An ATM/CHK2 Signaling Pathway Induces Nuclear Translocation of SRPK2 in Cisplatin-Treated HeLa Cells

Chemotherapeutic agents are frequently used to treat various cancers, but the mechanisms mediating the cellular response to the drugs are still not fully understood. We previously reported that the nuclear translocation of serine/arginine protein kinases (SRPKs), triggered by the exposure of cells to DNA damage-inducers, plays a pivotal role in drug responsiveness. Here, we investigated the mechanism linking the nuclear accumulation of SRPK2 to the cisplatin treatment of HeLa cells. We present experimental evidence that nuclear SRPK2 acts downstream of Chk2 in the ATM/Chk2 cascade. The inhibition of ATM or Chk2 kinase activity by specific low-molecular-weight inhibitors restricted SRPK2 to the cytoplasm and conferred tolerance to cisplatin treatment. A similar effect was achieved by treating cells with SRPIN340, a selective SRPK1/2 inhibitor, thus confirming previous findings that kinase activity is indispensable for the nuclear import of SRPKs. These data add to previous findings that support a decisive role of SRPKs in coordinating cellular response to DNA damage.

Download Full-text

Novel Pathways Linking Mammalian Septins and the DNA Damage Response

10.31219/osf.io/ft54u ◽

2016 ◽

Author(s):

Timothy M. Errington

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Cellular Response ◽

Nuclear Translocation ◽

Cytoskeletal Proteins ◽

Nuclear Accumulation ◽

Damage Response ◽

Induction Of Apoptosis ◽

End Resection ◽

Damaged Dna

A cellular response to damaged DNA, known as the DNA damage response (DDR), has evolved to combat damage that occurs from exposure to genotoxic agents or byproducts of normal cellular metabolism. Upon recognition of DNA damage, the cell arrests the cell cycle and repairs damaged DNA to maintain genome integrity. However, if the damage is severe, cells undergo apoptosis or initiate cellular senescence. The DDR is a highly coordinated event linking many pathways involved in various cellular processes. A previous study from this lab implicated mammalian septins in the DDR, although through an unknown mechanism. These cytoskeletal proteins function as signaling platforms and diffusion barriers and associate with various proteins including the adaptor proteins SOCS7 and NCK. In response to multiple types of DNA damage, NCK relocalizes from the cytoplasm to the nucleus, using the nucleocytoplasmic shuttling protein SOCS7. The nuclear accumulation of NCK in response to UV irradiation is dependent on the kinase activity of ATR, a member of the PIKK family that is activated early in the DDR. Depletion of NCK results in elevated phosphorylation of the transcription factor p53 and an early induction of apoptosis. Depletion of SOCS7, which blocks the nuclear accumulation of NCK, also increases phosphorylation of p53 and also results in an early induction of apoptosis. This indicates the anti-apoptotic role of NCK is dependent on its nuclear translocation during the DDR. Another septin interacting protein was identified using a proteomic approach. This novel nuclease called Septin Associated Nuclease 1 (SAN1) possesses unique 5’ exonuclease activity mediated by a FEN1-related nuclease domain necessary for the proper repair of DNA interstrand crosslinks (ICL). Depletion of SAN1 results in a low rate of homologous recombination (HR) due to a decrease in end-resection of double-strand breaks generated during ICL repair. Additionally, SAN1 is mostly localized in the cytoplasm but accumulates in the nucleus following treatment with ICL-inducing agents. However, in the absence of septins, SAN1 becomes mislocalized and distributed throughout the cell. Depletion of septins also results in a decrease in HR and end-resection. This suggests septins are necessary for the proper response to ICL by regulating SAN1 localization and activation. Taken together, these data demonstrate that mammalian septins play a role in the DDR and highlight an unexpected link between cytoskeletal elements and DNA damage signaling.

Download Full-text

Nuclear Translocation of SRPKs Is Associated with 5-FU and Cisplatin Sensitivity in HeLa and T24 Cells

Cells ◽

10.3390/cells10040759 ◽

2021 ◽

Vol 10 (4) ◽

pp. 759

Author(s):

Ioanna Sigala ◽

Maria Koutroumani ◽

Anastasia Koukiali ◽

Thomas Giannakouros ◽

Eleni Nikolakaki

Keyword(s):

