Functional Importance of the Anaphase-Promoting Complex-Cdh1-Mediated Degradation of TMAP/CKAP2 in Regulation of Spindle Function and Cytokinesis▿ †

Cytoskeleton-associated protein 2 (CKAP2), also known as tumor-associated microtubule-associated protein (TMAP), is a novel microtubule-associated protein that is frequently upregulated in various malignances. However, its cellular functions remain unknown. A previous study has shown that its protein level begins to increase during G1/S and peaks at G2/M, after which it decreases abruptly. Ectopic overexpression of TMAP/CKAP2 induced microtubule bundling related to increased microtubule stability. TMAP/CKAP2 overexpression also resulted in cell cycle arrest during mitosis due to a defect in centrosome separation and subsequent formation of a monopolar spindle. We also show that degradation of TMAP/CKAP2 during mitotic exit is mediated by the anaphase-promoting complex bound to Cdh1 and that the KEN box motif near the N terminus is necessary for its destruction. Compared to the wild type, expression of a nondegradable mutant of TMAP/CKAP2 significantly increased the occurrence of spindle defects and cytokinesis failure. These results suggest that TMAP/CKAP2 plays a role in the assembly and maintenance of mitotic spindles, presumably by regulating microtubule dynamics, and its destruction during mitotic exit serves an important role in the completion of cytokinesis and in the maintenance of spindle bipolarity in the next mitosis

A cytoskeleton-associated protein, TMAP/CKAP2, is involved in the proliferation of human foreskin fibroblasts

Previously, we reported the cloning of a cytoskeleton-associated protein, TMAP/CKAP2, which was up-regulated in primary human gastric cancers. Although TMAP/CKAP2 has been found to be expressed in most cancer cell lines examined, the function of CKAP2 is not known. In this study, we found that TMAP/CKAP2 was not expressed in G0/G1 arrested HFFs, but that it was expressed in actively dividing cells. After initiating the cell cycle, TMAP/CKAP2 levels remained low throughout most of the G1 phase, but gradually increased between late G1 and G2/M. Knockdown of TMAP/CKAP2 reduced pRB phosphorylation and increased p27 expression, and consequently reduced HFF proliferation, whereas constitutive TMAP/CKAP2 expression increased pR13 phosphorylation and enhanced proliferation. Our results show that this novel cytoskeleton-associated protein is expressed cell cycle dependently and that it is involved in cell proliferation. (c) 2006 Elsevier Inc. All rights reserved.N

Chloride channel conductance is required for NGF-induced neurite outgrowth in PC12 cells

