Decision letter: Sam68/KHDRBS1 is critical for colon tumorigenesis by regulating genotoxic stress-induced NF-κB activation

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Nuclear factor kappa B (NF-κB)-mediated transcription is an important mediator for cellular responses to DNA damage. Genotoxic agents trigger a 'nuclear-to-cytoplasmic' NF-κB activation signaling pathway; however, the early nuclear signaling cascade linking DNA damage and NF-κB activation is poorly understood. Here we report that Src-associated-substrate-during-mitosis-of-68kDa/KH domain containing, RNA binding, signal transduction associated 1 (Sam68/KHDRBS1) is a key NF-κB regulator in genotoxic stress-initiated signaling pathway. Sam68 deficiency abolishes DNA damage-stimulated polymers of ADP-ribose (PAR) production and the PAR-dependent NF-κB transactivation of anti-apoptotic genes. Sam68 deleted cells are hypersensitive to genotoxicity caused by DNA damaging agents. Upregulated Sam68 coincides with elevated PAR production and NF-κB-mediated anti-apoptotic transcription in human and mouse colon cancer. Knockdown of Sam68 sensitizes human colon cancer cells to genotoxic stress-induced apoptosis and genetic deletion of Sam68 dampens colon tumor burden in mice. Together our data reveal a novel function of Sam68 in the genotoxic stress-initiated nuclear signaling, which is crucial for colon tumorigenesis. https://doi.org/10.7554/eLife.15018.001 eLife digest Cells use signaling pathways to detect and respond to harmful conditions by switching on genes that keep the cell healthy. One important pathway is the nuclear factor kappa B (NF-κB) signaling pathway, which is activated by many stimuli. These stimuli may come from infections from outside the cell or may originate inside the cell, as seen for DNA damage caused by irradiation, chemicals or rapid DNA replication in cancer cells. Most of a cell’s DNA is located in the cell nucleus. However, NF-κB proteins are normally located outside the nucleus, in the cell’s cytoplasm. Damage to DNA triggers a signal from the nucleus to the cytoplasm. This signal activates the NF-κB proteins, which move into the nucleus and turn on genes that help the cell to recover from the damage. These genes include those that prevent the cell from self-destructing. In one step of the NF-κB activation process, chain-like molecules called polymers are made from a compound called poly(ADP-ribose), or PAR for short. However, few other details are known about how the damaged DNA in the nucleus signals to the cytoplasm. A protein called Sam68, which is found in the cell nucleus, has been linked to DNA damage signaling. Fu, Sun et al. now present evidence that suggests that if mouse cells lack Sam68, they do not produce PAR polymers in response to DNA damage. In addition, these cells could not trigger the PAR-dependent signaling cascade that is essential for activating NF-κB and for turning on the protective genes. Consequently, cells that lacked Sam68 were extremely sensitive to agents that cause DNA damage, such as chemicals and irradiation. The NF-κB pathway is regulated incorrectly in some cancers, but is also activated by DNA damage caused by cancer treatments. Therefore, Fu, Sun et al. also explored the role of Sam68 in cancer. Reducing the levels of Sam68 made human colon cancer cells more likely to self-destruct when they were exposed to DNA-damaging agents. Furthermore, removing Sam68 from mice that spontaneously grow colon cancer caused their tumors to develop more slowly than mice that retained Sam68 in their cells. Overall, the findings presented by Fu, Sun et al. suggest that Sam68 regulates the signal from the nucleus to the cytoplasm that activates NF-κB proteins in response to DNA damage. Sam68 also appears to be important for helping colon cancer cells grow and survive. Future challenges will be to understand how Sam68 regulates the production of