Dna Damage ◽

Kinase Activity ◽

Nuclear Translocation ◽

Nuclear Accumulation ◽

Catalytic Core ◽

Cytotoxic Effects ◽

Atm Kinase ◽

T24 Cells ◽

Unique Structural Feature ◽

Cisplatin Sensitivity

Serine/arginine protein kinases (SRPKs) phosphorylate Arg/Ser dipeptide-containing proteins that play crucial roles in a broad spectrum of basic cellular processes. The existence of a large internal spacer sequence that separates the bipartite kinase catalytic core and anchors the kinases in the cytoplasm is a unique structural feature of SRPKs. Here, we report that exposure of HeLa and T24 cells to DNA damage inducers triggers the nuclear translocation of SRPK1 and SRPK2. Furthermore, we show that nuclear SRPKs did not protect from, but on the contrary, mediated the cytotoxic effects of genotoxic agents, such as 5-fluorouracil (5-FU) and cisplatin. Confirming previous data showing that the kinase activity is essential for the entry of SRPKs into the nucleus, SRPIN340, a selective SRPK1/2 inhibitor, blocked the nuclear accumulation of the kinases, thus diminishing the cytotoxic effects of the drugs. ATR/ATM-dependent phosphorylation of threonine 326 and serine 408 in the spacer domain of SRPK1 was essential for the redistribution of the kinase to the nucleus. Substitution of either of these two residues to alanine or inhibition of ATR/ATM kinase activity abolished nuclear localization of SRPK1 and conferred tolerance to 5-FU treatment. These findings suggest that SRPKs may play an important role in linking cellular signaling to DNA damage in eukaryotic cells.

Download Full-text

DNase II mediates a parthanatos-like developmental cell death pathway in Drosophila primordial germ cells

Nature Communications ◽

10.1038/s41467-021-22622-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Lama Tarayrah-Ibraheim ◽

Elital Chass Maurice ◽

Guy Hadary ◽

Sharon Ben-Hur ◽

Alina Kolpakova ◽

...

Keyword(s):

Dna Damage ◽

Cell Death ◽

Germ Cells ◽

Nuclear Translocation ◽

Primordial Germ Cells ◽

Nuclear Accumulation ◽

Dependent Manner ◽

Dnase Ii ◽

Cell Death Pathway ◽

Parp 1

AbstractDuring Drosophila embryonic development, cell death eliminates 30% of the primordial germ cells (PGCs). Inhibiting apoptosis does not prevent PGC death, suggesting a divergence from the conventional apoptotic program. Here, we demonstrate that PGCs normally activate an intrinsic alternative cell death (ACD) pathway mediated by DNase II release from lysosomes, leading to nuclear translocation and subsequent DNA double-strand breaks (DSBs). DSBs activate the DNA damage-sensing enzyme, Poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) and the ATR/Chk1 branch of the DNA damage response. PARP-1 and DNase II engage in a positive feedback amplification loop mediated by the release of PAR polymers from the nucleus and the nuclear accumulation of DNase II in an AIF- and CypA-dependent manner, ultimately resulting in PGC death. Given the anatomical and molecular similarities with an ACD pathway called parthanatos, these findings reveal a parthanatos-like cell death pathway active during Drosophila development.

Download Full-text

Nuclear ErbB2 represses DEPTOR transcription to inhibit autophagy in breast cancer cells

Cell Death and Disease ◽

10.1038/s41419-021-03686-9 ◽

2021 ◽

Vol 12 (4) ◽

Author(s):

Yanli Bi ◽

Longyuan Gong ◽

Pengyuan Liu ◽

Xiufang Xiong ◽

Yongchao Zhao

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Transcriptional Repression ◽

Breast Cancer Cells ◽

Kinase Activity ◽

Nuclear Translocation ◽

Extracellular Signals ◽

Consensus Binding Sequence ◽

Binding Sequence

AbstractErbB2, a classical receptor tyrosine kinase, is frequently overexpressed in breast cancer cells. Although the role of ErbB2 in the transmission of extracellular signals to intracellular matrix has been widely studied, the functions of nuclear ErbB2 remain largely elusive. Here, we report a novel function of nuclear ErbB2 in repressing the transcription of DEPTOR, a direct inhibitor of mTOR. Nuclear ErbB2 directly binds to the consensus binding sequence in the DEPTOR promoter to repress its transcription. The kinase activity of ErbB2 is required for its nuclear translocation and transcriptional repression of DEPTOR. Moreover, the repressed DEPTOR by nuclear ErbB2 inhibits the induction of autophagy by activating mTORC1. Thus, our study reveals a novel mechanism for autophagy regulation by functional ErbB2, which translocates to the nucleus and acts as a transcriptional regulator to suppress DEPTOR transcription, leading to activation of the PI3K/AKT/mTOR pathway to inhibit autophagy.

Download Full-text

Delta-secretase (AEP) mediates tau-splicing imbalance and accelerates cognitive decline in tauopathies

Journal of Experimental Medicine ◽

10.1084/jem.20180539 ◽

2018 ◽

Vol 215 (12) ◽

pp. 3038-3056 ◽

Cited By ~ 7

Author(s):

Zhi-Hao Wang ◽

Pai Liu ◽

Xia Liu ◽

Shan Ping Yu ◽

Jian-Zhi Wang ◽

...

