The Rad9-Hus1-Rad1 checkpoint clamp regulates interaction of TopBP1 with ATR

TopBP1 serves as an activator of the ATR-ATRIP complex in response to the presence of incompletely replicated or damaged DNA. This process involves binding of ATR to the ATR-activating domain of TopBP1, which is located between BRCT domains VI and VII. TopBP1 displays increased binding to ATR-ATRIP in Xenopus egg extracts containing checkpoint-inducing DNA templates. We show that an N-terminal region of TopBP1 containing BRCT repeats I-II is essential for this checkpoint-stimulated binding of TopBP1 to ATR-ATRIP. The BRCT I-II region of TopBP1 also binds specifically to the Rad9-Hus1-Rad1 (9-1-1) complex in Xenopus egg extracts. This binding occurs via the C-terminal domain of Rad9 and depends upon phosphorylation of its Ser-373 residue. Egg extracts containing either a mutant of TopBP1 lacking the BRCT I-II repeats or a mutant of Rad9 with an alanine substitution at Ser-373 are defective in checkpoint regulation. Furthermore, an isolated C-terminal fragment from Rad9 is an effective inhibitor of checkpoint signaling in egg extracts. These findings suggest that interaction of the 9-1-1 complex with the BRCT I-II region of TopBP1 is necessary for binding of ATR-ATRIP to the ATR-activating domain of TopBP1 and the ensuing activation of ATR.     

Direct Role for the Replication Protein Treslin (Ticrr) in the ATR Kinase-mediated Checkpoint Response

TopBP1 (topoisomerase IIβ-binding protein 1) is a dual replication/checkpoint protein. Treslin/Ticrr, an essential replication protein, was discovered as a binding partner for TopBP1 and also in a genetic screen for checkpoint regulators in zebrafish. Treslin is phosphorylated by CDK2/cyclin E in a cell cycle-dependent manner, and its phosphorylation state dictates its interaction with TopBP1. The role of Treslin in the initiation of DNA replication has been partially elucidated; however, its role in the checkpoint response remained elusive. In this study, we show that Treslin stimulates ATR phosphorylation of Chk1 both in vitro and in vivo in a TopBP1-dependent manner. Moreover, we show that the phosphorylation state of Treslin at Ser-1000 is important for its checkpoint activity. Overall, our results indicate that, like TopBP1, Treslin is a dual replication/checkpoint protein that directly participates in ATR-mediated checkpoint signaling.     

Interaction between Rad9–Hus1–Rad1 and TopBP1 activates ATR–ATRIP and promotes TopBP1 recruitment to sites of UV-damage

The checkpoint clamp Rad9–Hus1–Rad1 (9–1–1) interacts with TopBP1 via two casein kinase 2 (CK2)-phosphorylation sites, Ser-341 and Ser-387 in Rad9. While this interaction is known to be important for the activation of ATR-Chk1 pathway, how the interaction contributes to their accumulation at sites of DNA damage remains controversial. Here, we have studied the contribution of the 9–1–1/TopBP1 interaction to the assembly and activation of checkpoint proteins at damaged DNA. UV-irradiation enhanced association of Rad9 with chromatin and its localization to sites of DNA damage without a direct interaction with TopBP1. TopBP1, as well as RPA and Rad17 facilitated Rad9 recruitment to DNA damage sites. Similar to Rad9, TopBP1 also localized to sites of UV-induced DNA damage. The DNA damage-induced TopBP1 redistribution was delayed in cells expressing a TopBP1 binding-deficient Rad9 mutant. Pharmacological inhibition of ATR recapitulated the delayed accumulation of TopBP1 in the cells, suggesting that ATR activation will induce more efficient accumulation of TopBP1. Taken together, TopBP1 and Rad9 can be independently recruited to damaged DNA. Once recruited, a direct interaction of 9–1–1/TopBP1 occurs and induces ATR activation leading to further TopBP1 accumulation and amplification of the checkpoint signal. Thus, we propose a new positive feedback mechanism that is necessary for successful formation of the damage-sensing complex and DNA damage checkpoint signaling in human cells.     

