Identification of the proteome complement of humanTLK1 reveals it binds and phosphorylates NEK1 regulating its activity

Abstract

The Tousled Like kinases (TLKs) are involved in numerous cellular functions, including the DNA Damage Response (DDR), but only a handful of substrates have been identified thus far. Through a novel proteomic screen, we have now identified 165 human proteins interacting with TLK1, and we have focused this work on NEK1 because of its known role in the DDR, upstream of ATR and Chk1. TLK1 and NEK1 were found to interact by coIP, and their binding is strengthened following exposure of cells to H2O2. Following incubation with doxorubicin, TLK1 and NEK1 relocalize with nuclear repair foci along with γH2AX. TLK1 phosphorylated NEK1 at T141, which lies in the kinase domain, and caused an increase in its activity. Following DNA damage, addition of the TLK1 inhibitor, THD, or overexpression of NEK1-T141A mutant impaired ATR and Chk1 activation, indicating the existence of a TLK1>NEK1>ATR>Chk1 pathway. Indeed, overexpression of the NEK1-T141A mutant resulted in an altered cell cycle response after exposure of cells to oxidative stress, including bypass of G1 arrest and implementation of an intra S-phase checkpoint.

Targeting the TLK1/NEK1 DDR axis with Thioridazine suppresses outgrowth of androgen independent prostate tumors

“…b ), an indicator of its activation, and consistent with the G1‐arrest shown above. In contrast, the simultaneous addition of THD suppressed Chk1 phosphorylation by abrogating the TLK1 > NEK1 > ATR > Chk1 pathway as we previously showed . Bicalutamide also caused an increase in pH2AX (Fig.…”

“…But since expression of TLK1B is largely controlled at the post‐transcriptional level it may have been largely underestimated in transcriptomics analyses of PCa. Recently we have identified the proteome complement of TLK1/1B, and have identified the protein kinase Nek1 as one of its principal targets . TLK1/1B activates Nek1, by phosphorylating T141, and we established that this mechanism is an early DDR mediator upstream of ATR and Chk1 upon oxidative damage or replication arrest, extending earlier studies on Nek1 .…”

“…Recently we have identified the proteome complement of TLK1/1B, and have identified the protein kinase Nek1 as one of its principal targets . TLK1/1B activates Nek1, by phosphorylating T141, and we established that this mechanism is an early DDR mediator upstream of ATR and Chk1 upon oxidative damage or replication arrest, extending earlier studies on Nek1 . It seemed possible that a similar pathway becomes activated upon AR inhibition by ADT, leading to a prosurvival cell cycle arrest.…”

“…b ), but was reduced in the THD and in the combination group. Finally, it seemed possible that bypass of the DDR elicited by bicalutamide via THD‐mediated inhibition of the TLK1/Nek1 axis would cause the emergency activation of the P53 > p21 pathway, as shown for treatment of LNCaP‐C42B cells with a Chk1 inhibitor . Indeed, there was a strong increase in p21 expression in the combination treatment group (Fig.…”

“…All PCa cell lines were obtained from the ATCC, recently authenticated, and cultured according to their guidelines. LNCaP cells stably overexpressing Nek1 (wt and mut) were generated as previously described …”