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Article
Phosphoproteomics Analysis Reveals a Potential Role of CHK1 in Regulation of Innate Immunity through IRF3
Zhen Chen, Chao Wang, Caoqi Lei, Xu Feng, Chen Li, Sung Yun Jung, Jun Qin, and Junjie Chen
J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.9b00829 • Publication Date (Web): 21 Apr 2020
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Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Phosphoproteomics Analysis Reveals a Potential Role of CHK1 in Regulation of
Innate Immunity through IRF3
Zhen Chen1, Chao Wang1, Caoqi Lei1,2, Xu Feng1, Chen Li2, Sung Yun Jung3, Jun Qin3,
and Junjie Chen1,*
Anderson Cancer Center, Houston, TX 77030, USA
2Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan
University, Wuhan 430072, China
3Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston,
TX 77030, USA
*Corresponding author:
E-mail: [email protected]
ABSTRACT
Inhibitors of checkpoint kinase 1 (CHK1), a central component of DNA damage and cell
cycle checkpoint response, represent a promising new cancer therapy, but the global
9
10 cellular functions they regulate through phosphorylation are poorly understood. To
11
12 elucidate the CHK1-regulated phosphorylation network, we performed a global
14
15 quantitative phosphoproteomics analysis, which revealed 142 phosphosites whose
16
17 phosphorylation levels were significantly different following treatment with the CHK1
18
19 inhibitor SCH 900776. Bioinformatics analysis identified phosphoproteins that function in

22 ATR-CHK1 signaling, DNA replication, and DNA repair. Furthermore, IRF3
23
24 phosphorylation at S173 and S175 was significantly reduced following treatment with
25
26 SCH 900776. Our findings indicate that the CHK1-dependent regulation of IRF3
27
28 phosphorylation at S173 and S175 may play a role in the induction of innate immune
30
31 response after replication stress or DNA damage, which suggests a potential function of
32
33 CHK1 in the innate immune response. Data are available via ProteomeXchange with
34
35 identifier PXD015125.

Keywords
CHK1, Chk1 kinase inhibitor, Phosphoproteomics, IRF3, innate immunity
INTRODUCTION
4
5 Checkpoint kinase 1 (CHK1), a serine/threonine protein kinase that is conserved from
7
8 yeast to human, mainly functions to control cell cycle progression. CHK1 activation
9
10 initiates cell cycle arrest, DNA repair, and cell death to prevent damaged cells from
11
12 progressing through the cell cycle 1. Of the two main kinase signaling pathways
14
15 activated by DNA damage or replication stress, ATR-CHK1 and ATM-CHK2 pathways,
16
17 the ATR-CHK1 signaling pathway is activated more strongly when DNA replication is
18
19 impeded 2. Based on our current understanding, when DNA replication is blocked or

22 stalled, replication protein A (RPA)-coated single-strand DNA (ssDNA) recruits and
23
24 activates ATR, which subsequently activates CHK1. In CHK1, the SQ/TQ motif sites
25
26 S317, S345, and S366 are target of ATM/ATR-dependent phosphorylation 3. The CHK1
27
28 autophosphorylation site, S296, is phosphorylated following CHK1 activation. Activated
30
31 CHK1 kinase acts on both nuclear and cytoplasmic substrates to regulate cell cycle
32
33 progression and other functions 4.
34
35 The best known CHK1 substrates are the CDC25 family phosphatases. In
37
38 mammalian cells, CDC25 has three isoforms, all of which are phosphorylated by CHK1.
39
40 CDC25A phosphorylation by CHK1 promotes CDC25A’s proteasome-mediated
41
42 degradation and leads to the inhibition of CDK1 and CDK2, thereby resulting in cell
cycle arrest at G1/S transition, S phase, or G2/M transition 5-6. CDC25B phosphorylation
46
47 by CHK1 leads to CDC25B’s sequestration from the centrosome and the inhibition of
48
49 centrosomal CDK1 7. CDC25C phosphorylation by CHK1 promotes CDC25C’s binding
50
51 to 14-3-3 proteins, inhibits the activation of CDK1-cyclin B1, and leads to G2 arrest 8. In
53
54 addition to these CDC25 isoforms, the replication initiation protein treslin has also been
found to bind to CHK1 via its C-terminal region and is phosphorylated by CHK1.
4
5 Disruption of treslin-CHK1 interaction increases DNA replication initiation 9, which
7
8 suggests that CHK1 normally acts to prevent DNA replication. Furthermore, CHK1 has
9
10 been shown to play roles in spindle checkpoint control through its regulation of Aurora B
11
12 kinase and BubR1 10.
14
15 Inhibition of the DNA damage response can enhance the anticancer efficacy of
16
17 chemotherapy and radiotherapy and selectively kill certain cancer cells. Because the
18
19 ATR-CHK1 pathway is critical for the survival of highly proliferative cells as well as cells
20
21
22 enduring a variety of DNA-damaging events, the ATR-CHK1 pathway may be an ideal
23
24 therapeutic target, as its inhibition, either alone or in combination with chemotherapeutic
25
26 agents that induce diverse genotoxic lesions, may selectively kill highly proliferative
27
28 cancer cells that under constant oncogenic stress 11. Thus, many groups have been
30
31 devoted to the development of CHK1 inhibitors 12. UCN-01, the first CHK1 inhibitor to be
32
33 developed, has limited clinical application owing to its lack of specificity and short half-
34
35 life 13-14. AZD7762, an ATP-competitive drug and potent CHK1/2 dual inhibitor, was in a
37
38 clinical trial that was terminated owing to the drug’s cardiac toxicity and multiple adverse
39
40 effects 15-16. More recently, researchers have developed several selective CHK1
41
42 inhibitors, including LY2603618, CHIR-124, SCH 900776, PF-00477736, and
LY2606368. Of these, LY2603618 was the first to be developed 17 and is the most
46
47 highly selective 18; however, clinical trials of the drug have not yet produced promising
48
49 results. CHIR-124 disrupts the S and G2-M checkpoints by interfering with CHK1
50
51 intracellular signaling and enhances the antitumor activity of topoisomerase I poisons 19.
53
54 Combined with DNA antimetabolites agents, such as hydroxyurea (HU), SCH 900776
(also called MK-8776) induces double strand breaks and cell death 20. PF-00477736
4
5 abrogates DNA damageinduced cell cycle arrest and enhances the cytotoxicity of
7
8 clinically proven chemotherapeutic agents such as gemcitabine and carboplatin 21.
9
10 LY2606368, a CHK1/CHK2 dual inhibitor, preferentially inhibits CHK1 in vitro, which
11
12 results in increased CDC25A activity and leads to the activation of CDK2 22. Several
14
15 other CHK1 inhibitors, including CCT244747 23, SAR-020106 24, MCL1020 25, and
16
17 MU380 26 are being developed and tested preclinically. Although many CHK1 inhibitors
18
19 have shown promising results in vitro, they have not yielded impressive outcomes in
21
22 clinical trials 12. Improving the clinical application of CHK1 inhibitors requires further
23
24 investigation, such as developing a new generation of CHK1 inhibitors that have fewer
25
26 side effects and establishing inhibitor-specific biomarkers that can be used to
28
29 personalize treatment. However, the biological functions and global cell signaling
30
31 network regulated by CHK1 remain unclear, and this prevents the further development
32
33 of suitable CHK1 inhibitors for clinical applications and the optimization of CHK1
34
35 inhibitorbased combination therapies.
37
38 To elucidate the way in which cells respond to CHK1 inhibition, we conducted a
39
40 global quantitative phosphoproteomics analysis to identify phosphosites whose
phosphorylation levels are significantly different after CHK1 inhibition. We cultured
44
45 HEK293A cells in SILAC (stable isotope labeling by amino acids in cell culture) media.
46
47 We then treated the cells with HU with or without a CHK1 inhibitor (SCH 900776). SCH
48
49 900776 was shown as a functionally optimal CHK1 inhibitor with minimal intrinsic
51
52 antagonistic properties 20. Our quantitative phosphoproteomics analysis identified
53
54 19,921 phosphosites. The phosphorylation levels of 142 of these phosphosites were

