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.
Keywords: ATR; Nek1; TLK1; cell cycle checkpoint activation; regulation of DNA damage response; replication stress.