the PAR polymer in this response and to explore whether Sam68 can be targeted for treating cancer. https://doi.org/10.7554/eLife.15018.002 Introduction Nuclear factor kappa B (NF-κB) is a crucial transcription factor in a variety of pathophysiological conditions (Dietz and Bahr, 2004; Grilli et al., 1993; Harhaj and Dixit, 2012; Hayden and Ghosh, 2008; Natoli, 2010; Smale, 2011; Vallabhapurapu and Karin, 2009; Wan and Lenardo, 2010; Wertz and Dixit, 2010; Wertz et al., 2004). NF-κB responds to genotoxic threats (e.g. DNA damaging agents and γ-irradiation) via the activation of the inhibitor of NF-κB kinase (IKK) and NF-κB liberation from IκB proteins, similar to the canonical pathway activated by external stimuli (Janssens et al., 2005; Perkins, 2007; Scheidereit, 2006; Wu and Miyamoto, 2007). NF-κB signaling pathway has emerged as an important mediator for cellular responses to DNA damage, in particular NF-κB-conferred anti-apoptotic transcription facilitates the cell 'escape' from the lethal effects of DNA damage (Janssens et al., 2005; Perkins, 2007; Scheidereit, 2006; Wu and Miyamoto, 2007) and initiates cell cycle checkpoint control to promote cellular recovery from damage (McCool and Miyamoto, 2012; Miyamoto, 2011). Besides ataxia telangiectasia mutated (ATM) and IKKγ, two known crucial regulators of the genotoxic stress-activated NF-κB signaling pathway (Li et al., 2001; Piret et al., 1999), poly (ADP-ribose) polymerase 1 (PARP1) was recently revealed to be indispensable for the signaling cascade that links nuclear DNA damage recognition to cytoplasmic IKK activation (Stilmann et al., 2009). Sequential post-translational modifications, including phosphorylation, ubiquitination and SUMOylation, of these signaling regulators are critical for NF-κB activation following DNA damage (Huang et al., 2003; Mabb et al., 2006; Wu et al., 2006), in particular, PARP1-catalyzed poly (ADP-ribosyl)ation (PARylation) has emerged as a vital means for rapid assembly of the signaling complexes that are critical for DNA damage-initiated NF-κB activation (Mabb et al., 2006; Stilmann et al., 2009). Although these studies have considerably advanced our understanding of the cellular response to DNA damage, the genotoxic stress-initiated ‘‘nuclear-to-cytoplasmic’’ NF-κB signaling pathway remains poorly understood, in particular the early signaling networks linking DNA lesion recognition in the nucleus to subsequent activation of IKK and liberation of NF-κB in the cytoplasm. Sam68 (Src-associated substrate during mitosis of 68 kDa, also named KH domain containing, RNA binding, signal transduction associated 1 [KHDRBS1], and encoded by KHDRBS1 gene), an RNA-binding protein that preferentially resides in the nucleus, plays versatile functions in an increasing number of cellular processes (Bielli et al., 2011; Cheung et al., 2007; Fu et al., 2013; Glisovic et al., 2008; Henao-Mejia et al., 2009; Huot et al., 2012; Iijima et al., 2011; Lukong and Richard, 2003; Matter et al., 2002; Paronetto et al., 2009; Rajan et al., 2008a, 2008b; Ramakrishnan and Baltimore, 2011; Richard, 2010; Sette, 2010; Yang et al., 2002). Through its KH (heteronuclear ribonucleoprotein particle K homology) domain, Sam68 is capable of binding single- and double-stranded DNA in addition to RNA (Lukong and Richard, 2003). Of note, Sam68 was identified as a PAR-binding protein in alkylating agent treated cells (Gagne et al., 2008) and a putative substrate of ATM, ATM and Rad3-related (ATR), and DNA-dependent protein kinase (DNA-PK) (Beli et al., 2012), which suggests that Sam68 could be an important molecule in the cellular response to DNA damage. Although emerging evidence suggests the involvement of Sam68 in multiple signaling pathways, it has not been extensively investigated yet whether Sam68, an almost strictly nuclear protein, participates in the signal communication network of nuclear-initiated signaling