Page 6 of 51

1
2
3 significantly changed after the cells were treated with HU plus SCH 900776. The quality
4
5 of this global phosphoproteomics analysis was confirmed by its identification of several
7
8 key proteins involved in the ATR-CHK1 signaling pathway. Furthermore, we found that
9
10 IRF3 phosphorylation at S173 and S175 was significantly reduced after treatment with
11
12 HU and SCH 900776, indicating that CHK1 may have a previously unknown function in
14
15 regulating innate immunity through IRF3 phosphorylation.
MATERIALS AND METHODS
Cell Culture and Transfection
27
28 HEK293A and THP-1 cells were purchased from ATCC (Manassas). Mouse lung
30
31 fibroblasts (MLFs) were isolated as described in previous study 27.
32
33 For plasmid transfection, cells were seeded in 6-well plates. The next day, 2 µg
34
35 DNA was mixed with polyethylenimines, and Opti-MEM (ThermoFisher Scientific), and
37
38 then the mixture was added into one well of each plate. After incubation for 18-24 h, the
39
40 cells were collected or treated as indicated.
Plasmids and Antibodies
46
47 The homemade plasmids were generated with PCR and subcloned into the pDONR201
48
49 vector using Gateway Technology (Invitrogen) for use as the entry clones and then
50
51 recombined into destination vectors for the expression of tagged fusion proteins. PCR-
53
54 mediated site-directed mutagenesis was used to generate serial point mutations as
indicated. ISRE-Luc reporter was a gift from Dr. Pinglong Xu (Zhejiang University,
4
5 Hangzhou). The other constructs were purchased from Addgene.
7
8 AntiCHK1 (2360S), antiCHK1 S345 (2348S), antiCHK1 S296 (2349S), and
9
10 anti IRF3 (4302S) antibodies were purchased from Cell Signaling Technology and used
at 1:1000 dilution. Antiα-tubulin (T6199) and antiFlag M2 (F3165) monoclonal
14
15 antibodies were purchased from Sigma-Aldrich and used at 1:5000 dilution. Antic-Myc
(9E10) monoclonal antibody was purchased from Santa Cruz Biotechnology and used
19
20 at 1:1000 dilution.

24 SILAC and Protein Digestion
26
27 HEK293A cells were labeled by passaging the cells 8 times in DMEM media for SILAC
28
29 (A33822; Thermo Fisher Scientific) containing L-arginine (Arg 0) and L-lysine (Lys 0;
30
31 “light”) or containing L-arginine-U-13C615N4 (Arg 10) and L-lysine-U-13C615N2 (Lys 8
33
34 “heavy”). Labeling efficiency was tested periodically using MS.
35
36 Heavy labeled HEK293A cells were treated with 2 mM HU and 1 μM SCH
37
38 900776. After 1 h, the treated cells were harvested and combined with the light labeled
control cells, which were treated with only 2 mM HU for 1 h. A biological repeat was
42
43 conducted using the same procedure but with reverse SILAC labeling.
44
45 Cells were subjected to lysis in NETN buffer at 4°C for 20 min. Crude lysates
46
47 were subjected to centrifugation at 14,000 rpm for 30 min at 4°C. Supernatants were
49
50 taken as the soluble fraction. Proteins in the pellet were extracted by sonication and
51
52 taken as the chromatin fraction.
Protein lysates were reduced in 5 mM dithiothreitol for 1 h at 56°C and then
4
5 subjected to alkylation with 20 mM iodoacetamide for 45 min at room temperature in the
7
8 dark. The treated proteins were precipitated in 80% acetone at -20°C overnight, and the
9
10 precipitants were resuspended in 8 M urea. The protein concentrations were
11
12 determined using the Bradford method. Then 2 mg of the proteins were diluted to 0.8 M
14
15 urea with 50 mM NH4HCO3, pH 8.5, and digested with trypsin (1:50) for 18 h at 37°C.
16
17 The tryptic peptides were desalted using a Sep-Pak C18 column (Cen-Med Enterprise
19 Inc).
Phosphopeptide Enrichment
25
26 Phosphopeptide enrichment was performed as described previously 28 with slight
27
28 modifications. Desalted and dried peptides were suspended in a solution with 300 mM
30
31 KCl, 5 mM KH2PO4, 50% acetonitrile (ACN), and 6% trifluoroacetic acid (TFA). TiO2
32
33 beads were weighed at a ratio of 10:1 to protein, and resuspended in loading buffer
34
35 (80% ACN, 6% TFA) at a concentration of 100 μL loading buffer per sample. The TiO2
37
38 beads and peptides were incubated in a ThermoMixer at 2,000 rpm for 5 min at 40°C.
39
40 The beads with the bounded peptides were washed with wash buffer (60% ACN, 1%
41
42 TFA) 5 times to remove the non-specifically binding peptides. For the second
enrichment, the solution was incubated with new TiO2 beads following the same
46
47 process described above. After the final wash, 100 μL of transfer buffer (80% ACN,
48
49 0.5% acetic acid) was added to each sample, which was then incubated in a
50
51 ThermoMixer at 2,000 rpm for 30 seconds at room temperature to resuspend the beads.
53
54 The beads were transferred on top of a C18 (single layer) StageTip. Phosphopeptides
were eluted 2 times with elution buffer (40% ACN, 15% NH4OH [25%, HPLC grade])
and then vacuum dried.
Peptide Identification and Quantification
11
12 Enriched peptides were fractionated using a micro-pipette tip packed with high pH C18
14
15 (Reprosil-Pur Basic C18, 3 μm) as described previously 29. Vacuum-dried peptides were
16
17 dissolved with ammonium bicarbonate buffer (10 mM, pH 10) and transfered to the C18
18
19 pipette tips. Given an ACN gradient of 5%35% in 10 mM NH4HCO3, pH 10, buffer, 12
21
22 fractions for each sample were collected and vacuum dried. The peptides were
23
24 reconstituted in HPLC solvent A (2.5% ACN, 0.1% formic acid), delivered onto an
25
26 EASY-nLC II liquid chromatography pump (Thermo Fisher Scientific), and eluted with
28
29 ACN gradient by increasing concentrations of solvent B (97.5% ACN, 0.1% formic acid)
30
31 from 6% to 30% in 30 mins. The eluates directly entered Orbitrap Elite MS (Thermo
32
33 Fisher Scientific), setting in positive ion mode and data-dependent manner with full MS
34
35 scan from 350-1250 m/z, resolution at 60,000, automatic gain control target at 1×106.
37
38 The top 10 precursors were then selected for MS/MS analysis.