Keyword(s):

Cognitive Decline ◽

Kinase Activity ◽

Nuclear Translocation ◽

Memory Deficits ◽

Mrna Splicing ◽

Splicing Factors ◽

Synaptic Functions ◽

Tau Isoforms ◽

Exon 10

SRPK2 is abnormally activated in tauopathies including Alzheimer’s disease (AD). SRPK2 is known to play an important role in pre–mRNA splicing by phosphorylating SR-splicing factors. Dysregulation of tau exon 10 pre–mRNA splicing causes pathological imbalances in 3R- and 4R-tau, leading to neurodegeneration; however, the role of SRPK2 in these processes remains unclear. Here we show that delta-secretase (also known as asparagine endopeptidase; AEP), which is activated in AD, cleaves SRPK2 and increases its nuclear translocation as well as kinase activity, augmenting exon 10 inclusion. Conversely, AEP-uncleavable SRPK2 N342A mutant increases exon 10 exclusion. Lentiviral expression of truncated SRPK2 increases 4R-tau isoforms and accelerates cognitive decline in htau mice. Uncleavable SRPK2 N342A expression improves synaptic functions and prevents spatial memory deficits in tau intronic mutant FTDP-17 transgenic mice. Hence, AEP mediates tau-splicing imbalance in tauopathies via cleaving SRPK2.

Download Full-text

Role of P53 Mutations in the Radiosensitivity Status of Tumor Cells

Tumori Journal ◽

10.1177/030089169808400501 ◽

1998 ◽

Vol 84 (5) ◽

pp. 517-520 ◽

Cited By ~ 15

Author(s):

Vincenzo Chiarugi ◽

Lucia Magnelli ◽

Marina Cinelli

Keyword(s):

Dna Damage ◽

Cellular Response ◽

P53 Protein ◽

Cell Cycle Control ◽

P53 Mutations ◽

Wild Type ◽

Bladder Tumors ◽

Variable Effect ◽

Wild Type P53

Wild-type p53 is involved in cellular response to DNA damage including cell cycle control, DNA repair and activation of apoptosis. Accumulation of p53 protein following DNA damage may initiate the apoptotic process, resulting in cell death. DNA damage induced by radiation is an example of apoptotic stimulus involving p53. Regulation of apoptosis by p53 can occur through transcriptional regulation of pro-apoptotic (e.g. bax) and anti-apoptotic (e.g. bel-2) factors. Although wild-type p53 usually sensitizes cells to radiation therapy, p53 mutations have a variable effect on radiation response. For example p53 mutations in bone or breast tumors have been found to be associated with resistance to chemotherapeutic drugs or ionizing radiation. Mutated p53 has has been reported to increase sensitivity to radiation and drugs in colorectal and bladder tumors. The present brief commentary tries to find an explanation at molecular level of these conflicting results.

Download Full-text

The role of NBS1 in the modulation of PIKK family proteins ATM and ATR in the cellular response to DNA damage

Cancer Letters ◽

10.1016/j.canlet.2006.01.026 ◽

2006 ◽

Vol 243 (1) ◽

pp. 9-15 ◽

Cited By ~ 20

Author(s):

Junqing Zhou ◽

Chang UK Lim ◽

Jian Jian Li ◽

Lu Cai ◽

Ying Zhang

Keyword(s):

Dna Damage ◽

Cellular Response

Download Full-text

DNA-PKcs promotes chromatin decondensation to facilitate initiation of the DNA damage response

Nucleic Acids Research ◽

10.1093/nar/gkz694 ◽

2019 ◽

Vol 47 (18) ◽

pp. 9467-9479 ◽

Cited By ~ 10

Author(s):

Huiming Lu ◽

Janapriya Saha ◽

Pauline J Beckmann ◽

Eric A Hendrickson ◽

Anthony J Davis

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Ataxia Telangiectasia ◽

Cellular Response ◽

Kinase Activity ◽

Human Cell Line ◽

Cell Cycle Checkpoint ◽

Chromatin Decondensation ◽

Damage Site ◽

Damage Response

Abstract The DNA damage response (DDR) encompasses the cellular response to DNA double-stranded breaks (DSBs), and includes recognition of the DSB, recruitment of numerous factors to the DNA damage site, initiation of signaling cascades, chromatin remodeling, cell-cycle checkpoint activation, and repair of the DSB. Key drivers of the DDR are multiple members of the phosphatidylinositol 3-kinase-related kinase family, including ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related (ATR), and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). ATM and ATR modulate multiple portions of the DDR, but DNA-PKcs is believed to primarily function in the DSB repair pathway, non-homologous end joining. Utilizing a human cell line in which the kinase domain of DNA-PKcs is inactivated, we show here that DNA-PKcs kinase activity is required for the cellular response to DSBs immediately after their induction. Specifically, DNA-PKcs kinase activity initiates phosphorylation of the chromatin factors H2AX and KAP1 following ionizing radiation exposure and drives local chromatin decondensation near the DSB site. Furthermore, loss of DNA-PKcs kinase activity results in a marked decrease in the recruitment of numerous members of the DDR machinery to DSBs. Collectively, these results provide clear evidence that DNA-PKcs activity is pivotal for the initiation of the DDR.

Download Full-text