Tousled-Like Kinase-Dependent Phosphorylation of Rad9 Plays a Role in Cell Cycle Progression and G2/M Checkpoint Exit

Abstract

Genomic integrity is preserved by checkpoints, which act to delay cell cycle progression in the presence of DNA damage or replication stress. The heterotrimeric Rad9-Rad1-Hus1 (9-1-1) complex is a PCNA-like clamp that is loaded onto DNA at structures resulting from damage and is important for initiating and maintaining the checkpoint response. Rad9 possesses a C-terminal tail that is phosphorylated constitutively and in response to cell cycle position and DNA damage. Previous studies have identified tousled-like kinase 1 (TLK1) as a kinase that may modify Rad9. Here we show that Rad9 is phosphorylated in a TLK-dependent manner in vitro and in vivo, and that T355 within the C-terminal tail is the primary targeted residue. Phosphorylation of Rad9 at T355 is quickly reduced upon exposure to ionizing radiation before returning to baseline later in the damage response. We also show that TLK1 and Rad9 interact constitutively, and that this interaction is enhanced in chromatin-bound Rad9 at later stages of the damage response. Furthermore, we demonstrate via siRNA-mediated depletion that TLK1 is required for progression through S-phase in normally cycling cells, and that cells lacking TLK1 display a prolonged G2/M arrest upon exposure to ionizing radiation, a phenotype that is mimicked by over-expression of a Rad9-T355A mutant. Given that TLK1 has previously been shown to be transiently inactivated upon phosphorylation by Chk1 in response to DNA damage, we propose that TLK1 and Chk1 act in concert to modulate the phosphorylation status of Rad9, which in turn serves to regulate the DNA damage response.

Tousled kinase activator, gallic acid, promotes homologous recombinational repair and suppresses radiation cytotoxicity in salivary gland cells

“…The transfection of siRNA TLK1 slows S-phase kinetics 7, 28 , and the decrease in homology-driven repair in NS-SV-AC shRNA-TLK1 cells alludes to the requirement of replicated sister chromatids in HR. Our findings corroborate a contributory role of TLK1 in HR.…”

“…Rad9, a substrate of TLK1, forms a complex with Rad1 and Hus1 to detect and coordinate DNA repair 4 . TLK1 phosphorylates Rad9 S328 and T355 within its C-terminal tail, and phosphorylated Rad9 is required to overcome cell arrest in late stages of DNA damage response to radiation 28 . Rad9 interacts with Rad51, a protein that is instrumental for strand invasion into homologous sequences in sister chromatids during HR 29 The preferential stimulation of HR by GA is not surprising given the interaction of TLK1 with Rad9.…”

Modulation of cell morphogenesis by tousled‐like kinase in the Drosophila follicle cell

“…In both mammalian cells and Drosophila , TLK phosphorylates Anti‐Silencing Function 1 (ASF1), a Histone H3/H4 chaperone and a chromatin assembly factor involved in DNA repair and chromatin assembly (Sillje and Nigg, ; Carrera et al, ). Rad9, a component of the heterotrimeric Rad9/Rad1/Hus1 complex involved in DNA damage response, is phosphorylated by Tlk (Canfield et al, ; Sunavala‐Dossabhoy and De Benedetti, ; Kelly and Davey, ). In addition, Tlk phosphorylates a splicing factor P element Somatic Inhibitor (PSI) to modulate pre‐mRNA splicing in Drosophila (Taliaferro et al, ).…”

“…These findings demonstrate critical roles of Tlk in cell cycle progression. Furthermore, TLK1B, a human homolog of Tlk, is involved in double‐strand break repair and DNA damage response, suggesting a role of Tlk in cell cycle checkpoint (Canfield et al, ; Sunavala‐Dossabhoy and De Benedetti, ; Kelly and Davey, ). By using C. elegans and Drosophila as models, roles of Tlk in chromosome segregation have been demonstrated in vivo (Li et al, ; Yeh et al, ).…”

“…Scale bars ¼ 20 mm. strand break repair and DNA damage response, suggesting a role of Tlk in cell cycle checkpoint (Canfield et al, 2009;Sunavala-Dossabhoy and De Benedetti, 2009;Kelly and Davey, 2013). By using C. elegans and Drosophila as models, roles of Tlk in chromosome segregation have been demonstrated in vivo Yeh et al, 2010).…”