42 MS Data Analysis
44
45 The MS/MS spectra were used to search MaxQuant software program (version 1.5.2.8;
46
47 Max Planck Institute of Biochemistry). Database searching included all entries from the
48
49 human Uniprot database (October 2016, 20,161 entries). Enzyme specificity was set to
51
52 tryptic with 2 missed cleavages. Carboxyamidomethyl for cysteine (+57.021 Da) was set
53
54 as static modification; heavy SILAC labeling, phosphorylation for serine, threonine, and

3 tyrosine residues, and oxidation for methionine residues were set as variable
4
5 modifications. Mass tolerance was set to 20 ppm for peptide and 0.5 Da for MS/MS.
7
8 The identified peptides were filtered using a false discovery rate < 1% based on the
9
10 target-decoy method. The common contaminants were excluded.
11
12 Peptides and proteins quantification was done by MaxQuant. Further data
14
15 analysis of the proteomics results was done with Microsoft Excel and R statistical
16
17 computing software. Phosphopeptides with a median fold-change greater than 1.5
18
19 between the cells treated with or without SCH 900776 were considered to be
20
21
22 differentially expressed. The localization probability cut-off for phosphorylation sites was
23
24 setting at 0.75. Conserved motif analysis was done by WebLogo. Function annotation
25
26 was done using Ingenuity Pathway Analysis software (QIAGEN) and reference mining.
27
Pulldown Assay
32
33 In the pulldown assay, 1  107 cells were harvested and lysed with NETN buffer. Then
34
35 the cell lysates were incubated with 20 L of conjugated S-beads for 2 h at 4°C. The
37
38 beads were washed three times with NETN buffer, boiled in 2 Laemmli buffer, and
39
40 then subjected to Western blot analysis 30.
In Vitro Kinase Assay
46
47 Recombinant WT IRF3, S173A mutant, S175A mutant, and S173A/S175A double
49
50 mutant forms were expressed in bacteria fused with GST protein tag and purified using
51
52 glutathione sepharose beads (17-0756-05; GE Healthcare). Active recombinant CHK1
53
54 (SRP5278; Sigma-Aldrich) and γ-32P-ATP (NEG002A100UC; PerkinElmer) were mixed

