Regulation of Membrane Targeting of the G Protein-coupled Receptor Kinase 2 by Protein Kinase A and Its Anchoring Protein AKAP79

The beta 2 adrenergic receptor (beta 2AR) undergoes desensitization by a process involving its phosphorylation by both protein kinase A (PKA) and G protein-coupled receptor kinases (GRKs). The protein kinase A-anchoring protein AKAP79 influences beta 2AR phosphorylation by complexing PKA with the receptor at the membrane. Here we show that AKAP79 also regulates the ability of GRK2 to phosphorylate agonist-occupied receptors. In human embryonic kidney 293 cells, overexpression of AKAP79 enhances agonist-induced phosphorylation of both the beta 2AR and a mutant of the receptor that cannot be phosphorylated by PKA (beta 2AR/PKA-). Mutants of AKAP79 that do not bind PKA or target to the beta 2AR markedly inhibit phosphorylation of beta 2AR/PKA-. We show that PKA directly phosphorylates GRK2 on serine 685. This modification increases Gbeta gamma subunit binding to GRK2 and thus enhances the ability of the kinase to translocate to the membrane and phosphorylate the receptor. Abrogation of the phosphorylation of serine 685 on GRK2 by mutagenesis (S685A) or by expression of a dominant negative AKAP79 mutant reduces GRK2-mediated translocation to beta 2AR and phosphorylation of agonist-occupied beta 2AR, thus reducing subsequent receptor internalization. Agonist-stimulated PKA-mediated phosphorylation of GRK2 may represent a mechanism for enhancing receptor phosphorylation and desensitization

TopBP1 recruits Brg1/Brm to repress E2F1-induced apoptosis, a novel pRb-independent and E2F1-specific control for cell survival

TopBP1 (DNA topoisomerase IIβ binding protein I) contains multiple BRCT domains and is involved in replication and the DNA damage checkpoint. Through its BRCT domain, TopBP1 interacts with and represses exclusively E2F1 but not other E2F factors. This regulation of E2F1 transcriptional activity is mediated by a pRb-independent, but Brg1/Brm-dependent mechanism. TopBP1 recruits Brg1/Brm, a central component of the SWI/SNF chromatin-remodeling complex, to E2F1-responsive promoters and represses the activities of E2F1, but not E2F2 or E2F3. This regulation is crucial in the control of E2F1-dependent apoptosis during normal cell growth and DNA damage. Interestingly, TopBP1 is induced by E2F and interacts with E2F1 during G1/S transition. Thus, TopBP1 functions as a critical modulator and serves as a negative feedback regulator of E2F1 by inhibiting E2F1-dependent apoptosis during G1/S transition as well as DNA damage to promote cell survival

Regulation of E2F1 by BRCT Domain-Containing Protein TopBP1

The E2F transcription factor integrates cellular signals and coordinates cell cycle progression. Our prior studies demonstrated selective induction and stabilization of E2F1 through ATM-dependent phosphorylation in response to DNA damage. Here we report that DNA topoisomerase IIβ binding protein 1 (TopBP1) regulates E2F1 during DNA damage. TopBP1 contains eight BRCT (BRCA1 carboxyl-terminal) motifs and upon DNA damage is recruited to stalled replication forks, where it participates in a DNA damage checkpoint. Here we demonstrated an interaction between TopBP1 and E2F1. The interaction depended on the amino terminus of E2F1 and the sixth BRCT domain of TopBP1. It was specific to E2F1 and was not observed in E2F2, E2F3, or E2F4. This interaction was induced by DNA damage and phosphorylation of E2F1 by ATM. Through this interaction, TopBP1 repressed multiple activities of E2F1, including transcriptional activity, induction of S-phase entry, and apoptosis. Furthermore, TopBP1 relocalized E2F1 from diffuse nuclear distribution to discrete punctate nuclear foci, where E2F1 colocalized with TopBP1 and BRCA1. Thus, the specific interaction between TopBP1 and E2F1 during DNA damage inhibits the known E2F1 activities but recruits E2F1 to a BRCA1-containing repair complex, suggesting a direct role of E2F1 in DNA damage checkpoint/repair at stalled replication forks

MCPH1/BRIT1 cooperates with E2F1 in the activation of checkpoint, DNA repair and apoptosis