pathways. Moreover, aberrant expression of Sam68 has been acknowledged in multiple cancers and elevated Sam68 expression correlates with tumor progression and poor prognosis in cancer patients (Chen et al., 2012; Liao et al., 2013; Song et al., 2010; Zhang et al., 2009). Overexpression of Sam68 has been proposed as a prognostic marker (Chen et al., 2012; Liao et al., 2013; Song et al., 2010; Zhang et al., 2009), however, the precise function of Sam68 in cancer development and survival remains obscure. Here we report that Sam68 is an important regulator in genotoxic stress-initiated early signaling in the nucleus, which leads to NF-κB activation. Sam68 deletion diminishes DNA damage-stimulated PARP1 activation and PAR production, as well as the PAR-dependent NF-κB signaling and transactivation of an array of anti-apoptotic genes. As a consequence, Sam68 knockout cells are hypersensitive to genotoxicity caused by γ-irradiation and DNA damaging chemicals. Moreover, downregulation of Sam68 substantially sensitizes human colorectal cancer cells to spontaneous and genotoxic stress-induced cell death and retards colon tumor growth and survival in genetically susceptible Apcmin716/+ mice. Hence our data reveal a crucial function of Sam68 in the genotoxic stress-initiated 'nuclear to cytoplasmic' NF-κB transactivation and the involvement of Sam68 in the development and survival of colon cancer. Results Sam68 is essential for DNA damage-induced NF-κB activation To examine the potential role of Sam68 in nuclear-initiated NF-κB activation, we first compared the genotoxic stress-induced NF-κB signaling in immortalized wild-type and Sam68 knockout (KO) mouse embryonic fibroblasts (MEFs). As expected, Camptothecin (CPT), a DNA-damaging chemical that inhibits DNA topoisomerase I (Stilmann et al., 2009), stimulated a rapid degradation of IκBα, a prerequisite for NF-κB liberation and transactivation, in a dose- and time-dependent manner in wild-type MEFs (Figure 1A–B and Figure 1—figure supplement 1A). In contrast, DNA damage-induced IκBα degradation was remarkably attenuated in Sam68 KO MEFs (Figure 1A–B) and MEFs without poly(ADP-ribose)polymerase 1 (PARP1) (Figure 1—figure supplement 1B), a recently identified key nuclear regulator of DNA damage-induced NF-κB activation (Stilmann et al., 2009). Moreover, CPT treatment triggered remarkable nuclear translocation of NF-κB in wild-type MEFs (Figure 1C); additionally, the derived nuclear extracts formed high-affinity binding complexes with immunoglobin (Ig) κB double-stranded DNA, which was further confirmed by cold oligonucleotide competition and super shift assays (Figure 1D). In striking contrast, NF-κB nuclear accumulation and binding capacity to Ig κB DNA were almost abolished in the CPT-stimulated Sam68 KO MEFs (Figure 1C–D). To ascertain that Sam68 deficiency solely results in the impaired genotoxic stress-induced NF-κB activation in MEFs, we examined the NF-κB signaling in wild-type MEFs with Sam68 knockdown by small interference RNAs (siRNAs) and Sam68 KO MEFs supplemented with exogenous Sam68. CPT-triggered IκBα degradation was substantially reduced in Sam68-specific siRNA-expressing wild-type MEFs, compared to scrambled non-specific siRNA-transfected cells (Figure 1—figure supplement 1C), whereas ectopic expression of green fluorescent protein (GFP) tagged Sam68, but not GFP alone, markedly restored CPT-induced IκBα degradation in Sam68 KO MEFs (Figure 1—figure supplement 1D–E). Together, these results suggest that Sam68 could execute an essential function in the nuclear-initiated NF-κB signaling pathway. Figure 1 with 2 supplements see all Download asset Open asset Sam68 is required for the nuclear-initiated NF-κB signaling in response to DNA damage. (A and B) Whole cell lysates from wild-type (WT) and Sam68 knockout (KO) mouse embryonic fibroblasts (MEFs) treated with indicated concentrations of CPT for 2 hr (A) or 10 μM of CPT for indicated periods (B) were immunoblotted (IB) for IκBα and Sam68, with β-actin as a loading control. Right, the IκBα levels, normalized to β-actin and untreated controls, were quantified from three independent experiments. (C) Cytosolic and nuclear fractions derived from WT and Sam68 KO MEFs stimulated with 10 μM of CPT for indicated periods were IB for indicated proteins. Caspase-3 (Casp3) and PARP1 served as loading controls and cytosolic and nuclear markers, respectively. Right, the p65 levels in the nucleus, normalized to PARP1 and untreated controls, were quantified from three independent experiments. (D) Nuclear extracts of WT and Sam68 KO MEFs treated with (+) or without (−) CPT (10 μM, 2 hr) were analyzed by EMSA with 32P-labeled immunoglobin (Ig) κB or OCT1 oligonucleotides. In some cases, EMSA was performed in the presence of 100-fold unlabeled Ig κB or OCT1 oligonucleotide competitors (lanes 5–6) or p65 antibody (Ab) (lane 7). Ig κB DNA binding complexes are labeled with filled triangles, and the supershifted band and nonspecific band are labeled with an open triangle and an asterisk, respectively. (E) WT and Sam68 KO MEFs were stimulated with 10 μM of CPT for indicated periods, and immunoprecipitants (IP) with IKKγ antibody were immunoblotted for indicated proteins. (F and G) Whole cell lysates (Input) from WT and Sam68 KO MEFs stimulated with 10 μM of CPT for indicated periods were IB directly or after IP with PARP1 antibody (F) or Sam68 antibody (G) for indicated proteins. Data are representative of at least three independent experiments. Results in (A, B and C) are expressed as mean and s.e.m. *p<0.05, **p<0.01, ***p<0.001 by Student’s t tests. https://doi.org/10.7554/eLife.15018.003 Sam68 participates in DNA damage-initiated early NF-κB signaling in the nucleus Sam68 is an almost strictly nuclear protein; its nuclear import is conferred by a nuclear localization signal (NLS) in the C-terminus (Fu et al., 2013; Ishidate et al., 1997; Lukong and Richard, 2003). Consistently, the ΔC (deletion of C-terminal NLS) mutant of Sam68 switches its preferred subcellular localization to the cytoplasm, in contrast to the strict nuclear accumulation observed in wild-type Sam68 (Figure 1—figure supplement 1D). Sam68 KO MEFs reconstituted with wild-type Sam68, but not GFP control, displayed restored CPT-triggered NF-κB activation signaling including IκBα degradation, phosphorylation of p65, and p65 nuclear accumulation (Figure 1—figure supplement 1E–F). In contrast, supplementing with the Sam68 (ΔC) mutant failed to restore CPT-triggered IκBα degradation (Figure 1—figure supplement 1E), which indicates an indispensable role of the Sam68 nuclear function in DNA damage-initiated NF-κB activation. Indeed, CPT treatment-induced SUMOylation of IKKγ, a pivotal post-translational modification on IKKγ in the nucleus that subsequently leads to activation of IKKβ and liberation of NF-κB in the cytoplasm (Huang et al., 2003; Mabb et al., 2006), was substantially attenuated in Sam68 KO MEFs in comparison to wild-type controls (Figure 1E). Moreover, assembly of the PARP1-IKKγ signal complex has been established as a prerequisite for cytoplasmic NF-κB activation in the cellular response to genotoxic stresses (Gibson and Kraus, 2012; McCool and Miyamoto, 2012; Miyamoto, 2011). As expected, PARP1 complexed with IKKγ following CPT treatment, whereas there was no detectable PARP1-IKKγ interaction in the absence of genotoxic stress in wild-type MEFs (Figure 1F). In striking contrast, the CPT-induced PARP1-IKKγ interaction was abolished in Sam68 KO MEFs (Figure 1F). Moreover, strong inducible interactions among Sam68, PARP1, and IKKγ were observed in wild-type MEFs, at 20 min after CPT treatment (Figure 1F–G). Of note, immune-depletion of Sam68 using antibodies abolished the CPT-induced PARP1-IKKγ interaction in wild-type MEFs (Figure 1—figure supplement 