with recombinant proteins in a kinase buffer (25 mM Tris, pH 7.5, 5 mM β-
4
5 glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, 10 mM MgCl2). The samples
7
8 were incubated at 30°C for 20 minutes, and then stopped via boiling. The proteins were
9
10 separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gel
11
12 was dried and imaged by using the Phosphorlmager software program.
14
Real-Time PCR
18
19 The THP-1 or MLFs cells were treated with DNA damaging reagents, etoposide or
camptothecin, and then incubated with CHK1 inhibitor at different concentrations. Total
23
24 RNA was isolated from cells using Trizol reagent. After reverse-transcription with oligo
25
26 (dT) primer using a RevertAidTM First Strand cDNA Synthesis Kit (Fermentas), the
27
28 samples were subjected to qPCR analysis to measure the mRNA expression levels of
30
31 the tested genes. Data are presented as the relative abundance of the indicated
32
33 mRNAs normalized to that of GAPDH or Gapdh. qPCR was performed using the
34
35 following primers:
37
38 Human GAPDH: Forward- GACAAGCTTCCCGTTCTCAG;
39
40 Reverse- GAGTCAACGGATTTGGTCGT.
41
42 Human IFNB1: Forward- CTTGGATTCCTACAAAGAAGCAGC;
43
44
45 Reverse- TCCTCCTTCTGGAACTGCTGCA.
46
47 Human ISG56: Forward- TCATCAGGTCAAGGATAGTC;
48
49 Reverse- CCACACTGTATTTGGTGTCTAGG.
50
51 Human CXCL1: Forward- AGCTTGCCTCAATCCTGCATCC;
53
54 Reverse- TCCTTCAGGAACAGCCACCAGT.
Human CXCL2: Forward- AATGGCAAATCCAACTGACCA;
4
5 Reverse- CTGTTCCTGTAAGGGCAGG.
7
8 Human CXCL10: Forward- GGTGAGAAGAGATGTCTGAATCC;
9
10 Reverse- GTCCATCCTTGGAAGCACTGCA.
11
12 Murine Gapdh: Forward- ACGGCCGCATCTTCTTGTGCA;
14
15 Reverse- ACGGCCAAATCCGTTCACACC
16
17 Murine Ifnb: Forward- TCCTGCTGTGCTTCTCCACCACA;
18
19 Reverse- AAGTCCGCCCTGTAGGTGAGGTT.
20
21
22 Murine Cxcl1: Forward- TCCAGAGCTTGAAGGTGTTGCC;
23
24 Reverse- AACCAAGGGAGCTTCAGGGTCA.
25
26 Murine Cxcl2: Forward- CATCCAGAGCTTGAGTGTGACG;
27
28 Reverse- GGCTTCAGGGTCAAGGCAAACT.
30
31 Murine Isg56: Forward- TACAGGCTGGAGTGTGCTGAGA;
32
33 Reverse- CTCCACTTTCAGAGCCTTCGCA.
34
35 Murine Cxcl10: Forward- ATCATCCCTGCGAGCCTATCCT;
37
38 Reverse- GACCTTTTTTGGCTAAACGCTTTC.
Luciferase Reporter Assay
43
44
45 293T Cells were transfected with 5 x ISRE-Luc reporter, a IRF3-responsive IFNβ
46
47 Luciferase reporter, which bears an ORF coding firefly luciferase, along with the pRL-Luc
48
49 with Renilla luciferase as the internal control for transfection and other expression vectors
50
51 as specified. After 48 h of transfection, cells were lysed by passive lysis buffer (Promega).
Luciferase assays were performed using a dual-luciferase assay kit (Promega), quantified
4
5 with Monolight 3010 (Becton Dickinson).
7
RESULTS
16
17
18
19 Global Phosphoproteomics Analysis of CHK1-dependent Phosphorylation Events
20
21
22 The way in which CHK1 regulates a variety of cellular responses, including DNA
23
24 replication and cell cycle checkpoint control, remains unknown. To improve our
25
26 understanding of CHK1-dependent phosphorylation, we performed a quantitative
27
28 phosphoproteomics analysis to assess cells’ responses to CHK1 inhibition, focusing on
30
31 changes in protein phosphorylation.
32
33 We first sought to assess ATR-dependent CHK1 phosphorylation and CHK1
34
35 activation in HEK293A cells treated with different concentrations of the CHK1 inhibitor
37
38 SCH 900776 alone or in combination with 2 mM HU (to activate CHK1 kinase) for 1 h.
39
40 Western blot analysis revealed that the cells treated with SCH 900776 alone had no
41
42 CHK1 autophosphorylation at S296, regardless of the SCH 900776 concentration, but
43
44
45 that ATR-dependent CHK1 phosphorylation at S345 increased dramatically with
46
47 increasing SCH 900776 concentration (Figure 1A). These data suggest that CHK1
48
49 normally inhibits its own activation by ATR; thus, cells under normal, unstressed
50
51 conditions show no ATR-dependent CHK1 phosphorylation or CHK1
53
54 autophosphorylation. However, cells treated with SCH 900776 may augment

3 endogenous replication stress and/or release its potential negative feedback regulation
4
5 on the ATR-CHK1 pathway and thus display increased ATR-dependent CHK1
7
8 phosphorylation following treatment with increasing concentrations of SCH 900776.
9
10 In cells treated with HU alone, both ATR-dependent CHK1 phosphorylation at
11
12 S345 and CHK1 autophosphorylation at S296 increased, indicating that HU-induced
14
15 replication stress induces the activation of the ATR-CHK1 pathway. In cells treated with
16
17 HU plus SCH 900776, CHK1 autophosphorylation at S296 gradually decreased with
18
19 increasing SCH 900776 concentration, which indicates that CHK1 kinase activity is

22 gradually inhibited with rising concentration of SCH 900776 in the cell. However, CHK1
23
24 phosphorylation at S345 did not change, indicating that HU fully activates ATR under
25
26 these conditions. Given these findings, for the phosphoproteomics analysis, we treated
27
28 cells with 1 μM SCH 900776 for 1 h to maximize the drug’s inhibitory effect.
30
31 Following the workflow shown in Figure 1B, the SILAC “light” or “heavy”
32
33 reagents labeled HEK293A cells were treated with 2 mM HU alone or in combination
34
35 with 1 μM SCH 900776 for 1 h. Then, the cells were mixed, disrupted and separated
37
38 into a soluble fraction (supernatant) and chromatin fraction (pellet). The extracted
39
40 protein lysates were digested with trypsin, and the phosphopeptides were enriched
41
42 using TiO2 beads. The samples were analyzed by LTQ Orbitrap Elite mass
43
44
45 spectrometry (MS; ThermoFisher Scientific), and the raw MS data were searched and
46
47 quantified with MaxQuant software program 31. A biological repeat was conducted with
48
49 reverse SILAC labeling. Analysis of phosphopeptide MS data with MaxQuant identified
50
51 19,921 phosphosites in 5,323 unique proteins (Figure 1C, Table S1, and Table S2).Of
53
54 these phosphosites, 7,346 were identified in soluble fraction samples, 7,993 were
identified in chromatin fraction samples, and 4,582 were identified in both chromatin and
4
5 soluble fractions (Figure 1D). Quantification analysis with MaxQuant revealed 15,030
7
8 quantified phosphopeptides. The fold change was plotted to the MS signal intensity in
9
10 the chromatin or soluble fraction (Figure 2A, and 2B). Using a fold-change greater than
11
12 1.5 in two biological repeats and a phosphorylation site localization probability higher
14
15 than 0.75 as cutoff criteria, our analysis revealed 142 phosphosites whose
16
17 phosphorylation levels were significantly different following SCH 900776 treatment
18
19 (Table S3). Among the altered phosphosites in the chromatin fraction, 65 had higher
20
21
22 phosphorylation levels in cells treated with HU plus SCH 900776 than in control cells
23
24 treated with HU alone and thus may be negatively and indirectly regulated by CHK1. In
25
26 contrast, 19 of the phosphosites had lower phosphorylation levels and are likely direct
27
28 or indirect CHK1 substrates. Among the altered phosphosites in the soluble fraction, 38
30
31 and 20 had higher and lower phosphorylation levels, respectively (Figure 2C).
Functional Analysis of Proteins Whose Phosphorylation Levels Changed in
37
38 Response to CHK1 Inhibition
39
40 To identify the function(s) and/or pathway(s) influenced by CHK1, we used Ingenuity
41
42 Pathway Analysis and a reference mining strategy to conduct a functional analysis of
the proteins our quantitative phosphoproteomics analysis revealed to have changed
46
47 phosphorylation levels following treatment with SCH 900776. Cellular location analysis
48
49 revealed that around 85% of the proteins in the chromatin fraction and 50% of those in
50
51 the soluble fraction were nucleus proteins (Figure 2D). Gene ontology enrichment
53
54 analysis of the regulated phosphoproteins revealed that most of the regulated