The Rad4(TopBP1) ATR-activation domain functions in G1/S phase in a chromatin-dependent manner

DNA damage checkpoint activation can be subdivided in two steps: initial activation and signal amplification. The events distinguishing these two phases and their genetic determinants remain obscure. TopBP1, a mediator protein containing multiple BRCT domains, binds to and activates the ATR/ATRIP complex through its ATR-Activation Domain (AAD). We show that Schizosaccharomyces pombe Rad4(TopBP1) AAD-defective strains are DNA damage sensitive during G1/S-phase, but not during G2. Using lacO-LacI tethering, we developed a DNA damage-independent assay for checkpoint activation that is Rad4(TopBP1) AAD-dependent. In this assay, checkpoint activation requires histone H2A phosphorylation, the interaction between TopBP1 and the 9-1-1 complex, and is mediated by the phospho-binding activity of Crb2(53BP1). Consistent with a model where Rad4(TopBP1) AAD-dependent checkpoint activation is ssDNA/RPA-independent and functions to amplify otherwise weak checkpoint signals, we demonstrate that the Rad4(TopBP1) AAD is important for Chk1 phosphorylation when resection is limited in G2 by ablation of the resecting nuclease, Exo1. We also show that the Rad4(TopBP1) AAD acts additively with a Rad9 AAD in G1/S phase but not G2. We propose that AAD-dependent Rad3(ATR) checkpoint amplification is particularly important when DNA resection is limiting. In S. pombe, this manifests in G1/S phase and relies on protein-chromatin interactions.     

Intra-S phase checkpoint kinase Chk1 dissociates replication proteins Treslin and TopBP1 through multiple mechanisms during replication stress

Replication stress impedes DNA polymerase progression causing activation of the ataxia telangiectasia and Rad3-related signaling pathway, which promotes the intra-S phase checkpoint activity through phosphorylation of checkpoint kinase 1 (Chk1). Chk1 suppresses replication origin firing, in part, by disrupting the interaction between the preinitiation complex components Treslin and TopBP1, an interaction that is mediated by TopBP1 BRCT domain-binding to two cyclin-dependent kinase (CDK) phosphorylation sites, T968 and S1000, in Treslin. Two nonexclusive models for how Chk1 regulates the Treslin–TopBP1 interaction have been proposed in the literature: in one model, these proteins dissociate due to a Chk1-induced decrease in CDK activity that reduces phosphorylation of the Treslin sites that bind TopBP1 and in the second model, Chk1 directly phosphorylates Treslin, resulting in dissociation of TopBP1. However, these models have not been formally examined. We show here that Treslin T968 phosphorylation was decreased in a Chk1-dependent manner, while Treslin S1000 phosphorylation was unchanged, demonstrating that T968 and S1000 are differentially regulated. However, CDK2-mediated phosphorylation alone did not fully account for Chk1 regulation of the Treslin–TopBP1 interaction. We also identified additional Chk1 phosphorylation sites on Treslin that contributed to disruption of the Treslin–TopBP1 interaction, including S1114. Finally, we showed that both of the proposed mechanisms regulate origin firing in cancer cell line models undergoing replication stress, with the relative roles of each mechanism varying among cell lines. This study demonstrates that Chk1 regulates Treslin through multiple mechanisms to promote efficient dissociation of Treslin and TopBP1 and furthers our understanding of Treslin regulation during the intra-S phase checkpoint.     

Dynamic changes in C-Raf phosphorylation and 14-3-3 protein binding in response to growth factor stimulation: differential roles of 14-3-3 protein binding sites

Phosphorylation events play a crucial role in Raf activation. Phosphorylation of serines 259 and 621 in C-Raf and serines 364 and 728 in B-Raf has been suggested to be critical for association with 14-3-3 proteins. To study the functional consequences of Raf phosphorylations at these positions, we developed and characterized phosphospecific antibodies directed against 14-3-3 binding epitopes: a monoclonal phosphospecific antibody (6B4) directed against pS621 and a polyclonal antibody specific for B-Raf-pS364 epitope. Although 6B4 detected both C- and B-Raf in Western blots, it specifically recognizes the native form of C-Raf but not B-Raf. Contrary to B-Raf, a kinase-dead mutant of C-Raf was found to be only poorly phosphorylated in the Ser-621 position. Moreover, serine 259 to alanine mutation prevented the Ser-621 phosphorylation suggesting an interdependence between these two 14-3-3 binding domains. Direct C-Raf.14-3-3 binding studies with purified proteins combined with competition assays revealed that the 14-3-3 binding domain surrounding pS621 represents the high affinity binding site, whereas the pS259 epitope mediates lower affinity binding. Raf isozymes differ in their 14-3-3 association rates. The time course of endogenous C-Raf activation in mammalian cells by nerve growth factor (NGF) has been examined using both phosphospecific antibodies directed against 14-3-3 binding sites (6B4 and anti-pS259) as well as phosphospecific antibodies directed against the activation domain (anti-pS338 and anti-pY340/pY341). Time course of Ser-621 phosphorylation, in contrast to Ser-259 phosphorylation, exhibited unexpected pattern reaching maximal phosphorylation within 30 s of NGF stimulation. Phosphorylation of tyrosine 340/341 reached maximal levels subsequent to Ser-621 phosphorylation and was coincident with emergence of kinase activity. Taken together, we found substantial differences between C-Raf.14-3-3 binding epitopes pS259 and pS621 and visualized for the first time the sequence of the essential C-Raf phosphorylation events in mammalian cells in response to growth factor stimulation.     

