated. ETAA1 predicted disorder was calculated using IUPred2A (31). The AAD region is indicated by the red box. B, schematic of the ETAA1 AAD, with the critical tryptophan (W) and predicted coiled coil (CC) indicated. Predicted coiled coil per residue scores were calculated using Paircoil2 (29). C–F, Purified ATR–ATRIP complexes were incubated with GST or the indicated GST-ETAA1 AADs, an ATR substrate, and [γ-³²P]ATP. Reaction products were separated by SDS-PAGE and detected by Coomassie staining and immunoblotting. Substrate phosphorylation was detected by autoradiography. MW, molecular weight. G, quantification of three experiments as shown in F. Statistical significance was calculated using a one-way analysis of variance and Tukey's multiple comparisons test. Error bars are mean ± S.D.

Figure 2.

Identification of the TOPBP1 ATR activation domain. A, schematic of TOPBP1 with the AAD indicated. BRCT domains are shown as dark blue boxes. TOPBP1 predicted disorder was calculated using IUPred2A (31). The AAD region is indicated by the red box. B, schematic of the TOPBP1 AAD, with the critical tryptophan (W) and predicted coiled coil (CC) indicated. Predicted coiled coil per residue scores were calculated using Paircoil2 (29). C and D, purified ATR–ATRIP complexes were incubated with GST or the indicated GST-TOPBP1 AADs, an ATR substrate, and [γ-³²P]ATP. Reaction products were separated by SDS-PAGE and detected by Coomassie staining and immunoblotting. Substrate phosphorylation was detected by autoradiography. MW, molecular weight.

Figure 3.

Mutation of a conserved phenylalanine disrupts ETAA1- and TOPBP1-dependent ATR activation. A, sequence alignment of ETAA1 and TOPBP1 predicted coiled coil residues flanking ETAA1 Phe-198 and TOPBP1 Phe-1071. B and C, purified ATR–ATRIP complexes were incubated with GST or the indicated GST-ETAA1 or GST-TOPBP1 AADs, an ATR substrate, and [γ-³²P]ATP. Reaction products were separated by SDS-PAGE and detected by Coomassie staining. Substrate phosphorylation was detected by autoradiography. D–I, empty vector or the indicated ETAA1 or TOPBP1 AAD proteins were expressed in U2OS cells. γH2AX (D, E, G, and H) or MCM2 pSer-108 (F and I) was visualized in FLAG-positive nuclei. Scale bars = 10 μm. A.U., arbitrary units. γH2AX and MCM2 pSer-108 intensity is normalized to the FLAG expression level. Statistical significance was calculated using a one-way analysis of variance and Tukey's multiple comparisons test. Error bars are mean ± S.D.

Figure 4.

Mutation of the AADs does not alter AAD oligomerization. A and B, the indicated ETAA1 (A) or TOPBP1 (B) AADs were eluted from a Superdex^TM 200 Increase 10/300 GL column while measuring UV absorbance at 280 nm. Equal amounts of fractions corresponding to peaks were separated by SDS-PAGE and visualized by Coomassie staining. The elution volume of molecular mass standards is indicated. MW, molecular weight.

Figure 5.

The predicted coiled coils promote the ETAA1 and TOPBP1 AAD–ATR interaction. A and B, empty vector or the indicated ETAA1 (A) or TOPBP1 (B) AADs were immunoprecipitated (IP) from HEK293T cell nuclear extracts. Immunoprecipitated proteins were separated by SDS-PAGE and detected by immunoblotting. MW, molecular weight. C and D, purified GST or recombinant GST-ETAA1 (C) or GST-TOPBP1 (D) proteins bound to GSH beads were incubated with HEK293T cell nuclear extracts expressing FLAG-ATR and HA-ATRIP. Bound proteins were eluted, separated by SDS-PAGE, and detected by Coomassie staining (CB) or immunoblotting.