Biomedical subjects

Xiu Fen Liu

Publications and source records attributed to Xiu Fen Liu.

3 recordsLinked to original sources

PubMed2006

Inhibition of protein kinase C zeta blocks the attachment of stable microtubules to kinetochores leading to abnormal chromosome alignment.

The attachment of spindle microtubules to kinetochores is crucial for accurate segregation of chromosomes to daughter cells during mitosis. While a growing number of proteins involving this step are being identified, its molecular mechanisms are still not clear. Here we show that protein kinase C zeta (PKCzeta) is localized at the mitotic spindle during mitosis and plays a role in stable kinetochore-microtubule attachment. Striking staining for PKCzeta was observed at the mitotic spindle and spindle poles in cells at prometaphase and metaphase. PKCzeta molecules at these stages were phosphorylated at Thr-410, as detected by a phosphospecific antibody. PKCzeta was also detected at the spindle midzone and the midbody during anaphase and telophase, respectively, and PKCzeta at these stages was no longer phosphorylated at Thr-410. The polarity determinants Par3 and Par6, which are known to associate with PKCzeta, were also localized to the spindles and spindle poles at prometaphase and metaphase. Knockdown of PKCzeta by RNA interference affected normal chromosome alignment leading to generation of cells with aberrant nuclei. A specific PKCzeta inhibitor strongly blocked the formation of cold-sensitive stable kinetochore microtubules, and thus prevented microtubule-kinetochore attachment. Treatment of cells with the PKCzeta inhibitor also dislocated the minus-end directed motor protein dynein from kinetochores, but not the mitotic checkpoint proteins Mad2 and CENP-E. Prolonged exposure to the PKCzeta inhibitor eventually resulted in cell death. These results suggest a critical role of PKCzeta in spindle microtubule-kinetochore attachment and subsequent chromosomal separation.

Adaptor Proteins, Signal Transducing↗

PubMed2006

High expression of a cytokeratin-associated protein in many cancers.

We have described previously a cDNA library made from membrane-bound polysomal mRNA prepared from breast and prostate cancer cell lines. The library is highly enriched for cDNAs encoding membrane proteins, secreted proteins, and cytokeratins. To characterize this library, 25,277 cDNA clones were sequenced and aligned with various databases; 1,439 clones did not align with known genes. From this set of clones we identified a previously uncharacterized gene encoding a 334-aa protein. Although protein structural motif prediction programs indicate that the gene encodes a membrane protein comprising a signal sequence, a series of leucine-rich repeats, and a single transmembrane domain with a cytoplasmic tail, confocal microscopy of MCF7 breast cancer cells demonstrates that the protein is not directly associated with the plasma membrane or intracellular membranes but instead colocalizes with intermediate filaments and cytokeratins within the cell. Immunofluorescence studies also show that protein expression is increased greatly in mitotic MCF7 cells, and immunohistochemistry demonstrates its expression in human breast cancer cells. Analysis of mRNA levels in 25 different normal tissues by RT-PCR shows that this gene is expressed highly in normal prostate and salivary gland, very weakly in colon, pancreas, and intestine, and not at all in other tissues. RT-PCR studies on human cancer samples show that the RNA is expressed highly in many cancer cell lines and cancer specimens, including 26 of 33 human breast cancers, 3 of 3 prostate cancers, 3 of 3 colon cancers, and 3 of 3 pancreatic cancers. We name the protein CAPC, cytokeratin-associated protein in cancer.

Amino Acid Sequence↗

PubMed2006

Nucleotide exchange factor ECT2 regulates epithelial cell polarity.

Cell polarity regulates diverse biological events such as localization of embryonic determinants and establishment of tissue and organ architecture. Epithelial cell polarity is regulated by the polarity complex Par6/Par3/atypical protein kinase C (aPKC). We previously found that the nucleotide exchange factor ECT2 associates with this polarity complex and regulates aPKC activity, but the role of ECT2 in cell polarity is still unclear. Here we show that expression of a dominant negative (ECT2-N2) or constitutively active (ECT2-DeltaN5) form of ECT2 inhibits normal cyst formation of MDCK cells in 3-dimensional collagen gels. Central lumens were not observed in cysts formed by cells expressing either ECT2-DeltaN5 or ECT2-N2. Apical localization of ZO-1 and basolateral localization of beta-catenin were no longer observed in these cells. Interestingly, cells expressing ECT2-N2 did form normal cysts when cultured in the basement membrane matrix Matrigel instead of collagen gels. Addition of a major Matrigel component, laminin, partially rescued the normal cyst formation inhibited by ECT2-N2 in 3-dimensional collagen gels. Thus, signaling through laminin might override the defects of signaling through collagen and ECT2. Whereas ECT2-N2 inhibited the lumen formation of MDCK cysts, caspase-3, which is reportedly involved in lumen formation through apoptosis, was activated at various locations of cells in the cysts. It is likely that perturbation of ECT2 signaling inhibits the establishment of epithelial cell polarity leading to the inhibition of selected elimination of cells at the center of cysts. Thus, ECT2 appears to play a critical role in epithelial cell polarity.

Animals↗