5 phosphorylation of these proteins may help cells rapidly develop treatment resistance
7
8 by regulating the expression of many other proteins to ensure cell survival. In addition,
9
10 many of the regulated phosphoproteins are cell cycle proteins, which agrees well with
11
12 the notion that the cell cycle is tightly controlled by CHK1-dependent replication and that
14
15 this replication is tightly linked to DNA repair and cell cycle progression 4. The analysis
16
17 also showed that many other phosphoproteins are involved in DNA repair. Thus, the
18
19 functional groups found to be enriched in the gene ontology analysis confirmed the
2 quality of our phosphoproteomics analysis (Figure 2E).
Complex Regulation of the ATR-CHK1 Pathway and DNA Replication in Response
27
28 to Replication Stress
30
31 Interestingly, a group of key proteins known to participate in ATR-CHK1 signaling were
32
33 not only identified as phosphoproteins but also found to have changed phosphorylation
34
35 levels after treatment with HU plus SCH 900776 (Figure 3A). These newly identified
37
38 phosphorylation sites and the changes in their phosphorylation levels provide insights
39
40 into the intricate regulation of the ATR-CHK1 signaling pathway under replication stress.
41
42 The RPA complex not only is essential for DNA replication but also controls DNA
43
44
45 repair and DNA damage checkpoint activation. This complex binds to and stabilizes
46
47 ssDNA intermediates. RPA-coated ssDNA is responsible for activating the ATR-CHK1
48
49 pathway 32. The level of RPA1 phosphorylation at T180 increases dramatically after
50
51 ultraviolet damage 33. Interestingly, the RPA1 phosphorylation site T180 was induced in
53
the soluble fraction after treatment with HU plus SCH 900776, indicating that CHK1
4
5 inhibits this site in response to replication stress.
7
8 ATRIP, which may recognize RPA-coated ssDNA, is an ATR binding partner and
9
10 is required for checkpoint signaling after DNA damage 34. ATRIP is also required for
11
12 ATR expression 35. ATRIP phosphorylation at S518 was significantly inhibited in the
14
15 chromatin fraction following treatment with HU plus SCH 900776, suggesting that
16
17 ATRIP phosphorylation at this site is CHK1-dependent, implying that CHK1 has
18
19 feedback regulation on the ATR-ATRIP complex.
20
21
22 Claspin, another component involved in the DNA damage/replication checkpoint
23
24 in mammalian cells, is phosphorylated in response to replication stress and binds
25
26 directly to CHK1 36. Claspin has been shown to regulate CHK1 in response to DNA
27
28 replication stress and is required for replication checkpoint control 37. Whereas Claspin
30
31 phosphorylation at S810, S839, S846, and S1289 was induced in the chromatin fraction
32
33 after treatment with HU plus SCH 900776, its phosphorylation at S720 was inhibited in
34
35 both the chromatin and soluble fractions, indicating that multiple kinases phosphorylate
37
38 and regulate Claspin following DNA damage. Besides Claspin, the Timeless-Tipin
39
40 complex is also involved in promoting ATR signaling 38. Specifically, this complex
41
42 mediates CHK1 phosphorylation by ATR in response to DNA damage or replication
43
44
45 stress 39. Timeless phosphorylation at S1087 and S1149 and Tipin phosphorylation at
46
47 S220 and S222 were activated in the chromatin fraction following treatment with HU
48
49 plus SCH 900776. Thus, our phosphoproteomics analysis uncovered several CHK1-
50
51 dependent phosphorylation sites on Timeless-Tipin, Claspin, ATRIP, and RPA, all of
which act closely with ATR and CHK1 to facilitate ATR-dependent CHK1
4
5 phosphorylation and activation at sites of DNA damage or replication stress.
7
8 PCNA acts as a scaffold to recruit proteins involved in DNA replication or DNA
9
10 repair. We found that CHK1 phosphorylates and regulates the PCNA-binding proteins
11
12 WIZ and SALL1 40 (Figure 3A). In the chromatin fraction, WIZ phosphorylation at S1134
14
15 was induced, whereas SALL1 phosphorylation at S1198 was inhibited, after treatment
16
17 with HU plus SCH 900776. CHK1 also regulated RFC1, another PCNA-loading protein
18
19 41, as RFC1 phosphorylation at S190, T193, and S312 was induced following SCH
20
21
22 900776 treatment (Figure 3A). Moreover, the Mcm2-7 complex serves as a DNA
23
24 replication helicase in eukaryotes. The MCM complex functions in both DNA replication
25
26 initiation and elongation 42-43. In our dataset, MCM2 phosphorylation at S27 and S139
27
28 and MCM4 phosphorylation at S131 were induced following SCH 900776 treatment
30
31 (Figure 3A).
32
33 We also identified many other proteins involved in DNA damage or DNA
34
35 replication whose phosphorylation levels increased following treatment with HU plus
37
38 SCH 900776, which suggests that a key function of CHK1 in the replication checkpoint
39
40 is to prevent these phosphorylation events, probably indirectly through its inhibitory
41
42 functions on cell cycle progression. Using protein-protein interaction information
43
44
45 obtained from RegPhos 2.0 44, we built a network of these phosphoproteins (Figure
46
47 3B).
48
49 That our unbiased quantitative phosphoproteomics analysis revealed the
50
51 enrichment of these phosphorylation sites on proteins known to be involved in DNA
53
54 replication and replication checkpoint control not only confirms the reliability of our