2), suggesting that PARP1 interacts with Sam68 and IKKγ simultaneously. Hence our results suggest that Sam68 participates in the early nuclear signaling cascade in DNA damage-initiated NF-κB activation. Sam68 controls DNA damage-induced PARylation Beyond its indispensable role in DNA repair (Gibson and Kraus, 2012), emerging evidence demonstrates that PARP1-mediated PARylation is one of the most crucial post-translational modifications orchestrating DNA damage-initiated NF-κB signaling (Stilmann et al., 2009). The inducible Sam68-PARP1 interaction following genotoxic stress led us to examine whether Sam68 impacts DNA break-stimulated PAR synthesis. As expected, CPT treatment induced a wide array of PARylated proteins peaking at 20 min post stimulation in wild-type MEFs, which were diminished by one-hour pretreatment with two independent PARP inhibitors, Olaparib and PJ-34, thus confirming the PAR specificity (Figure 2A and Figure 2—figure supplement 1A). Strikingly, genotoxic stress-induced PAR synthesis was markedly dampened in Sam68 KO MEFs (Figure 2A and Figure 2—figure supplement 1A). Consistently, genotoxic stress-induced PARylation of the known PARP1 substrates including PARP1 itself, NBS1, and Ku70 was also substantially attenuated in Sam68 KO MEFs (Figure 2—figure supplement 1B). PAR that is synthesized after DNA damage can be rapidly degraded by PAR glycohydrolase (PARG), therefore we examined the possibility that PARG is hyper-activated in the absence of Sam68, thus degrading the formed PAR chains immediately after genotoxic stress. However, down-regulation of PARG by siRNA in Sam68 KO MEFs failed to restore CPT-induced PAR production to a similar level to that in wild-type MEFs (Figure 2—figure supplement 2), which suggests that the attenuated PAR formation in the absence of Sam68 is not caused by a rapid degradation of PAR chains. Furthermore, CPT and γ-irradiation stimulated PAR production was markedly reduced in primary colonic epithelial cells (CECs) derived from Khdrbs1-/- (Sam68 knockout) mice, compared to Khdrbs1+/- (Sam68 heterozygote) controls (Figure 2B–D), thus supporting the critical function of Sam68 in controlling DNA damage-stimulated PAR production. Figure 2 with 4 supplements see all Download asset Open asset Sam68 facilitates PARP1-catalyzed PARylation in response to DNA damage. (A) Wild-type (WT) and Sam68 knockout (KO) mouse embryonic fibroblasts (MEFs) pretreated with Olaparib (10 μM), PJ-34 (10 μM), or DMSO for 1 hr, were stimulated with 10 μM of CPT for indicated periods, and whole cell lysates were immunoblotted (IB) for indicated proteins, with β-actin as a loading control. (B and C) Primary colonic epithelial cells (CECs) isolated from Khdrbs1+/- and Khdrbs1-/- mice were stimulated with 10 μM of CPT (B) or 10 Gy of γ-irradiation (IR) (C). Whole cell lysates were derived at indicated periods post stimulation and IB as in (A). Right, the PAR levels, normalized to β-actin and untreated controls, were quantified from three independent experiments. (D) Immunofluorescence micrographs of PARylated proteins (PAR) in CECs treated as in (C), with nuclei counterstained by DAPI. Scale bar, 10 μm. Percentage of CECs (>100 cells from 5–8 random fields) with PAR staining was quantified (right). (E) WT and Sam68 KO MEFs were γ-irradiated (IR) at 10 Gy and the chromatin, soluble (Sol. fr.), and insoluble (Ins. fr.) subcellular fractions were derived at indicated time points post γ-irradiation and IB for Sam68, Caspase-3 (Casp-3), and Histone H3 (H3). (F) WT MEFs were stimulated with 10 μM of CPT for indicated periods and the chromatin fractions were derived and IB as in (E). Right, the Sam68 and PARP1 levels in the chromatin fractions, normalized to H3 and untreated controls, were quantified from three independent experiments. (G) Whole cell lysates from WT and Sam68 KO MEFs stimulated with 10 μM of CPT for 20 min, were IB directly