PP6 Regulatory Subunit R1 Is Bidentate Anchor for Targeting Protein Phosphatase-6 to DNA-dependent Protein Kinase

Department of Radiation Oncology, University of Virginia Health System, Charlottesville, Virginia 22908, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 02/2012; 287(12):9230-9. DOI: 10.1074/jbc.M111.333708
Source: PubMed


DNA-dependent protein kinase (DNA-PK) becomes activated in response to DNA double strand breaks, initiating repair by the
non-homologous end joining pathway. DNA·PK complexes with the regulatory subunit SAPSR1 (R1) of protein phosphatase-6 (PP6).
Knockdown of either R1 or PP6c prevents DNA-PK activation in response to ionizing radiation-induced DNA damage and radiosensitizes
glioblastoma cells. Here, we demonstrate that R1 is necessary for and bridges the interaction between DNA-PK and PP6c. Using
R1 deletion mutants, DNA-PK binding was mapped to two distinct regions of R1 spanning residues 1–326 and 522–700. Either region
expressed alone was sufficient to bind DNA-PK, but only deletion of residues 1–326, not 522–700, eliminated interaction of
R1 with DNA-PK. We assign 1–326 as the dominant domain and 522–700 as the supporting region. These results demonstrate that
R1 acts as a bidentate anchor to DNA-PK and recruits PP6c. Targeting the dominant interface with small molecule or peptidomimetic
inhibitors could specifically prevent activation of DNA-PK and thereby sensitize cells to ionizing radiation and other genotoxic

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    • "Sit4, the homolog in yeast, is required for G1/S progression and equal chromosome segregation in yeast (Sutton et al., 1991). The human PP6 has been shown to play a role in DNA damage response, cell cycle, apoptosis and pre-mRNA splicing by acting on DNA-dependent protein kinase (DNA-PK), histone γ-H2AX, Aurora A, NF-κB and the U1 small nuclear ribonucleic protein (snRNP) (Douglas et al., 2014, 2010; Hammond et al., 2013; Hosing et al., 2012; Kajihara et al., 2014; Kamoun et al., 2013; Stefansson and Brautigan, 2006, 2007; Zeng et al., 2010; Zhong et al., 2011). However, the role of PP6 in reproductive cells remains unclear. "
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    ABSTRACT: Dynamic protein phosphorylation and dephosphorylation, mediated by a conserved cohort of protein kinases or phosphatases, regulate cell cycle progression. Among the well-known PP2A-like protein phosphatases, PP6 has been analyzed in mammalian mitosis recently identifying Aurora A as its key substrate. However, the functions of PP6 in meiosis are still entirely unknown. To identify the physiological role of PP6 in female gametogenesis, Ppp6c(F/F) mice were first generated and crossed with Zp3-Cre mice to selectively disrupt Ppp6c expression in oocytes. Here we report for the first time that PP6c was dispensable for oocyte meiotic maturation but essential for MII exit after fertilization, since depletion of PP6c caused abnormal MII spindle and disrupted MII cytokinesis, resulting in zygotes with high risk of aneuploidy, defective early embryonic development, thus severe subfertility. We also revealed that PP6 inactivation interfered with MII spindle formation and MII exit due to increased Aurora A activity, and Aurora A inhibition with MLN8237 could rescue the PP6c depletion phenotype. In conclusion, our findings uncover a hitherto unknown role for PP6 as an indispensable regulator of oocyte meiosis and female fertility.
    Journal of Cell Science 09/2015; 142(21). DOI:10.1242/jcs.173179 · 5.43 Impact Factor
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    • "In animal cells PP6 has been proposed to exist as a heterotrimeric enzyme comprised of the PP6 catalytic subunit, one of the three subunits containing a helical-repeat SAPS domain [13] plus one of three ankyrin repeat domain (ANKRD) subunits [19]. In some cell lines the levels of PP6c and SAPS appear to be co-dependent in response to knockdown by RNAi, suggesting the proteins do not survive separately, but only as complexes [20]. Our results indicate PP6c can associate with other proteins, such as E-cadherin, without participation of SAPS. "
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    ABSTRACT: Epithelial tissues depend on intercellular homodimerization of E-cadherin and loss of E-cadherin is central to the epithelial to mesenchymal transition seen in multiple human diseases. Signaling pathways regulate E-cadherin function and cellular distribution via phosphorylation of the cytoplasmic region by kinases such as casein kinases but the protein phosphatases involved have not been identified. This study shows protein Ser/Thr phosphatase-6 catalytic subunit (PP6c) is expressed in epithelial tissue and its mRNA and protein are robustly up-regulated in epithelial cell lines at high vs. low density. PP6c accumulates at adherens junctions, not tight junctions, co-immunoprecipitates with E-cadherin-catenin complexes without a canonical SAPS subunit, and associates directly with the E-cadherin cytoplasmic tail. Inducible shRNA knockdown of PP6c dispersed E-cadherin from the cell surface and this response was reversed by chemical inhibition of casein kinase-1 and prevented by alanine substitution of Ser846 in murine E-cadherin. PP6c associates with E-cadherin in adherens junctions and is required to oppose casein kinase-1 to maintain cell surface localization of E-cadherin. There is feedback signaling to enhance PP6c transcription and boost protein levels in high density epithelial cells.
    BMC Cell Biology 09/2013; 14(1):42. DOI:10.1186/1471-2121-14-42 · 2.34 Impact Factor
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    ABSTRACT: Unlabelled: Recent whole genome melanoma sequencing studies have identified recurrent mutations in the gene encoding the catalytic subunit of serine/threonine phosphatase 6 (PPP6C/PP6C). However, the biochemical, functional, and clinical ramifications of these mutations are unknown. Sequencing PP6C from patients with melanoma (233 primary and 77 metastatic specimens) with extended prospective clinical outcome revealed a large number of hotspot mutations in patients with both primary and metastatic melanoma. Despite minimal association between stage and presence of PP6C mutations in patients with primary melanoma, a subpopulation of cells within each tumor did contain PP6C mutations, suggesting PP6C mutation is an early, but non-tumor-initiating event in melanoma. Among patients with primary melanoma with PP6C mutations, patients with stop mutations had significantly shorter recurrence-free survival compared with patients without stop mutations. In addition, PP6C mutations were independent of commonly observed BRAF and NRAS mutations. Biochemically, PP6C mutations could be classified as those that interact with PP6C regulatory subunits and those that do not. Mutations that did not bind to PP6C regulatory subunits were associated with increased phosphorylation of Aurora kinase, a PP6C substrate, and mitotic defects. However, both classes of PP6C mutations led to increased sensitivity to Aurora kinase inhibition. Together, these data support for the first time that PP6C mutations are molecularly, biochemically, and clinically heterogeneous. Implications: PP6C mutations have distinct functional and clinical consequences in melanoma, and confer sensitivity to Aurora A kinase inhibitors.
    Molecular Cancer Research 12/2013; 12(3). DOI:10.1158/1541-7786.MCR-13-0422 · 4.38 Impact Factor
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