Microcephalin (MCPH1) has a crucial role in the DNA damage response by promoting the expression of Checkpoint kinase 1 (CHK1) and Breast cancer susceptibility gene 1 (BRCA1); however, the mechanism of this regulation remains unclear. Here, we show that MCPH1 regulates CHK1 and BRCA1 through the interaction with E2F1 on the promoters of both genes. MCPH1 also regulates other E2F target genes involved in DNA repair and apoptosis such as RAD51, DDB2, TOPBP1, p73 and caspases. MCPH1 interacts with E2F1 on the p73 promoter, and regulates p73 induction and E2F1-induced apoptosis as a result of DNA damage. MCPH1 forms oligomers through the second and third BRCT domains. An MCPH1 mutant containing only its oligomerization domain has a dominant-negative role by blocking MCPH1 binding to E2F1. It also inhibits p73 induction in DNA damage and E2F1-dependent apoptosis. Taken together, MCPH1 cooperates with E2F1 to regulate genes involved in DNA repair, checkpoint and apoptosis, and might participate in the maintenance of genomic integrity

Targeting TopBP1 at a Convergent Point of Multiple Oncogenic Pathways for Cancer Therapy

The progression of many solid tumours is driven by deregulation of multiple common pathways, particularly Rb, PI(3)K/Akt and p53. Prior studies identified TopBP1 as a key mediator for the oncogenic gain-of-function activities of mutant p53 (mutp53) in cancer. In Akt-hyperactive cancer, TopBP1 forms oligomers and represses E2F1-dependent apoptosis. Here we perform a molecular docking screening and identify a lead compound, calcein, capable of blocking TopBP1 oligomerization and p53 binding, resulting in re-activation of E2F1-dependent apoptosis and blockade of mutp53 gain-of-function. Calcein AM, the cell-permeable derivative of calcein, shows significant antitumour activity in a wide spectrum of cultured cancer cells harbouring high TopBP1 levels. These biochemical findings are recapitulated in breast cancer xenograft models. Thus, our study provides proof-of-concept evidence for targeting TopBP1, a convergent point of multiple pathways, as a cancer therapy

Targeting TopBP1 at a Convergent Point of Multiple Oncogenic Pathways for Cancer Therapy

The progression of many solid tumours is driven by deregulation of multiple common pathways, particularly Rb, PI(3)K/Akt and p53. Prior studies identified TopBP1 as a key mediator for the oncogenic gain-of-function activities of mutant p53 (mutp53) in cancer. In Akt-hyperactive cancer, TopBP1 forms oligomers and represses E2F1-dependent apoptosis. Here we perform a molecular docking screening and identify a lead compound, calcein, capable of blocking TopBP1 oligomerization and p53 binding, resulting in re-activation of E2F1-dependent apoptosis and blockade of mutp53 gain-of-function. Calcein AM, the cell-permeable derivative of calcein, shows significant antitumour activity in a wide spectrum of cultured cancer cells harbouring high TopBP1 levels. These biochemical findings are recapitulated in breast cancer xenograft models. Thus, our study provides proof-of-concept evidence for targeting TopBP1, a convergent point of multiple pathways, as a cancer therapy

Associations of Warfarin Use with Risks of Ischemic Cerebrovascular Events and Major Bleeding in Patients with Hyperthyroidism-Related Atrial Fibrillation

The use of oral anticoagulants for patients with new-onset hyperthyroidism-related atrial fibrillation (AF) is controversial. We aimed to evaluate the clinical benefits of warfarin therapy in this population. This retrospective cohort study used a data-cut of Taiwan Health and Welfare Database between 2000 and 2016. We compared warfarin users and nonusers among AF patients with hyperthyroidism. We used 1:2 propensity score matching to balance covariates and Cox regression model to calculate hazard ratios (HRs). The primary outcome was risk of ischemic stroke/transient ischemic attack (TIA), and the secondary outcome was major bleeding. After propensity score matching, we defined 90 and 168 hyperthyroidism-related AF patients with mean (SD) age of 59.9 ± 13.5 and 59.2 ± 14.6 in the warfarin-treated group and untreated group separately. The mean (SD) CHA2DS2-VASc scores for the two groups were 2.1 ± 1.6 and 1.8 ± 1.5, respectively. Patients with hyperthyroidism-related AF receiving warfarin had no significant risk of ischemic stroke/TIA (adjusted HR: 1.16, 95% confidence interval [CI]: 0.52–2.56, p = 0.717) compared to nonusers. There was a comparable risk of major bleeding between those receiving warfarin or not (adjusted HR: 0.91, 95% CI: 0.56–1.47, p = 0.702). The active-comparator design also demonstrated that warfarin use had no significant association with the risk of stroke/TIA versus aspirin use (adjusted HR: 2.43; 95% CI: 0.68–8.70). In conclusion, anticoagulation therapy did not have a statistically significant benefit on ischemic stroke/TIA nor risk of bleeding, among patients with new-onset hyperthyroidism-related AF under a low CHA2DS2-VASc score, by comparing those without use