datasets but also reveals a complex regulation of the ATR-CHK1-dependent pathway
4
5 that is beyond our current understanding. Further investigation of these phosphorylation
7
8 events and their potential functions in DNA replication, DNA repair, and cell cycle
9
10 checkpoint control will improve our understanding of this complex process and its
11
12 importance for genome maintenance.
14
15
16
17 Motif Analysis of the Changed Phosphosites
18
19 We used WebGestalt 45 to assess the way these phosphoproteins are regulated and
20
21
22 identify upstream kinases besides CHK1. This analysis identified 15 phosphoproteins
23
24 from the chromatin fraction and 13 phosphoproteins from the soluble fraction that are
25
26 deposited in the database as substrates regulated by CDK1/2 (Figure 4A). The
27
28 phosphorylation levels of these phosphoproteins all increased after treatment with HU
30
31 plus SCH 900776. CDKs are central regulators that drive cell cycle progression from S
32
33 to G2 and M phase. CDKs are activated by CDC25A and CDC25C phosphatases,
34
35 which remove the inhibitory phosphorylation from CDK1 and CDK2 46. Both CDC25A
37
38 and CDC25C are known substrates of CHK1, and their CHK1-dependent
39
40 phosphorylation promotes their degradation, which leads to the inhibition of cell cycle
41
42 progression via CDK inhibition. Another CHK1 downstream protein kinase is WEE1,
43
44
45 which prevents cell-cycle progression via its inhibitory phosphorylation of CDK1 and
46
47 CDK2 47. Our unbiased phosphoproteomics analysis confirmed these modes of
48
49 regulation. Inhibition of CHK1 increased the activities of CDK1 and CDK2 and thus led
50
51 to the induction of the phosphorylation of many CDK1 and CDK2 downstream proteins
53
54 (Figure 4A). We used the software program WebLogo 48 to perform motif analyses of

these phosphosites. A (S/T)P motif, which has “P” at the +1 position right after the
4
5 serine/threonine phosphorylation site, was identified by aligning the phosphosite and the
7
8 nearby protein sequence. This motif is consistent with the reported substrate motif of
9
10 the CDK-cyclin complex 49-50. It is suggested that inhibition of CHK1 leads to the release
11
12 of CDKs and promotes mitotic entry even in the presence of DNA damage 51. This
14
15 uncontrolled cell cycle progression is believed to interfere with the mechanisms
16
17 underlying the therapeutic efficacy of combinations of DNA-damaging chemotherapeutic
18
19 agents with CHK1 inhibitors, which should trigger cell death in highly proliferative cancer
cells and enhance the therapeutic index of standard chemotherapy.
23
24 Several Ser/Thr sites in CDC25A and CDC25C have been reported to be
25
26 phosphorylated by CHK1 9, 52. We aligned these CHK1 substrate phosphorylation sites
27
28 and the nearby sequences and found a (R/K)XX(S/T) motif in these CHK1 substrates,
30
31 with a conserved arginine/lysine at the -3 position before the phosphorylated sites
32
33 (Figure S1). We analyzed the phosphosites inhibited by HU plus SCH 900776
34
35 treatment, since these sites are potential direct phosphorylation sites of CHK1. A
37
38 (K/R)XXS motif was visualized in both chromatin and soluble fractions (Figure 4B),
39
40 which indicates that many of these sites are likely directed phosphorylated by CHK1 in
41
42 response to replication stress. These newly identified phosphosites and corresponding
phosphoproteins may play important roles in ATR-CHK1 signaling and warrant further
46
47 investigation.
48
CHK1-dependent IRF3 Phosphorylation at S173 and S175 Following Replication
54 Stress
Interestingly, our quantitative phosphoproteomics analysis revealed that two serine sites
4
5 in IRF3, S173 and S175, were inhibited following treatment with SCH 900776. SCH
7
8 900776 significantly inhibited IRF3 phosphorylation at S175 in both the chromatin and
9
10 soluble fractions (Figure 5A). The IRF3 S173 site was significantly downregulated in
11
12 the chromatin fraction. Owing to the relatively poor MS signal, the phosphopeptide
14
15 containing this site in the soluble fraction was not quantifiable.
16
17 The sequence surrounding the IRF3 S173 and S175 sites is shown in Figure S2.
18
19 The S173 site is highly conserved evolutionally. The sequence surrounding S175

24 phosphorylation substrates, CDC25A and CDC25C. Pulldown experiments confirmed
25
26 the binding between CHK1 and IRF3 (Figure 5B). Next, we purified recombinant IRF3
27
28 proteins with GST tag from bacteria, including IRF3 WT, IRF3 S173A mutant, IRF3
30
31 S175A mutant, and IRF3 S173A/S175A double mutant. Then we conducted in vitro
32
33 kinase assay with these forms. The results indicated CHK1 could directly phosphorylate
34
35 IRF3, and the major CHK1 phosphorylation site of IRF3 could be S173 (Figure 5C).
37
38 Further experiments are needed to define the kinase-substrate relationship between
39
40 these two proteins.
41
CHK1 Modulates the Transcriptional Regulatory Activity of IRF3
46
47 IRF3 is a critical player in the induction of innate immune response to cytoplasmic DNA
48
49 after DNA damage. Having established that CHK1 phosphorylated IRF3 in vitro, we
50
51 further analyzed the roles of CHK1 in the regulation of the expression of inflammatory
53
54 cytokines after DNA damage. THP-1 cells and mouse lung fibroblasts (MLFs) were first