TopBP1 functions with 53BP1 in the G1 DNA damage checkpoint

TopBP1 is a checkpoint protein that colocalizes with ATR at sites of DNA replication stress. In this study, we show that TopBP1 also colocalizes with 53BP1 at sites of DNA double‐strand breaks (DSBs), but only in the G1‐phase of the cell cycle. Recruitment of TopBP1 to sites of DNA replication stress was dependent on BRCT domains 1–2 and 7–8, whereas recruitment to sites of DNA DSBs was dependent on BRCT domains 1–2 and 4–5. The BRCT domains 4–5 interacted with 53BP1 and recruitment of TopBP1 to sites of DNA DSBs in G1 was dependent on 53BP1. As TopBP1 contains a domain important for ATR activation, we examined whether it contributes to the G1 cell cycle checkpoint. By monitoring the entry of irradiated G1 cells into S‐phase, we observed a checkpoint defect after siRNA‐mediated depletion of TopBP1, 53BP1 or ATM. Thus, TopBP1 may mediate the checkpoint function of 53BP1 in G1.     

Structure and function of the Rad9-binding region of the DNA-damage checkpoint adaptor TopBP1

TopBP1 is a scaffold protein that coordinates activation of the DNA-damage-checkpoint response by coupling binding of the 9-1-1 checkpoint clamp at sites of ssDNA, to activation of the ATR–ATRIP checkpoint kinase complex. We have now determined the crystal structure of the N-terminal region of human TopBP1, revealing an unexpected triple-BRCT domain structure. The arrangement of the BRCT domains differs significantly from previously described tandem BRCT domain structures, and presents two distinct sites for binding phosphopeptides in the second and third BRCT domains. We show that the site in the second but not third BRCT domain in the N-terminus of TopBP1, provides specific interaction with a phosphorylated motif at pSer387 in Rad9, which can be generated by CK2.     

Rim,a component of the presynaptic active zone and modulator of exocytosis,binds 14-3-3 through its N terminus

Rim1, a brain-specific Rab3a-binding protein, localizes to the presynaptic cytomatrix and plays an important role in synaptic transmission and synaptic plasticity. Rim2, a homologous protein, is more ubiquitously expressed and is found in neuroendocrine cells as well as in brain. Both Rim1 and Rim2 contain multiple domains, including an N-terminal zinc finger, which in Rim1 strongly enhances secretion in chromaffin and PC12 cells. The yeast two-hybrid technique identified 14-3-3 proteins as ligands of the N-terminal domain. In vitro protein binding experiments confirmed a high-affinity interaction between the N terminus of Rim1 and 14-3-3. The N-terminal domain of Rim2 also bound 14-3-3. The binding domains were localized to a short segment just C-terminal to the zinc finger. 14-3-3 proteins bind to specific phosphoserine residues. Alkaline phosphatase treatment of N-terminal domains of Rim1 and Rim2 almost completely inhibited the binding of 14-3-3. Two serine residues in Rim1 (Ser-241 and Ser-287) and one serine residue in Rim2 (Ser-335) were required for 14-3-3 binding. Incubation with Ca2+/calmodulin-dependent protein kinase II greatly stimulated the interaction of recombinant N-terminal Rim but not the S241/287A mutant with 14-3-3, again indicating the importance of the phosphorylation of these residues for the binding. Rabphilin3, another Rab3a effector, also bound 14-3-3. Serine-to-alanine mutations identified Ser-274 as the likely phosphorylated residue to which 14-3-3 binds. Because the phosphorylation of this residue had been shown to be stimulated upon depolarization in brain slices, the interaction of 14-3-3 with Rabphilin3 may be important in the dynamic function of central nervous system neurons.     

