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. 2017 May 19;16(10):915-926.
doi: 10.1080/15384101.2017.1314421. Epub 2017 Apr 20.

Identification of the Proteome Complement of humanTLK1 Reveals It Binds and Phosphorylates NEK1 Regulating Its Activity

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Identification of the Proteome Complement of humanTLK1 Reveals It Binds and Phosphorylates NEK1 Regulating Its Activity

Vibha Singh et al. Cell Cycle. .
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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.

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

Figures

Figure 1
Figure 1
. TLK1B binds NEK1. (A) NEK1 was pulled-out with GST-TLK1B, but not GST, from 0.3 mg of Hek293 cell extract. (B) Immunoprecipitated NEK1 was treated with CIP to establish the mobility of the phosphorylated form. (C) The interaction between TLK1 and NEK1 is strengthened upon DNA damage. Hek293 cells were exposed to 0.2 mM H2O2 for the indicated time. Extracts were prepared and immunoprecipitated with TLK1 antiserum and probed for coIP with NEK1 serum, or for TLK1 (bottom). EXT (cell extract).
Figure 2.
Figure 2.
Colocalization of TLK1, NEK1, and γH2AX at sites of DNA damage following incubation with doxorubicin. A. Cells treated with or without doxorubicin (3uM) and then processed for IF as described in. Cells were imaged on the Perkin elmer Mantra scope (20x). Raw files were obtained using the InForm. Colocalization layover was applied using InForm software which generates a pseudo pixel (yellow) where the 2 fluors colocalize. Antibodies used were: Rabbit anti-NEK1, Rabbit anti-TLK1, mouse anit γH2AX. B. Microscopy was performed following treatment with doxorubicin. Imaged were obtained at 60X and analyzed with Nikon software. Colocalization of NEK1 and TLK1 is shown in pink.
Figure 3.
Figure 3.
TLK1 phosphorylates in vitro preferentially NEK1N-terminal fragment (NT). 3 µg of Ni-Sepharose purified recombinant NEK1-CT and NEK1-NT proteins expressed in bacteria following induction with IPTG were phosphorylated in a kinase reaction using 50 ng recombinant TLK1B and γP32-ATP. The samples were separated by SDS/PAGE and either exposed to film (autoradiography) or immunoblotted with anti-His antibody.
Figure 4.
Figure 4.
TLK1B addition stimulates the NEK1 kinase activity in vitro. (A) Kinase reactions (see methods) contained 100 ng/µl of the indicated recombinant proteins and 250 ng/µl β-casein as the substrate. The supplemented reaction with NEK1 contained 5 ng/µl TLK1B. After separation on a 10% PAGE, the gel was stained with coomassie blue (CB), dried and exposed to XR film for 4 h. (B) Phosphorylation of NEK1 by TLK1B occurs on T141. In kinase reactions, 1 µg of recombinant NEK1 or the NEK1-T141A mutant were incubated in absence or presence of 50 ng TLK1B. After separation on an 8% PAGE, the gel was stained with coomassie blue (CB), dried and exposed to XR film for 4 h. Note that the autophosphorylation of NEK1 was similar and weak for both wt and T141A NEK1 proteins. (C) The NEK1-T141A mutant kinase is not activated by incubation with TLK1B. Reactions were like in (A) but in addition we tested the activity of NEK1-T141A kinase.
Figure 5.
Figure 5.
(A and B). The activity of NEK1 is cell cycle regulated. DU145 cells were arrested enriched in G1/S with HU, synchronously released in S after washing with fresh medium, and then arrested in M with nocodazole (see panel B). Cell extracts from equal cell numbers were prepared and NEK1 was isolated by IP (see methods) and tested for activity using β-casein as the substrate (panel A). C) The activity of NEK1 increases following addition of doxorubicin, but reduced by concomitant addition of THD. Cells were treated as described in Methods, and NEK1 was IP'd and tested for activity in vitro using β-casein.
Figure 6.
Figure 6.
Expression of some dominant KD mutants of TLK1B result in loss of phosphorylated NEK1. (Left) Expression of TLK1B mutants in stably transfected 293T cells. (Right) WB of NEK1, where the main 2 mobility forms of the protein are indicated as NEK1 and pNEK1.
Figure 7.
Figure 7.
(A) The activation of ATR (autophosphorylation at T1989) after 6h incubation with hydroxyurea (HU) is strongly reduced by concomitant addition of the TLK1 inhibitor, thioridazine (THD, 10 µM). (B) Activation of ATR (pATR-T1989) is impaired following 1h incubation with 0.2 mM H2O2 in cells expressing NEK1-T141A in contrast to wt-NEK1 expressing cells. Where indicated, siRNA to nek1 (si1) was added 24h before H2O2. (C) Activation of Chk1 (pChk1-S345) is reduced in cells overexpressing NEK1-T141A.
Figure 8.
Figure 8.
(A) Expression of endogenous NEK1 in control H3k293 (293) cells and in and stably transfected NEK1 overexpressing constructs. The control and NEK1 overexpressing cells (siRNA resistant constructs) were treated for 18h with 2 different siRNAs, and cell extracts were prepared and immunoblotted for NEK1 and actin. (B) Hek293 controls and cells overexpressing wt-NEK1 or the NEK1-T141A mutants were treated (or not) with si1 to knockdown endogenous NEK1. Cells were then treated with H2O2 for 4h, as described in text, and then processed for cell cycle analysis with PtdIns staining.

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