Ji Liao

A PP4-Phosphatase Complex Dephosphorylates γ-H2AX Generated during DNA Replication

The histone H2A variant H2AX is rapidly phosphorylated in response to DNA double-stranded breaks to produce gamma-H2AX. gamma-H2AX stabilizes cell-cycle checkpoint proteins and DNA repair factors at the break site. We previously found that the protein phosphatase PP2A is required to resolve gamma-H2AX foci and complete DNA repair after exogenous DNA damage. Here we describe a three-protein PP4 phosphatase complex in mammalian cells, containing PP4C, PP4R2, and PP4R3beta, that specifically dephosphorylates ATR-mediated gamma-H2AX generated during DNA replication. PP4 efficiently dephosphorylates gamma-H2AX within mononucleosomes in vitro and does not directly alter ATR or checkpoint kinase activity, suggesting that PP4 acts directly on gamma-H2AX in cells. When the PP4 complex is silenced, repair of DNA replication-mediated breaks is inefficient, and cells are hypersensitive to DNA replication inhibitors, but not radiomimetic drugs. Therefore, gamma-H2AX elimination at DNA damage foci is required for DNA damage repair, but accomplishing this task involves distinct phosphatases with potentially overlapping roles.

Cytokinesis and cancer: Polo loves ROCK‘n’ Rho(A)

Cytokinesis is the last step of the M (mitosis) phase, yet it is crucial for the faithful division of one cell into two. Cytokinesis failure is often associated with cancer. Cytokinesis can be morphologically divided into four steps: cleavage furrow initiation, cleavage furrow ingression, midbody formation and abscission. Molecular studies have revealed that RhoA as well as its regulators and effectors are important players to ensure a successful cytokinesis. At the same time, Polo-like kinase 1 (Plk1) is an important kinase that can target many substrates and carry out different functions during mitosis, including cytokinesis. Recent studies are beginning to unveil a closer tie between Plk1 and RhoA networks. More specifically, Plk1 phosphorylates the centralspindlin complex Cyk4 and MKLP1/CHO1, thus recruiting RhoA guanine nucleotide-exchange factor (GEF) Ect2 through its phosphopeptide-binding BRCT domains. Ect2 itself can be phosphorylated by Plk1 in vitro. Plk1 can also phosphorylate another GEF MyoGEF to regulate RhoA activity. Once activated, RhoA-GTP will activate downstream effectors, including ROCK1 and ROCK2. ROCK2 is among the proteins that associate with Plk1 Polo-binding domain (PBD) in a large proteomic screen, and Plk1 can phosphorylate ROCK2 in vitro. We review current understandings of the interplay between Plk1, RhoA proteins and other proteins (e.g., NudC, MKLP2, PRC1, CEP55) involved in cytokinesis, with particular emphasis of its clinical implications in cancer.

Human RIF1 encodes an anti-apoptotic factor required for DNA repair

Human Rap1-interacting protein 1 (RIF1) contributes to the ataxia telangiectasia, mutated-mediated DNA damage response against the dexterous effect of DNA lesions and plays a critical role in the S-phase checkpoint. However, the molecular mechanisms by which human RIF1 conquers DNA aberrations remain largely unknown. We here showed that inhibition of RIF1 expression by small interfering RNA led to defective homologous recombination-mediated DNA double-strand break repair and sensitized cancer cells to camptothecin or staurosporine treatment. RIF1 underwent caspase-dependent cleavage upon apoptosis. We further found that RIF1 was highly expressed in human breast tumors, and its expression status was positively correlated with differentiation degrees of invasive ductal carcinoma of the breast. Our results suggest that RIF1 encodes an anti-apoptotic factor required for DNA repair and is a potential target for cancer treatment.

CDK5RAP2 is required for spindle checkpoint function

The combination of paclitaxel and doxorubicin is among the most successful chemotherapy regimens in cancer treatment. CDK5RAP2, when mutated, causes primary microcephaly. We show here that inhibition of CDK5RAP2 expression causes chromosome mis-segregation, fails to maintain the spindle checkpoint, and is associated with reduced expression of the spindle checkpoint proteins BUBR1 and MAD2 and an increase in chromatin-associated CDC20. CDK5RAP2 resides on the BUBR1 and MAD2 promoters and regulates their transcription. Furthermore, CDK5RAP2-knockdown cells have increased resistance to paclitaxel and doxorubicin, and this resistance is partially rescued upon restoration of CDK5RAP2 expression. Cancer cells cultured in the presence of paclitaxel or doxorubicin exhibit dramatically decreased CDK5RAP2 levels. These results suggest that CDK5RAP2 is required for spindle checkpoint function and is a common target in paclitaxel and doxorubicin resistance.

Protein phosphatase PP4 is overexpressed in human breast and lung tumors

Laboratory of Cancer Biology, College of Life Sciences, Capital Normal University, #105 Xi San Huan Road (N), Beijing 100037, China; Department of Pathology, Peking University School of Oncology, Beijing 100142, China; College of Life Science, Beijing Normal University, Beijing 100875, China; Division of Biology, City of Hope National Medical Center, Duarte, CA 91010, USA; Department of Radiation Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA; Division of Molecular and Clinical Pharmacology, City of Hope National Medical Center, Duarte, CA 91010, USA Cell Research (2008) 18:974-977. doi: 10.1038/cr.2008.274; published online 12 August 2008 npg Cell Research (2008) 18:974-977.

Protein phosphatase PP4 is involved in NHEJ-mediated repair of DNA double-strand breaks.