or after immunoprecipitation (IP) with PARP1 antibody for indicated proteins. (H) Recombinant PARP1 protein was incubated in reaction buffer containing damaged DNA or with purified GST or GST-Sam68 protein in the presence or absence of NAD+ or the PARP1 inhibitor PJ-34, as indicated. The reaction mixture was separated by SDS-PAGE and subjected to IB with the PAR, PARP1, and Sam68 antibodies. Results in (B, C, D and F) are expressed as mean and s.e.m. ns, non-significant difference and , *p<0.05, ***p<0.001 by Student’s t tests. Data are representative of at least three independent experiments. https://doi.org/10.7554/eLife.15018.006 Given the evidence that PARP1 is rapidly recruited to DNA damage sites (Krishnakumar and Kraus, 2010), we performed chromatin fractionation assays to examine the possibility that Sam68 can be recruited to DNA lesions. Remarkably, Sam68 was enriched in chromatin fractions in MEFs following γ-irradiation (Figure 2E) and CPT treatment (Figure 2F), and the kinetics of Sam68 enrichment was similar to that of PARP1 on damaged chromatin (Figure 2F), which supports the inducible interaction between Sam68 and PARP1 in the early DNA damage signaling (Figure 1F–G). Moreover, Sam68 and PARP1 were still substantially enriched in chromatin fractions in the PARP1-inhibited MEFs following γ-irradiation (Figure 2—figure supplement 3A), indicating that genotoxic stress-induced Sam68 enrichment in chromatin is not dependent on PAR formation. We further examined whether ectopic expression of PARP1 could rescue the defect in DNA damage-induced PAR formation caused by Sam68 loss. As expected, supplementing exogenous GFP-Sam68, compared to the GFP control, largely restored γ-irradiation-stimulated PAR production in Sam68 KO MEFs (Figure 2—figure supplement 3B). In contrast, PARP1 overexpression did not rescue the PAR chain formation in response to γ-irradiation under a Sam68 deleted condition (Figure 2—figure supplement 3B), which suggests that Sam68 is required for DNA damage-triggered PARP1 activation and is consistent with Sam68 being an essential upstream activator of PARP1. DNA-dependent PARP enzymes, i.e. PARP1 and PARP2, are activated during the cellular response to DNA damage, of which PARP1 is the most robust enzyme that catalyzes >90% of PAR formation in cells (Krishnakumar and Kraus, 2010). In contrast to the genotoxic stress-induced Sam68-PARP1 interaction (Figure 1F–G and Figure 1—figure supplement 2D), no detectable interaction between Sam68 and PARP2 was observed under either unstimulated or damaged conditions (Figure 1—figure supplement 2D). Provided the crucial funciton of PARP1 in DNA damage responses, we therefore examined whether Sam68 impacts PARP1-conferred PARylation following DNA damage. In line with the vigorous PAR production in the presence of genotoxic stress (Figure 2A), immunoprecipitated PARP1 was associated with Sam68 and various PARylated target proteins in wild-type MEFs (Figure In contrast, the PARylated were reduced an of PARP1 immunoprecipitated from Sam68 KO MEFs was (Figure supplementing with Sam68, in comparison to a GFP control, markedly the DNA damage-triggered PAR synthesis in Sam68 KO MEFs (Figure 2—figure supplement 3B), further supporting the key function of Sam68 in controlling DNA damage-initiated To the of Sam68 on PARP1-catalyzed PAR production, we PARP1 and Sam68 proteins in in PARylation DNA PARP1 with the addition of PAR in the presence of and DNA, as indicated by the formation of PARylated PARP1 (Figure and Figure 2—figure supplement and a in PARP1 protein (Figure and Figure 2—figure supplement 4 with Strikingly, of Sam68 protein, but not GST control, with PARP1 PAR production, which was by a of PARP1 protein (Figure and Figure 2—figure supplement with Of note, the of Sam68 protein from the PARylation reaction at a level (Figure with thus the possibility that Sam68 is a substrate of PARP1 in and