We have previously shown that in PC12 cells: (1) high extracellular KCl induces moesin phosphorylation, an event which was dependent on chloride channel activation, and (2) NGF induces moesin phosphorylation which is required for neurite outgrowth. These results suggest that NGF-induced intracellular signaling and neurite outgrowth is also mediated by activation of anion channels. Using a patch-clamp technique, we found that NGF treatment increased anionic conductance in PC12 cells, an effect which was completely blocked by NPPB, a chloride channel inhibitor. Also, the NGF-induced moesin phosphorylation was suppressed by NPPB. Additionally, NPPB and SITS, another chloride channel blocker, suppressed NGF-induced TrkA phosphorylation and subsequent PI3K/Akt phosphorylation and Rac1 activation in PC12 cells. Moreover, the chloride channel inhibitors also suppressed the neurite outgrowth and decreased the cell viability in response to long-term treatment of NGF. In summary, our results suggest that chloride ion flux plays an important role in TrkA-mediated signaling pathway during NGF-induced differentiation of PC12 cells. (C) 2010 Elsevier Ltd. All rights reserved.Jiang H, 1999, J BIOL CHEM, V274, P26209Lu CC, 1999, J GEN PHYSIOL, V114, P429Cunningham ME, 1997, J BIOL CHEM, V272, P10957Jiang H, 1997, J BIOL CHEM, V272, P6835VOETS T, 1995, PFLUG ARCH EUR J PHY, V431, P132GREENE LA, 1995, CURR OPIN NEUROBIOL, V5, P579DIKIC I, 1995, J BIOL CHEM, V270, P15125VANDERGEER P, 1995, CURR BIOL, V5, P404KAPLAN DR, 1994, J NEUROBIOL, V25, P1404Wang XQ, 2006, NEURON, V52, P321, DOI 10.1016/j.neuron.2006.08.035de Tassigny AD, 2008, EUR J HEART FAIL, V10, P39, DOI 10.1016/j.ejheart.2007.11.002Yin ZH, 2008, AM J PHYSIOL-CELL PH, V294, pC535, DOI 10.1152/ajpcell.00291.2007Heo KS, 2008, MOL CELLS, V26, P468Gayer CP, 2009, J BIOL CHEM, V284, P2001, DOI 10.1074/jbc.M804576200Song EJ, 2009, INT J BIOCHEM CELL B, V41, P539, DOI 10.1016/j.biocel.2008.04.022Rayasam GV, 2009, BRIT J PHARMACOL, V156, P885, DOI 10.1111/j.1476-5381.2008.00085.xMaeno E, 2000, P NATL ACAD SCI USA, V97, P9487Xiao GN, 2002, LIFE SCI, V70, P2233Ben-Ari Y, 2002, NAT REV NEUROSCI, V3, P728, DOI 10.1038/nrn920Small DL, 2002, NEUROSCI LETT, V334, P95Huang EJ, 2003, ANNU REV BIOCHEM, V72, P609, DOI 10.1146/annurev.biochem.72.121801.161629Nilius B, 2003, ACTA PHYSIOL SCAND, V177, P119Brauer M, 2003, AM J PHYSIOL-CELL PH, V285, pC22, DOI 10.1152/ajpcell.00289.2002Stein V, 2004, J COMP NEUROL, V468, P57, DOI 10.1002/cne.10983Wood ER, 2004, BIOORG MED CHEM LETT, V14, P953, DOI 10.1016/j.bmcl.2003.12.002Wei L, 2004, PFLUG ARCH EUR J PHY, V448, P325, DOI 10.1007/s00424-004-1277-2Jeon S, 2005, J BIOL CHEM, V280, P12181, DOI 10.1074/jbc.M408253200Shimazu K, 2005, J CELL PHYSIOL, V203, P501, DOI 10.1002/jcp.20309Zhang HN, 2006, APOPTOSIS, V11, P327, DOI 10.1007/10495-006-3980-2Guan YY, 2006, TRENDS PHARMACOL SCI, V27, P290, DOI 10.1016/j.tips.2006.04.008LOEB DM, 1994, J BIOL CHEM, V269, P8901STEPHENS RM, 1994, NEURON, V12, P691CANTLEY LC, 1994, J CELL SCI, P121OBERMEIER A, 1993, EMBO J, V12, P933JING SQ, 1992, NEURON, V9, P1067NIKODIJEVIC B, 1991, J NEUROSCI RES, V28, P192GREENE LA, 1976, P NATL ACAD SCI USA, V73, P2424

A cytoskeleton-associated protein, TMAP/CKAP2, is involved in the proliferation of human foreskin fibroblasts (vol 348, pg 222, 2006)

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Copy Number Gains at 8q24 and 20q11-q13 in Gastric Cancer Are More Common in Intestinal-Type than Diffuse-Type

<div><p>The present study was aimed at discovering DNA copy number alterations (CNAs) involved in the carcinogenesis of stomach and at understanding their clinicopathological significances in the Korean population. DNA copy numbers were analyzed using Agilent 244K or 400K array comparative genomic hybridization (aCGH) in fresh-frozen tumor and matched normal tissues from 40 gastric cancer patients. Some of the detected CNA regions were validated using multiplex ligation-dependent probe amplification (MLPA) in six of the 40 patients and customized Agilent 60K aCGH in an independent set of 48 gastric cancers. The mRNA levels of genes at common CNA regions were analyzed using quantitative real-time PCR. Copy number gains were more common than losses across the entire genome in tumor tissues compared to matched normal tissues. The mean number of alterations per case was 64 for gains and 40 for losses, and the median aberration length was 44016 bp for gains and 4732 bp for losses. Copy number gains were frequently detected at 7p22.1 (20%), 8q24.21 (27%–30%), 8q24.3 (22%–48%), 13q34 (20%–31%), and 20q11-q13 (25%–30%), and losses at 3p14.2 (43%), 4q35.2 (27%), 6q26 (23%), and 17p13.3 (20%–23%). CNAs at 7p22.1, 13q34, and 17p13.3 have not been reported in other populations. Most of the copy number losses were associated with down-regulation of mRNA levels, but the correlation between copy number gains and mRNA expression levels varied in a gene-dependent manner. In addition, copy number gains tended to occur more commonly in intestinal-type cancers than in diffuse-type cancers. In conclusion, the present study suggests that copy number gains at 8q24 and 20q11-q13 and losses at 3p14.2 may be common events in gastric cancer but CNAs at 7p22.1, 13q34, and 17p13.3 may be Korean-specific.</p></div