treated with the DNA-damaging reagents etoposide or camptothecin. Then, CHK1
4
5 inhibitor at different concentrations was added to the media (Figure 6A, and 6B) to
7
8 assess the effect of CHK1 on innate immune response. Quantitative polymerase chain
9
10 reaction PCR (qPCR) analysis revealed that etoposide treatment significantly
11
12 potentiated the transcription of IRF3 downstream genes such as CXCL1, CXCL2,
14
15 IFNB1, CXCL10, and ISG56 in THP-1 cells and MLFs. Upon treatment with SCH
16
17 900776, the etoposide-triggered transcription of the IRF3 downstream genes markedly
18
19 increased in a dose-dependent manner, indicating that CHK1 normally acts to inhibit
innate immune response following DNA damage.
23
24 To directly monitor the activation of IRF3, we performed luciferase assays using
25
26 the IFN-stimulated response element (ISRE) luciferase reporter. As shown in Figure
27
28 6C, the luciferase activity in cells expressing the constitutively active form of IRF3,
30
31 IRF3-5D, was significantly higher than that in cells expressing wild-type IRF3. Then, we
32
33 induced the expression of wild-type CHK1, kinase-dead CHK1, or MST1 in cells co-
34
35 expressing IRF3 and the ISRE luciferase reporter. MST1 was used as a control
37
38 because it is a negative regulator of IRF3 function that blocks cytosolic antiviral defense
39
40 through IRF3 phosphorylation 53. In cells overexpressing IRF3-5D, wild-type CHK1
41
42 functioned similar to MST1, which inhibited the activity of IRF3. This inhibition increased
as the CHK1 expression level increased. In contrast, kinase-dead CHK1 did not have
46
47 an inhibitory effect. These results indicate that the inhibition of IRF3 function may
48
49 depend on the kinase activity of CHK1.
50
51 We generated IRF3 with S173A/S175A mutations, IRF3 with S173D/S175D
53
54 mutations, IRF3-5D with S173A/S175A mutations, and IRF3-5D with S173D/S175D

mutations. We then transfected the ISRE luciferase reporter cells with these four
4
5 different IRF3 mutants and wild-type IRF3 or IRF3-5D. Compared with control cells,
7
8 cells overexpressing IRF3-5D with or without S173A/S175A mutations had significantly
9
10 higher luciferase activity. However, the luciferase activity in cells expressing IRF3 with
11
12 S173D/S175D mutations was dramatically lower than that in control cells (Figure 6D).
14
15 Second, we induced the co-expression of wild-type CHK1 or kinase-dead CHK1 and
16
17 different forms of IRF3 in ISRE luciferase reporter cells. Compared with cells expressing
18
19 wild-type CHK1, cells expressing kinase-dead CHK1 had significantly higher luciferase
24 and thus phosphorylate IRF3 at S173 and S175, may inhibit the function of IRF3.
DISCUSSION

35 phosphorylation levels changed significantly following treatment with HU plus CHK1
37
38 inhibitor SCH 900776. The functions of the proteins bearing these altered phosphosites
39
40 indicate that CHK1 and the replication checkpoint have diverse roles in controlling
41
42 transcription regulation, DNA replication, and DNA repair. We also found that SCH

45 900776 inhibited the phosphorylation of the transcriptional factor IRF3 at S173 and
46
47 S175, suggesting that CHK1 has a potential role in regulating innate immunity through
48
49 regulating IRF3 via phosphorylation.
50
51 Global phosphoproteomics analysis, by detecting changes in phosphorylation, is
53
54 a powerful technique for elucidating the kinase signaling pathways. As we reported

previously 54, the combination of CRISPR-Cas9-mediated knock out cells and global
4
5 quantitative phosphoproteomics analysis is an ideal approach to identifying downstream
7
8 phosphoproteins, including kinase substrates, regulated by AMPK kinase. In the present
9
10 study, it was difficult to generate stable CHK1 knock out cells, as CHK1 is essential for
11
12 cell survival. Because CHK1 is the key downstream kinase of the ATR-dependent
14
15 responsive pathway, many groups have developed CHK1 inhibitors for clinical
16
17 application. However, the mechanisms and cellular functions regulated by CHK1 remain
18
19 unclear. Therefore, in this study we combined global quantitative phosphoproteomics
20
21
22 analysis and kinase inhibitor treatment to study the phosphoproteome regulated by
23
24 CHK1.
25
26 We compared the list of genes with decreased phosphorylation level after Chk1
27
28 inhibitor treatment to the results reported by Blasius et al 55. With the motif analysis, the
30
31 -3 site should be conserved R/K were identified by both studies, which was also
32
33 discovered by another group, in which Gary et al. used the oriented peptide library
34
35 approach for the identification of preferred CHK1 phosphoryaltion sites 56. For the
37
38 function analysis of those identified genes in our study and the Blasius study, we both
39
40 concluded that many of these genes play important functions in transcription, RNA
41
42 process, or DNA replication/recombination/repair. We also compared the identified
43
44
45 genes in our list (32 unique genes) with those in the Blasius study (146 unique genes).
46
47 Only 4 genes were identified in both datasets. There are many reasons for the low
48
49 overlapping between these two datasets. One reason may due to the different
50
51 techniques used by these two studies. Blasius et al. used chemical genetics screen with
53
54 an analog-specific method, which attempted to capture direct phosphorylation

