C-Myb Regulates Cell Cycle-dependent Expression Of Erbin

Objectives: Erbin was originally identified as a protein that interacts with the receptor tyrosine kinase HER2 using a yeast two-hybrid strategy. It belongs to LAP [leucine-rich repeats and PSD-95/Dlg-A/ZO-1(PDZ) domains] family which plays roles in generating epithelial cell polarity and assembly of adherens/apical junctions. Erbin has 16 leucine-rich repeats (LRRs) in the N terminus and a single PDZ domain in the C terminus. The biological function for Erbin is not yet fully elucidated. It is reported that Erbin is localized at plasma membrane and cell-cell contacts and involved in proper basolateral localization of HER2. However, the functions of Erbin have not been extensively explored so far.Materials and Methods:Using immunofluorescent staining and confocal microscopy, Erbin expression was observed in human breast cancer cell line SKBR3. The expression of Erbin at both mRNA and protein level was analyzed by Western blot and real-time RT-PCR. The Erbin promoter activity was examined by luciferase assays. Binding of c-Myb to c-Myb-response-element in Erbin promoter is analyzed by chromatin immunoprecipitation (ChIP) and oligonucleotide pull-down. The functionanity of c-Myb site was determined by site-directed mutagenesis, c-Myb specific RNA interference and overexpression of c-Myb.Results:1. To explore the distribution of Erbin in human breast cancer cells, we stained SKBR3 cells by immunofluorescent method using a specific antibody against Erbin. The result shows that Erbin was diffusely distributed near the cytoplasmic membrane in SKBR3 cells. Interestingly, Erbin was strikingly accumulating in the mitotic cells. To clarify whether Erbin expression is associated with mitosis, we monitored the level of Erbin throughout the cell cycle by cell cycle synchronization. Using nocodazole (a microtubule inhibitor) MCF-7 cells were arrested at G2/M phase and released into cell cycle synchronously. Cell cycle arrest was confirmed by flow cytometric analysis of cellular DNA content. Cells at different stages of mitosis were lysed and whole-cell lysates subjected to SDS-PAGE and Western blot analysis with anti-Erbin antibody. We found that the expression of Erbin with mRNA and protein during mitosis, increased at the S phase and peaked at the G2/M phase. To determine if the cell cycle dependent accumulation of Erbin mRNA results from corresponding activation of the Erbin promoter, we constructed the plasmids that carry the core region of the Erbin promoter (pLuc-483). Hela cells were transiently co-transfected with this plasmids and pRL-TK and then synchronized by a double-thymidine block that arrests cells at G1/S boundary and nocodazole treatment for G2/M phase arrest, respectively. The arrested cells were harvested and assayed for luciferase activity. In G1 phase cells, the level of luciferase activity was low, but cells arrested in G2/M phase exhibited remarkably increased luciferase activities which were about 2– 4-fold greater than the cells arrested in G1 phase.2. Sequence analysis revealed that the proximal region of the Erbin promoter includes a consensus c-Myb site at position -86/-103.To clarify whether the regulatory elements in the Erbin promoter are responsible for the cell cycle-dependent transcription of Erbin, we introduced substitutional mutations in c-Myb consensus sequences.GTTGT was converted to ATCGC for c-Myb. Luciferase assays show that mutation of c-Myb binding site strikingly eliminated the luciferase activity, especially in G2/M phase. To further characterize the binding specificity of c-Myb to the c-Myb site in the Erbin promoter, we performed ChIP and oligonucleotide pull-down assays.The results prove that the binding of c-Myb to the c-Myb site in the Erbin promoter is specific.3. Ectopic overexpression of c-Myb led to the up-regulation of Erbin promoter activity and c-Myb silencing by small interfering RNA significantly decreased Erbin protein level. Moreover, transfection of c-Myb rescued Erbin expression that was impaired by c-Myb knockdown. It proves that c-Myb and the c-Myb response element in the Erbin promoter mediate the cell cycle-dependent expression of Erbin. These results unravel a critical role of c-Myb in promoting Erbin transcription in G2/M phaseConclusions:1. Erbin is expressed in a cell cycle dependent manner.2. The c-Myb positively regulates the expression of Erbin by binding to the c-Myb element in the promoter of Erbin.3. This study provides a clue for an unknown functional role of Erbin in cell cycle progression.