indicating that Sam68 a function for DNA-dependent PARP1 In the absence of PARP1, GST GST-Sam68 protein PARylation (Figure and indicating that Sam68 not the to Moreover, in the absence of damaged DNA, of Sam68 and PARP1 failed to detectable PAR chains from supplemented NAD+ (Figure 2—figure supplement which suggests that Sam68 DNA-dependent PARP1 activation and subsequent PAR production. The Sam68-PARP1 interaction is critical for genotoxic stress-induced PARylation and NF-κB activation various Sam68 (Figure 3A), we to understand the key in Sam68 for its interaction with PARP1. We the of the and Sam68 to PARP1, but not GFP control (Figure and Figure supplement 1A). In contrast, deletion of the of Sam68 almost abolished the of Sam68 to PARP1 (Figure and Figure supplement suggesting that the are critical for the Sam68-PARP1 Moreover, our assays using proteins a Sam68-PARP1 interaction (Figure In contrast to the strong between PARP1 and Sam68, Sam68 protein with PARP1 (Figure which further supports the critical role of of Sam68 for the Sam68-PARP1 To examine the of the Sam68-PARP1 we compared DNA damage-stimulated NF-κB signaling in Sam68 KO MEFs supplemented with or mutant Sam68. of Sam68, but not GFP control, restored genotoxic stress-induced PARylation (Figure and Figure supplement 1C), assembly of the PARP1-IKKγ signaling complex (Figure cytoplasmic degradation of IκBα, and nuclear translocation of p65 (Figure supplement 1C), consistent with our (Figure and Figure 1—figure supplement 1E–F). In contrast, Sam68 and Sam68 strict nuclear localization (Figure ectopic expression of Sam68 mutant failed to restore the DNA damage-triggered PARP1-IKKγ signal complex IκBα degradation, and p65 nuclear accumulation (Figure and Figure supplement Moreover, we in PARylation assays using Sam68 and Sam68 proteins, to examine the role of the Sam68-PARP1 interaction for PARP1 activation. with our (Figure of Sam68 with PARP1 substantially PAR formation in the presence of damaged DNA and NAD+ (Figure supplement with 7). However, the of Sam68 on PARP1 activation and PARylation under the condition was by Sam68 protein (Figure supplement with suggesting that the Sam68-PARP1 interaction is essential for Sam68 to PARP1 activation. our results that the of Sam68 is important for the between Sam68 and PARP1 and the Sam68-PARP1 interaction is critical for DNA damage-induced PARylation and PAR-dependent NF-κB signaling. Figure with 1 supplement see all Download asset Open asset of Sam68 is crucial for the Sam68-PARP1 interaction and genotoxic stress-induced NF-κB (A) of Sam68 protein or indicated ΔC and with The K domain and nuclear localization signal (NLS) are labeled in and respectively. (B) Whole cell lysates from cells indicated GFP or proteins were IB directly or after IP with GFP antibody for indicated proteins. (C) Whole cell (Input) containing PARP1 were IB directly or after with indicated GST or proteins for indicated proteins. (D) Sam68 KO MEFs GFP-Sam68, or proteins were stimulated with 10 μM of CPT for indicated periods, and the derived whole cell lysates were IB directly or after IP with PARP1 antibody for indicated proteins. (E) Immunofluorescence micrographs of Sam68 KO MEFs GFP or proteins, with nuclei counterstained by DAPI. Scale bar, 10 μm. Data in are representative of at least three independent experiments. Sam68 deletion genotoxic stress-induced NF-κB signaling in mouse primary cells To explore the of Sam68 in primary we examined the DNA damage-initiated NF-κB activation in known for their DNA from Khdrbs1+/- and Khdrbs1-/- mice. and IκBα degradation rapidly in Khdrbs1+/- but was attenuated in Khdrbs1-/- cells (Figure and Figure supplement 1A). In γ-irradiation and CPT triggered p65 nuclear translocation in Khdrbs1+/- whereas the nuclear accumulation of p65 was abolished in Khdrbs1-/- cells (Figure and Figure supplement 1B). Moreover, in Khdrbs1+/- γ-irradiation