ABSTRACT
LMO7 (LIM domain only 7) is a transcription regulator for expression of many Emery-Dreifuss muscular dystrophy-relevant genes, and binds to α-actinin and AF6/afadin at adherens junctions for epithelial cell-cell adhesion. In this study, we found that human LMO7 interacted with the spindle assembly checkpoint (SAC) protein MAD1. LMO7 colocalized with actin filaments at the cell membrane but did not colocalize with MAD1 at kinetochores in prometaphase. Our observations reveal that overexpression but not depletion of LMO7 caused a SAC defect, and that the LIM domain of LMO7 was a determinant of its ability to interfere with kinetochore localization of the SAC proteins MAD2 and BUBR1 and cause a SAC defect though the LIM peptide itself did neither bind to MAD1, MAD2 and BUBR1 nor localize to the actin filaments. However, overexpression of LMO7 or the LIM peptide did not interfere with kinetochore localization of MAD1. Additionally, overexpression of the LIM peptide prolonged mitotic timing and interfered with chromosome congression whereas that of LMO7b did not. Taken together, we conclude that LMO7 via its LIM domain acts to control mitosis progression and exerts an effect on the SAC.
Subject(s)
Actin Cytoskeleton/metabolism , Cell Cycle Proteins/metabolism , Cell Membrane/metabolism , LIM Domain Proteins/metabolism , M Phase Cell Cycle Checkpoints , Mitosis , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Animals , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cell Line, Tumor , Humans , Interphase , Kinetochores/metabolism , LIM Domain Proteins/antagonists & inhibitors , LIM Domain Proteins/chemistry , LIM Domain Proteins/genetics , Luminescent Proteins/chemistry , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Metaphase , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Prometaphase , Protein Domains , Protein Multimerization , Protein Transport , RNA Interference , Rats , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Spindle Poles/metabolism , Transcription Factors/antagonists & inhibitors , Transcription Factors/chemistry , Transcription Factors/geneticsABSTRACT
Aneuploidy is a common characteristic of human solid tumors. It has been proposed that a defect of the spindle assembly checkpoint (SAC) generates aneuploidy and might facilitate tumorigenesis. However, a direct link between the SAC proteins and tumorigenesis has not yet been elucidated. Here, we demonstrate the association of the SAC protein MAD1 with the RNA polymerase II complex and its role in gene expression. Furthermore, MAD1 binds to the E-cadherin promoter region. Knockdown of endogenous MAD1 by siRNA reduces E-cadherin expression and enhances the migration ability of non-metastatic breast cancer cells, indicating that reduced MAD1 expression is a new potential diagnostic symptom of tumor metastasis.
Subject(s)
Cadherins/metabolism , Cell Cycle Proteins/metabolism , Nuclear Proteins/metabolism , Cadherins/genetics , Cell Cycle Proteins/genetics , Cell Line, Tumor , Cell Movement/genetics , Gene Expression , Gene Expression Regulation, Neoplastic , HeLa Cells , Humans , Nuclear Proteins/genetics , Protein Binding , Protein Transport , RNA Polymerase II/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolismABSTRACT
PARK2, an ubiquitin ligase closely correlated with Parkinson's disease and cancer, has been shown to accumulate at centrosomes to ubiquitinate misfolded proteins accumulated during interphase. In the present study, we demonstrated that PARK2 can also localize to centrosomes in mitosis and that the protein does not fluctuate through the S- to M-phase. A C-terminal truncation of PARK2 resulted in a spindle assembly checkpoint defect, characterized by HeLa cells able to bypass mitotic arrest induced by nocodazole and form multinucleated cells when overexpressing the C-terminal truncated PARK2 protein. The spindle assembly checkpoint defect may be due to a change in a biochemical or structural property of PARK2 caused by the C-terminal truncation, resulting in a loss of self-interaction between PARK2 proteins.
Subject(s)
Spindle Apparatus , Ubiquitin-Protein Ligases/physiology , Blotting, Western , Circular Dichroism , HeLa Cells , Humans , Microscopy, Fluorescence , Solubility , Ubiquitin-Protein Ligases/chemistryABSTRACT
In cancer cells, loss of E-cadherin gene expression caused dysfunction of the cell-cell junction system, triggering cancer invasion and metastasis. Therefore, E-cadherin is an important tumor-suppressor gene. To understand how E-cadherin gene expression is regulated in cancer cells, we have used E-cadherin-positive and -negative expressing cells to find out the possible up- or down regulating transcription factors in human E-cadherin regulatory sequences. Functional analysis of human E-cadherin regulatory sequences constructs indicated that AML1, Sp1, and p300 may play important roles in promoting E-cadherin expression. In addition, we found there are four HNF3-binding sites in human E-cadherin regulatory sequences. The exogenous HNF3 can enhance the E-cadherin promoter activity in metastatic breast cancer cells and the metastatic breast cancer cells stably transfected with HNF3 showed re-expression of E-cadherin. The HNF3 stable transfectants changed from mesenchymal-like into epithelial morphology. The transwell assays showed the re-expressed E-cadherin reduced cell motility of metastatic breast cancer cells. These results suggested HNF3 may play important roles in the upregulation of the E-cadherin promoter, with the consequent re-expression of E-cadherin, thus reducing the metastatic potential of breast cancer cells. These findings suggested HNF3 plays important roles in the upregulation of the E-cadherin gene and may be able to reduce the motility of metastatic breast cancer cells.
