TLK1B mediated phosphorylation of Rad9 regulates its nuclear/cytoplasmic localization and cell cycle checkpoint

BMC Molecular Biology

Research Article

Sanket Awate¹  Arrigo De Benedetti¹ 

[1] Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, 1501 Kings Highway, 71130, Shreveport, LA, USA;

关键词: DNA damage response;    Replication stress;    TLK1;    TLK1B;    Rad9;    pRad9 S328;    WRN;    γH2AX;    9-1-1 complex;   

DOI  :  10.1186/s12867-016-0056-x

 received in 2015-10-05, accepted in 2016-01-26,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundThe Tousled like kinase 1B (TLK1B) is critical for DNA repair and survival of cells. Upon DNA damage, Chk1 phosphorylates TLK1B at S457 leading to its transient inhibition. Once TLK1B regains its kinase activity it phosphorylates Rad9 at S328. In this work we investigated the significance of this mechanism by overexpressing mutant TLK1B in which the inhibitory phosphorylation site was eliminated.Results and discussionThese cells expressing TLK1B resistant to DNA damage showed constitutive phosphorylation of Rad9 S328 that occurred even in the presence of hydroxyurea (HU), and this resulted in a delayed checkpoint recovery. One possible explanation was that premature phosphorylation of Rad9 caused its dissociation from 9-1-1 at stalled replication forks, resulting in their collapse and prolonged activation of the S-phase checkpoint. We found that phosphorylation of Rad9 at S328 results in its dissociation from chromatin and redistribution to the cytoplasm. This results in double stranded breaks formation with concomitant activation of ATM and phosphorylation of H2AX. Furthermore, a Rad9 (S328D) phosphomimic mutant was exclusively localized to the cytoplasm and not the chromatin. Another Rad9 phosphomimic mutant (T355D), which is also a site phosphorylated by TLK1, localized normally. In cells expressing the mutant TLK1B treated with HU, Rad9 association with Hus1 and WRN was greatly reduced, suggesting again that its phosphorylation causes its premature release from stalled forks.ConclusionsWe propose that normally, the inactivation of TLK1B following replication arrest and genotoxic stress functions to allow the retention of 9-1-1 at the sites of damage or stalled forks. Following reactivation of TLK1B, whose synthesis is concomitantly induced by genotoxins, Rad9 is hyperphosphorylated at S328, resulting in its dissociation and inactivation of the checkpoint that occurs once repair is complete.

【 授权许可】

CC BY   
© Awate and De Benedetti. 2016