Reversible phosphorylation is an essential posttranslational modification to turn on/off a protein function and to regulate many cellular activities, including DNA repair. A DNA double-strand break (DSB) is the most lethal form of DNA damage and is mainly fixed by the error-prone nonhomologous end joining (NHEJ)-mediated repair and by the high-fidelity homology recombination (HR)-mediated repair. We found previously that protein phosphatase PP4 is required for HR-mediated DSB repair. In this report, we showed that depletion of PP4C by siRNA compromised NHEJ-mediated repair of DSBs induced by the nuclease I-SceI. Both PP4C and its regulatory subunit PP4R2 physically interacted with the chromatin condensation factor KAP1 (KRAB-associated protein 1). Depletion of PP4C led to sustained phosphorylation of KAP1 at Ser824. Conversely, overexpression of PP4C resulted in a decrease of KAP1 phosphorylation. PP4 dephosphorylated pKAP1 in vitro. Inhibition of KAP1 expression resulted in a defect on NHEJ-mediated DSB repair, and co-depletion of PP4c and KAP1 did not have significant synergistic effect on NHEJ-mediated DSB repair. Taken together, our results suggest that PP4C and KAP1 are in the same epistasis group, and PP4 is involved in NHEJ-mediated DSB repair, possibly through regulating the phosphorylation status of KAP1.

Protein phosphatase PP6 is required for homology-directed repair of DNA double-strand breaks

DNA double-strand breaks (DSBs) are among the most lethal lesions associated with genome stability which, when destabilized, predisposes organs to cancers. DSBs are primarily fixed either with little fidelity by non-homologous end joining (NHEJ) repair or with high fidelity by homology-directed repair (HDR). The phosphorylated form of H2AX on serine 139 (g-H2AX) is a marker of DSBs. In this study, we explored if the protein phosphatase PP6 is involved in DSB repair by depletion of its expression in human cancer cell lines, and determined PP6 expression in human breast cancer tissues by immunohistochemistry staining. We found that bacterially-produced PP6c (the catalytic subunit of PP6)-containing heterotrimeric combinations exhibit phosphatase activity against g-H2AX in the in vitro phosphatase assays. Depletion of PP6c or PP6R2 led to persistent high levels of g-H2AX after DNA damage and a defective HDR. Chromatin immunoprecipitation assays demonstrated that PP6c was recruited to the region adjacent to the DSB sites. Expression of PP6c, PP6R2, and PP6R3 in human breast tumors was significantly lower than those in benign breast diseases. Taken together, our results suggest that g-H2AX is a physiological substrate of PP6, and PP6 is required for HDR and its expression may harbor a protective role during the development of breast cancer.

Structural mechanism of the phosphorylation-dependent dimerization of the MDC1 forkhead-associated domain

MDC1 is a key mediator of the DNA-damage response in mammals with several phosphorylation-dependent protein interaction domains. The function of its N-terminal forkhead-associated (FHA) domain remains elusive. Here, we show with structural, biochemical and cellular data that the FHA domain mediates phosphorylation-dependent dimerization of MDC1 in response to DNA damage. Crystal structures of the FHA domain reveal a face-to-face dimer with pseudo-dyad symmetry. We found that the FHA domain recognizes phosphothreonine 4 (pT4) at the N-terminus of MDC1 and determined its crystal structure in complex with a pT4 peptide. Biochemical analysis further revealed that in the dimer, the FHA domain binds in trans to pT4 from the other subunit, which greatly stabilizes the otherwise unstable dimer. We show that T4 is phosphorylated primarily by ATM upon DNA damage. MDC1 mutants with the FHA domain deleted or impaired in its ability to dimerize formed fewer foci at DNA-damage sites, but the localization defect was largely rescued by an artificial dimerization module, suggesting that dimerization is the primary function of the MDC1 FHA domain. Our results suggest a novel mechanism for the regulation of MDC1 function through T4 phosphorylation and FHA-mediated dimerization.

LSD1 is required for chromosome segregation during mitosis.

Dynamic methylation/demethylation of histones and non-histone proteins occurs during the cell cycle. Lysine-specific demethylase 1 (LSD1) exhibits diverse transcriptional activities through catalyzing demethylation of mono- and di-methylated histone H3 on lysine 4 (H3K4) and lysine 9 (H3K9). We show here that inhibition of LSD1 expression by siRNA leads to abnormal chromosomal segregation in unperturbed mitosis and abnormal centrosome duplication, and is associated with decreased protein levels of MAD2 and BUBR1. LSD1 positively regulates the BUBR1 and MAD2 promoter activity and maintains local monomethylation status of H3K9, which is a repressive histone mark for gene transcription. Expression of exogenous BUBR1 and MAD2 in LSD1-depleted cells partially rescues the defect of chromosome segregation. Our results suggest that LSD1 plays a role in chromosomal segregation during mitosis partially through transcriptional regulation of BUBR1 and MAD2.

HERC2/USP20 coordinates CHK1 activation by modulating CLASPIN stability

CLASPIN is an essential mediator in the DNA replication checkpoint, responsible for ATR (ataxia telangiectasia and Rad3-related protein)-dependent activation of CHK1 (checkpoint kinase 1). Here we found a dynamic signaling pathway that regulates CLASPIN turn over. Under unperturbed conditions, the E3 ubiquitin ligase HERC2 regulates the stability of the deubiquitinating enzyme USP20 by promoting ubiquitination-mediated proteasomal degradation. Under replication stress, ATR-mediated phosphorylation of USP20 results in the disassociation of HERC2 from USP20. USP20 in turn deubiquitinates K48-linked-polyubiquitinated CLASPIN, stabilizing CLASPIN and ultimately promoting CHK1 phosphorylation and CHK1-directed checkpoint activation. Inhibition of USP20 expression promotes chromosome instability and xenograft tumor growth. Taken together, our findings demonstrated a novel function of HERC2/USP20 in coordinating CHK1 activation by modulating CLASPIN stability, which ultimately promotes genome stability and suppresses tumor growth.