Biomedical subjects

Tang K Tang

Publications and source records attributed to Tang K Tang.

9 recordsLinked to original sources

PubMed2026

Centrosome migration and apical membrane formation during epithelial polarization in MDCK cysts.

Polarization is crucial for the proper functioning of epithelial cells. Early hallmarks include the trafficking and enrichment of polarity molecules to form the apical membrane (AM) or cell-cell junctions, and the apical positioning of the centrosome. However, the dependencies among polarity molecules, AM formation, and centrosome positioning remain poorly understood. When cultured in Matrigel, de novo polarization of a single epithelial cell is often coupled with cytokinesis. During mitotic exit, centrosomes move to the future AM site, raising questions about their role in polarization. We perturbed centrosomes and polarity regulators in Matrigel-cultured cells and manipulated polarity direction using suspension culture to examine the relationships among polarization features. Surprisingly, centrosomal microtubules may not be necessary for centrosome positioning or AM formation, but their absence reduces the efficiency of AM formation. The polarity regulator Par3, rather than AM component trafficking, influences centrosome positioning. In suspension culture, centrosomes migrate in the direction opposite to AM formation. Taken together, our findings define the hierarchical relationships among several polarization features and show that centrosome-based polarity is not universal in epithelial cells, providing new insights into the mechanisms of epithelial polarization.

Animals↗

PubMed2006

CPAP interacts with 14-3-3 in a cell cycle-dependent manner.

We have previously reported that CPAP (Centrosomal Protein 4.1-Associated Protein) carries a novel microtubule-destabilizing motif that may regulate microtubule dynamics at the centrosome. In this study, we searched for conserved sequence motifs in CPAP and identified two classical 14-3-3 binding sites. The interaction between CPAP and 14-3-3 was demonstrated by both yeast two-hybrid and co-immunoprecipitation experiments. Further analyses revealed that the 14-3-3 binding motif is located within the C-terminal domain of CPAP. Alkaline phosphatase treatment disrupted CPAP binding to 14-3-3, suggesting that phosphorylation is required for the interaction. Mutation of serine 1109 to alanine (S1109A) in the 14-3-3 binding motif completely abolished the association of CPAP with 14-3-3. Taking together, these results imply that phosphorylation of CPAP on serine 1109 is required for 14-3-3 binding. Furthermore, we observed that the interaction between CPAP and 14-3-3 was significantly reduced in mitotic cells, suggesting that 14-3-3 binding to CPAP is regulated during cell cycle progression. In summary, our results show a direct interaction between CPAP and 14-3-3, and this interaction appears to be phosphorylation and cell cycle dependent.

14-3-3 Proteins↗

PubMed2006

Dynamic localization and functional implications of Aurora-C kinase during male mouse meiosis.

Aurora-C was first identified during screening for kinases expressed in mouse sperm and eggs. Herein, we report for the first time the precise subcellular localization of endogenous Aurora-C during male meiotic division. The localization of Aurora-C was analyzed by immunofluorescence staining on chromosome spreads of mouse spermatocytes or in squashed seminiferous tubules. Aurora-C was first detected at clusters of chromocenters in diplotene spermatocytes and was concentrated at centromeres in metaphase I and II. Interestingly, Aurora-C was also found along the chromosome axes, including both the regions of centromeres and the chromosome arms in diakinesis. During the anaphase I/telophase I and anaphase II/telophase II transitions, Aurora-C was relocalized to the spindle midzone and midbody. A similar distribution pattern was also observed for Aurora-B during male meiotic divisions. Surprisingly, we detected no Aurora-C in mitotic spermatogonia. Furthermore, immunoprecipitation analyses revealed that INCENP associated with Aurora-C in the male testis. We propose that INCENP recruits Aurora-C (or some other factor(s) recruit INCENP and Aurora-C) to meiotic chromosomes, while Aurora-C may either work alone or cooperate with Aurora-B to regulate chromosome segregation during male meiosis.

Anaphase↗

PubMed2005

Depletion of CPAP by RNAi disrupts centrosome integrity and induces multipolar spindles.

We previously identified a novel microtubule-destabilizing motif in CPAP that can disassemble microtubules. To examine further the CPAP function in human cells, we used siRNA to knockdown its expression. Our results showed that CPAP depletion arrested cells in mitosis and induced apoptosis. Interestingly, more than 40% of these mitotic cells had multiple spindle poles. Furthermore, inhibition of the kinesin Eg5 in CPAP-depleted cells resulted in monopolar spindles, indicating that Eg5 function is required for multipolar spindle formation in the absence of CPAP. Together, our results reveal a structural role for CPAP to maintain centrosome integrity and normal spindle morphology during cell division.

Apoptosis↗

PubMed2005

Overexpression of an Aurora-C kinase-deficient mutant disrupts the Aurora-B/INCENP complex and induces polyploidy.

Aurora kinases are emerging as key regulators of centrosome function, chromosome segregation and cytokinesis. We previously isolated Aurora-C (Aie1), a third type of Aurora kinase, in a screen for kinases expressed in mouse sperm and eggs. Currently, we know very little about the precise localization and function of Aurora-C. Immunofluorescence analysis of ectopically expressed GFP-Aurora-C has revealed that Aurora-C is a new member of the chromosomal passenger proteins localizing first to the centromeres and then to the central spindles during cytokinesis. In order to study the potential role of Aurora-C, we examined the effects of a kinase-deficient (KD) mutant (AurC-KD) in HeLa Tet-Off cells under tetracycline control. Our results showed that overexpression of AurC-KD causes defects in cell division and induces polyploidy and apoptosis. Interestingly, AurC-KD overexpression also inhibits centromere/kinetochore localization of Aurora-B, Bub1, and BubR1, reduces histone H3 phosphorylation, and disrupts the association of INCENP with Aurora-B. Together, our results showed that Aurora-C is a chromosomal passenger protein, which may serve as a key regulator in cell division.