substrates, but may also capture non-specific substrates phosphorylated under this
4
5 condition. In our experiments, we applied global phosphoproteomics analysis, which
7
8 may identify both direct and indirect phosphorylation sites. Second, we both missed the
9
10 most studied CHK1 substrates CDC25A and CDC25C. In our experiment, we identified
11
12 five phosphorylated sites of CDC25A (including S124 which can be phosphorylated by
14
15 CHK1/CHK2), four phosphorylated sites of CDC25C (including S216 which can be
16
17 phosphorylated by CHK1/CHK2/BRSK1/MAPK14). However, none of them has been
18
19 quantified as putative substrates with phosphorylation level significantly changed after
20
21
22 CHK1 inhibitor treatment. There are some underlying technical reasons. For an
23
24 example, the abundance of some of those phosphorylation peptides were too low,
25
26 and/or the sensitivity, coverage, or the signal quality identified by mass spectrometry
27
28 were not good enough for reliable quantification analysis. There are also some
30
31 biological reasons that may prevent us from identifying some of these CHK1 substrates.
32
33 For example, it is known that CDC25A and CDC25C are rapidly degraded following
34
35 their phosphorylation by CHK1. This type of substrates may be challenging to identify
37
38 based on phosphoproteomics experiments. In summary, we believe that both studies
39
40 captured CHK1 substrates, however even the combined coverage with these two
41
42 techniques is still too low to uncover all or the majority of CHK1 substrates.
43
44
45 We identified many proteins are reported to be involved in the ATR-CHK1
46
47 signaling pathway and its regulation. We found that CHK1 phosphorylation at S345
48
49 increased with increasing SCH 900776 concentration. The CHK1 S345 site is
50
51 phosphorylated by ATR, which is the upstream regulator of CHK1. Our quantitative
53
54 phosphoproteomics analysis also identified many key proteins in the ATR-CHK1
signaling pathway, including RPA1, ATRIP, Claspin, Timeless, and Tipin. The
4
5 phosphorylation and activation of this group of proteins leads to the phosphorylation of
7
8 CHK1 at S317 and S345 by ATR to activate CHK1 kinase. In the present study, all
9
10 these proteins’ phosphorylation levels were significantly different following SCH 900776
11
12 treatment. With the exception of Claspin phosphorylation at S720, these proteins’
14
15 phosphorylation levels were all increased following treatment with SCH 900776. These
16
17 results indicate the presence of feedback regulation mechanism in the ATR-CHK1
18
19 signaling pathway. Given these findings, we hypothesize that CHK1 kinase is activated
20
21
22 following DNA damage-induced replication stress. The inhibition of CHK1 activity
23
24 evokes a feedback mechanism in which a group of upstream proteins are
25
26 phosphorylated to activate the ATR-CHK1 signaling pathway to ensure cell cycle control
27
28 and genome maintenance. Precisely how this feedback regulation is controlled requires
30
31 further investigation.
32
33 In our phosphoproteomics study, we also found that CHK1 regulates IRF3, one
34
35 of the well-characterized transcription factors involved in innate immune response.
37
38 Recent findings suggest that IRF3 phosphorylation at S173 inhibits IRF3 activity. Gao et
39
40 al. proposed that MEEK2 is the upstream kinase that phosphorylates IRF3 to trigger its
41
42 poly-ubiquitination and block its dimerization, translocation to the nucleus, and
43
44
45 transcriptional activity after viral infection 57. Our quantitative phosphoproteomics
46
47 analysis showed that CHK1 inhibition blocked IRF3 phosphorylation at both S173 and
48
49 S175. Results of our validation analyses may suggest that S173 and S175 are two
50
51 important phosphorylation sites for CHK1 kinase regulating IRF3 activity. When these
53
54 two serine sites are phosphorylated, IRF3 function may be inhibited. Our results support
a working hypothesis that CHK1 negatively regulates IRF3 function through regulating
4
5 IRF3 phosphorylation at S173 and S175. A model of the ATR-CHK1 signaling pathway
7
8 and the regulation of IRF3 phosphorylation at S173 and S175 is shown in Figure 7. It
9
10 would be interesting to know whether replication stress and/or CHK1 would also affect
11
12 IRF3 phosphorylation at other residues, which are also important for immune response.
14
15 As shown in Figure S3, we could not detect any significant difference in the two well-
16
17 known IRF3 phosphorylation sites, S386 and S396 under our experimental conditions.
18
19 These data indicate that replication stress and CHK1 may regulate the immune
20
21
22 response through a process that is distinct from other signaling pathways, which
23
24 regulate IRF3 activity through phosphorylation of IRF3 S385, S386, or S396 residues.
25
26 Future experiments are needed to confirm this regulation and uncover the detailed
27
28 underlying mechanisms.
30
31 CHK1 has been suggested to promote tumor growth and may contribute to
32
33 therapy resistance 58. CHK1 inhibitors have been developed and tested. However, the
34
35 outcomes from clinical trials are not very impressive, suggesting that further
37
38 improvement of CHK1 inhibitors and/or their clinical applications are much needed. Our
39
40 quantitative phosphoproteomics analysis elucidated a global phosphorylation network
41
42 regulated by CHK1 and revealed many new phosphorylation sites and regulatory
43
44
45 mechanisms. This phosphorylation dataset will be valuable for the discovery of new
46
47 proteins and functions affected by CHK1 inhibitors. This information will be beneficial for
48
49 designing new treatment strategies such as combination therapies for cancer treatment.

ASSOCIATED CONTENT
4
5 Supporting Information
6
7 The Supporting Information is available free of charge on the ACS Publications website.
9
10 Figure S1 Sequence analysis of two well-known CHK1 phosphosubstrates. CDC25A and
11
12 CDC25C were reported as CHK1 phosphosubstrates. The peptide sequence of six serine
13
14 sites from CDC25A and one serine site from CDC25C were analyzed. The motif assay
15
16
17 shows a conserved arginine at -3 site.
18
19 Figure S2 IRF3 S173 and S175 sequence analysis. The peptide sequence around S173
20
21 and S175 were compared among different species.
22
23 Figure S3 Western blotting analysis of the two well-known IRF3 phosphorylation sites,
25
26 S386 and S396 sites, when cells were treated mock treated or treated with HU and/or
27
28 CHK1 inhibitor.
29
30 Figure S4 Uncropped Western blot scans. (A) Uncropped Western blot scans from Figure
32
33 1A. (B) Uncropped Western blot scans from Figure 5B. (C) Uncropped Western blot scans
34
35 from Figure 5C. (D) Uncropped Western blot scan from Figure 6C.
Table S1 Complete Dataset of the phosphoproteomics data. All identified proteins for
41
42 soluble and chromatin fractions with two biological repeats are listed in the data sheet.
43
44 Table S2 Complete Dataset of the phosphopeptides data. All identified phosphopeptides
45
46 for soluble and chromatin fractions with two biological repeats are listed in the data sheet.
48
49 Table S3 Differential phosphosites lists. The significant changed phosphosites were
50
51 filtered from Table S2 with fold-change greater than 1.5 in both biological repeats.

AUTHOR INFORMATION
4
5 Corresponding Author
7
8 E-mail: [email protected]
9
Author Contributions
14
15 Z.C., C.W., C.L., S.Y.J., J.Q. and J.C. conceived the project. Z.C., C.W., C.L., X.F., and
16
17 C.L. performed the experiments. Z.C. and J.C. wrote the manuscript with input from all
18
19 authors.
Notes
25
26 The authors declare no competing financial interest.
27
28 The mass spectrometry proteomics data have been deposited to the ProteomeXchange
30
31 Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner
32
33 repository with the dataset identifier PXD015125.
ACKNOWLEDGEMENTS
39
40 We thank Drs. Yi Wang, Yin Ye, Jong Min Choi, and Antrix Jain for their kind help. We
41
42 also thank the Department of Scientific Publications at the University of Texas MD
43
44
45 Anderson Cancer Center for editing the manuscript. This work was supported internal MD
46
47 Anderson research support to J.C., who also received support from the Pamela and
48
49 Wayne Garrison Distinguished Chair in Cancer Research.

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