The Mre11-Rad50-Nbs1 Complex Mediates Activation of TopBP1 by ATM

The activation of ATR-ATRIP in response to double-stranded DNA breaks (DSBs) depends upon ATM in human cells and Xenopus egg extracts. One important aspect of this dependency involves regulation of TopBP1 by ATM. In Xenopus egg extracts, ATM associates with TopBP1 and thereupon phosphorylates it on S1131. This phosphorylation enhances the capacity of TopBP1 to activate the ATR-ATRIP complex. We show that TopBP1 also interacts with the Mre11-Rad50-Nbs1 (MRN) complex in egg extracts in a checkpoint-regulated manner. This interaction involves the Nbs1 subunit of the complex. ATM can no longer interact with TopBP1 in Nbs1-depleted egg extracts, which suggests that the MRN complex helps to bridge ATM and TopBP1 together. The association between TopBP1 and Nbs1 involves the first pair of BRCT repeats in TopBP1. In addition, the two tandem BRCT repeats of Nbs1 are required for this binding. Functional studies with mutated forms of TopBP1 and Nbs1 suggested that the BRCT-dependent association of these proteins is critical for a normal checkpoint response to DSBs. These findings suggest that the MRN complex is a crucial mediator in the process whereby ATM promotes the TopBP1-dependent activation of ATR-ATRIP in response to DSBs.     

TopBP1 and ATR colocalization at meiotic chromosomes: role of TopBP1/Cut5 in the meiotic recombination checkpoint

Mammalian TopBP1 is a BRCT domain-containing protein whose function in mitotic cells is linked to replication and DNA damage checkpoint. Here, we study its possible role during meiosis in mice. TopBP1 foci are abundant during early prophase I and localize mainly to histone gamma-H2AX-positive domains, where DNA double-strand breaks (required to initiate recombination) occur. Strikingly, TopBP1 showed a pattern almost identical to that of ATR, a PI3K-like kinase involved in mitotic DNA damage checkpoint. In the synapsis-defective Fkbp6(-/-) mouse, TopBP1 heavily stains unsynapsed regions of chromosomes. We also tested whether Schizosaccharomyces pombe Cut5 (the TopBP1 homologue) plays a role in the meiotic recombination checkpoint, like spRad3, the ATR homologue. Indeed, we found that a cut5 mutation suppresses the checkpoint-dependent meiotic delay of a meiotic recombination defective mutant, indicating a direct role of the Cut5 protein in the meiotic checkpoint. Our findings suggest that ATR and TopBP1 monitor meiotic recombination and are required for activation of the meiotic recombination checkpoint.     

Inhibition of integrin-linked kinase/protein kinase B/Akt signaling: mechanism for ganglioside-induced apoptosis

Ganglioside GT1b inhibits keratinocyte attachment to and migration on a fibronectin matrix by binding to alpha(5)beta(1) and preventing alpha(5)beta(1) interaction with fibronectin. The role of gangliosides in triggering keratinocyte apoptosis, however, is unknown. Addition of GT1b to keratinocyte-derived SCC12 cells, grown in serum-free medium but exposed to fibronectin, suppressed Bad phosphorylation, activated caspase-9, and inhibited cyclin D and E expression, resulting in cell cycle arrest at G(1) phase and initiation of apoptosis. The mechanism of GT1b activation of caspase-9 involved inhibition of beta(1) integrin serine/threonine phosphorylation and decreased phosphorylation of both integrin-linked kinase and protein kinase B/Akt at its Ser-473 site, leading to cytochrome c release from mitochondria. Consistently, blockade of GT1b function with anti-GT1b antibody specifically activated the Ser-473 site of Akt, markedly suppressing apoptosis. The ganglioside-induced inhibition of Akt phosphorylation was GT1b-specific and was not observed when cells were treated with other keratinocyte gangliosides, including GD3. These studies suggest that the modulation of keratinocyte cell cycle and survival by GT1b is mediated by its direct interaction with alpha(5)beta(1) and resultant inhibition of the integrin/integrin-linked kinase/protein kinase B/Akt signaling pathway.