Apoptosis↗

PubMed2004

Identification of a novel microtubule-destabilizing motif in CPAP that binds to tubulin heterodimers and inhibits microtubule assembly.

We have previously identified a new centrosomal protein, centrosomal protein 4.1-associated protein (CPAP), which is associated with the gamma-tubulin complex. Here, we report that CPAP carries a novel microtubule-destabilizing motif that not only inhibits microtubule nucleation from the centrosome but also depolymerizes taxol-stabilized microtubules. Deletion mapping and functional analyses have defined a 112-residue CPAP that is necessary and sufficient for microtubule destabilization. This 112-residue CPAP directly recognizes the plus end of a microtubule and inhibits microtubule nucleation from the centrosome. Biochemical and functional analyses revealed that this 112-residue CPAP also binds to tubulin dimers, resulting in the destabilization of microtubules. Using the tetracycline-controlled system (tet-off), we observed that overexpression of this 112-residue CPAP inhibits cell proliferation and induces apoptosis after G2/M arrest. The possible mechanisms of how this 112-residue motif in CPAP that inhibits microtubule nucleation from the centrosome and disassembles preformed microtubules are discussed.

Amino Acid Motifs↗

PubMed2002

Mutational analysis of the phosphorylation sites of the Aie1 (Aurora-C) kinase in vitro.

We previously reported two novel serine/threonine kinases, Aie1 (mouse) and AIE2 (human), both later referred to as aurora-C, a newly recognized member of the mitotic aurora kinase family. In the present study, we analyzed the phosphorylation sites of mouse Aie1 by site-directed mutagenesis. Our results showed that protein kinase A (PKA) phosphorylates Aie1 at a threonine residue located at amino acid position 171. The T171A and T175A mutants, in which threonines located at residues 171 and 175 were replaced by alanines, revealed a significant increase in their kinase activities to phosphorylate ACS-1 (Aurora-C substrate 1). In contrast, the double mutant T171A-T175A showed impaired kinase activity. In addition, we had previously identified a PEST-like motif located at the N terminus of Aie1. Mutation analysis in the present study revealed that the quadruple mutant in which the PEST-like motif was mutated significantly abrogated Aie1 kinase activity. This is the first report of the analysis of potential phosphorylation sites of mouse aurora-C in vitro.

Animals↗

PubMed2002

CPAP is a novel stat5-interacting cofactor that augments stat5-mediated transcriptional activity.

Stat5, a member of the signal transducer and activators of transcription (Stat) protein family, is a primary mediator of prolactin (PRL) signaling in the mammary gland. There are two distinct Stat5 genes, Stat5a and Stat5b. The Stat5a isoform has been demonstrated to have an essential role in mammary epithelial differentiation, whereas Stat5b is required for dimorphic sexual growth. To search for proteins that interact with the C terminus of Stat5a, a highly divergent region amongst Stat family members, we performed a yeast two-hybrid screen of HBL100 and primary breast adenocarcinoma libraries. This led to the identification of a protein that had previously been isolated as a centrosomal P4.1-associated protein (CPAP). CPAP was shown to specifically interact with Stat5a and Stat5b but not with Stat1 or Stat3. Both the tyrosine phosphorylated and unphosphorylated forms of Stat5, as well as Stat5a/Stat5b heterodimers, could associate with CPAP. CPAP was expressed in human breast cancer cell lines and the developing mammary gland as well as in other tissues. Indirect immunofluorescence and cellular fractionation studies revealed that CPAP was predominantly cytoplasmic, with low levels in the nucleus. Nuclear levels of CPAP increased substantially upon activation of the PRL pathway, most likely reflecting cotranslocation of this protein with activated Stat5. Furthermore, CPAP was found to augment Stat5-mediated transcription. Thus, we have identified CPAP as a novel coactivator of Stat5 proteins in the PRL (and probably other) pathways.

Active Transport, Cell Nucleus↗

PubMedDate not supplied

NuMA expression and function in mouse oocytes and early embryos.

Nuclear mitotic apparatus protein (NuMA), originally described as a nuclear protein, is an essential component in the formation and maintenance of mitotic spindle poles. In this study, we analyze the expression pattern and function of NuMA in mouse oocytes and early embryos. In germinal vesicle-stage oocytes, NuMA was detected both at the centrosome and in the nucleus. However, after nuclear maturation and extrusion of the first polar body, NuMA was concentrated at the broad meiotic spindle poles and at cytasters (centers of cytoplasmic microtubule asters) of mature metaphase II oocytes. Cold-induced depolymerization of microtubules appeared to disassociate NuMA foci from the cytoplasmic cytasters. During fertilization, NuMA was relocated into the re-formed male and female pronuclei. Microinjection of anti-NuMA antibody into 1 of 2 cells of 2-cell-stage embryos inhibited normal cell division. These results suggest that NuMA might play an important role in cell division during early embryonic mitosis.

Animals↗