Cloning and Characterization of a Novel Mammalian PP2C Isozyme

PP2C is a structurally diversified protein phosphatase family with a wide range of functions in cellular signal transduction. A novel PP2C subtype, designated PP2Cdelta, was identified from a rat cDNA clone, which encodes a protein of 392 amino acid residues. While PP2Cdelta shares approximately 30% sequence identity in its catalytic domain with the mammalian PP2C, it lacks a 90-residue carboxyl-terminal sequence conserved in mammalian PP2C. Northern blot analysis showed that PP2Cdelta is widely expressed in rat tissues. The transcription of the PP2Cdelta gene was activated in response to stress, such as the addition of ethanol to the culture medium or UV irradiation of cells. Recombinant PP2Cdelta purified from bacteria exhibited a potent Mn2+-dependent serine/threonine phosphatase activity. Unlike other members of the PP2C family, the activity of PP2Cdelta was inhibited, rather than stimulated, by Mg2+. Transfection with PP2Cdelta resulted in inhibition of cell growth, precluding generation of stable 293 or CHO transfectants. Using a modified tetracycline-regulated PP2Cdelta-GFP dicistronic expression cassette, it was revealed that overexpression of PP2Cdelta blocked cell cycle progression and arrested cells at early S phase, resulting in inhibition of DNA synthesis and leading to cell death. These results suggest that PP2Cdelta plays a role in regulation of cell cycle progression via dephosphorylation of its substrates whose appropriate phosphorylation states might be crucial for cell proliferation

DDX3X suppresses the susceptibility of hindbrain lineages to medulloblastoma

DEAD-Box Helicase 3 X-Linked (DDX3X) is frequently mutated in the Wingless (WNT) and Sonic hedghog (SHH) subtypes of medulloblastoma—the commonest malignant childhood brain tumor, but whether DDX3X functions as a medulloblastoma oncogene or tumor suppressor gene is not known. Here, we show that Ddx3x regulates hindbrain patterning and development by controlling Hox gene expression and cell stress signaling. In mice predisposed to Wnt- or Shh medulloblastoma, Ddx3x sensed oncogenic stress and suppressed tumor formation. WNT and SHH medulloblastomas normally arise only in the lower and upper rhombic lips, respectively. Deletion of Ddx3x removed this lineage restriction, enabling both medulloblastoma subtypes to arise in either germinal zone. Thus, DDX3X is a medulloblastoma tumor suppressor that regulates hindbrain development and restricts the competence of cell lineages to form medulloblastoma subtypes

An Integrated In Vitro and In Vivo High-Throughput Screen Identifies Treatment Leads for Ependymoma

SummaryUsing a mouse model of ependymoma—a chemoresistant brain tumor—we combined multicell high-throughput screening (HTS), kinome-wide binding assays, and in vivo efficacy studies, to identify potential treatments with predicted toxicity against neural stem cells (NSC). We identified kinases within the insulin signaling pathway and centrosome cycle as regulators of ependymoma cell proliferation, and their corresponding inhibitors as potential therapies. FDA approved drugs not currently used to treat ependymoma were also identified that posses selective toxicity against ependymoma cells relative to normal NSCs both in vitro and in vivo, e.g., 5-fluorouracil. Our comprehensive approach advances understanding of the biology and treatment of ependymoma including the discovery of several treatment leads for immediate clinical translation

Cloning and characterization of a novel mammalian PP2C isozyme

UI - 99074314NRC publication: Ye

Comparative developmental profile of the neuropeptide Y Y1 receptor gene and protein in the rat brain

NRC publication: Ye

Expression of the neuropeptide Y Y1 receptor in human embryonic kidney 293 cells: ligand binding characteristics, in situ hybridization and receptor autoradiography

NRC publication: Ye

Ontogenic profile of the expression of the mu opioid receptor gene in the rat telencephalon and diencephalon: an in situ hybridization study

NRC publication: Ye

Identification of a novel protein tyrosine phosphatase with sequence homology to the cytoskeletal proteins of the band 4.1 family

NRC publication: Ye

Genome-wide microarray comparison reveals downstream genes of Pax6 in the developing mouse cerebellum.

The Pax6 transcription factor is expressed in cerebellar granule cells and when mutated, as in the Sey/Sey mouse, produces granule cells with disturbed survival and migration and with defects in neurite extension. The impact of Pax6 on other genes in the context of cerebellar development has not been identified. In this study, we performed transcriptome comparisons between wildtype and Pax6-null whole cerebellar tissue at embryonic day (E) 13.5, 15.5 and 18.5 using Affymetrix arrays (U74Av2). Statistical analyses identified 136 differentially regulated transcripts (FDR 0.05, 1.2-fold change cutoff) over time in Pax6-null cerebellar tissue. In parallel we examined the Math1-null granuloprival cerebellum and identified 228 down-regulated transcripts (FDR 0.05, 1.2-fold change cutoff). The intersection of these two microarray datasets produced a total of 21 differentially regulated transcripts. For a subset of the identified transcripts, we used qRT-PCR to validate the microarray data and demonstrated the expression in the rhombic lip lineage and differential expression in Pax6-null cerebellum with in situ hybridisation analysis. The candidate genes identified in this way represent direct or indirect Pax6-downstream genes involved in cerebellar development