New Functions Of Erbin In Cell Cycle Regulation

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 play roles in generating epithelial cell polarity and assembly of adherens/apical junctions. It has 16 leucine-rich repeats (LRRs) in the N terminus and a 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.Recent studies also showed that Erbin suppressed ERK activation by interfering with the activation of Raf-1 by Ras-GTP and Nod2-dependent activation of NF-κB.In the first part, the full-length cDNA of Erbin was amplified by RT-PCR from MCF7 cells. His-tagged recombinant protein Erbin PDZ was overproduced in E. coli and purified by Ni-NTA column chromatography. Production of the antibody against PDZ domain of Erbin was achieved by injecting the recombinant protein into Balb/c mice and the serum was obtained. Specificity of these antibodies for Erbin were characterized by ELISA and Western blot using lysates of human breast cancer cell lines or transfected cells with pEGFP-N1-Erbin PDZ. It indicates that the antibody can be used for the detection of both denatured and natural Erbin protein. The above-mentioned data confirmed the specificity and sensitivity of the antibody. By using immunofluorescence staining with the antibody against the recombinant protein, the expression of endogenous Erbin was detected. The positive labeling was observed at the cytoplasmic membrane, consistent with the subcellular localization of Erbin. Interestingly, the immunohistochemical and immunofluorescence staining with this antibody showed that the expression of Erbin was remarkably increased in mitotic cells and diffusely distributed throughout the cytoplasm.To further elucidate the differential expression and localization of Erbin in mitotic cells, we detected the expression level of Erbin at different stage in cell cycle. The expression of Erbin at both mRNA and protein level was significantly increased at the S phase and peaked at the G2/M phase. To find the potential sites of intracellular action of Erbin during mitosis and interpret its cell cycle dependent expression, we inspected the localization of Erbin and tubulin in mitotic SKBR3 cells by confocal microscopy using double immunofluorescent staining. Surprisingly, Erbin appeared at the central chromosomal areas at prometaphase and then translocated to the spindle pole regions or associated with mitotic spindles at metaphase. Detailed observations revealed that Erbin was accumulated to the ingressing cleavage furrow with a dramatic concentration in the midbody region at anaphase and cytoplasmic bridge connecting two daughter cells at cytokinesis. To exclude the possibility that the antibody we used cross-reacted with some protein other than Erbin, we utilized GFP as a tracer to follow the movement of Erbin during mitosis. The Erbin-PDZ-GFP has a diffuse nuclear and cytoplasmic localization in interphase cells. However, it was remarkably accumulated at the central chromosomal areas in prometaphase cells and concentrated in distinct register with the spindles in metaphase and anaphase cells. In telophase it was aggregated at the contractile ring in the cleavage furrow and midbody region. It is completely in agreement with the findings in the detection of endogenous Erbin in SKBR3 cells. To further determine whether Erbin interacted withβ-tubulin during mitosis, we performed coimmunoprecipitation (co-IP) assays and found thatβ-tubulin was very efficiently co-precipitated with Erbin in the lysates of cells from G2/M phase, but not in the lysates of cells from S and G0/G1 phase.Increasing experimental data have demonstrated that PDZ domain containing proteins play central roles in organizing signal transduction complexes. To prove this, we observed the colocalization of Erbin with Ras, MEK and ERK in MCF-7 cells stably expressing Erbin PDZ-GFP. Superimposition of images clearly showed that all three signal molecules exhibited identical subcellular localization to that of Erbin in prophase and metaphase. Ras, MEK and ERK were dramatically co-precipitated by anti-Erbin antibody in the cell extracts from the mitotic phase. In contrast, only small amount of MEK and ERK was co-immunoprecipitated by anti-Erbin antibody in the cell lysates from G0/G1 and S phases. It supports the possibility that Erbin is involved in the assembly of supermolecular complexes during mitosis.Our data suggest the close association of Erbin/MEK/ERK signal transduction complexes on the mitotic microtubules. The coimmunoprecipitated proteins by anti-Erbin antibody should contain proteins that bind to Erbin directly, but may also include proteins that associated with other Erbin binding proteins. Thus, the questions regarding how Erbin binds to the mitotic microtubules and whether Erbin interacts with tubulin directly or indirectly need to be answered. To elucidate the mechanism of Erbin binding to mitotic microtubules, we conducted a global proteomic analysis of an Erbin protein complex by using immunoprecipitation, MALDI-TOF-MS and ESI-LC-MS/MS. Totally nineteen proteins were identified in the Erbin complex. Among the proteins co-precipitated, the ones belonging to motor protein family are prominent, including various non-muscle myosins and cytoplasmic dynein. The typical poleward localization of Erbin strongly suggests its association with dynein during mitosis. Using anti-Erbin antibody, dynein was efficiently precipitated from the cell extracts from G2/M phase but not from G0/G1 and S phases. Thus, compelling evidence demonstrates that Erbin interacts with the dynein protein in mitotic cells. Treatment with Erbin siRNA significantly affected the proliferation of MCF-7 cells.In summary, our data may provide an initial clue for the control of chromosome separation and cytokinesis organized by Erbin, that is, Erbin assemblies multiple molecules of Ras signaling pathway into a macromolecular complex. By simultaneously attaching to the signaling complexes and motor proteins, Erbin brings signaling proteins to mitotic apparatus and guide the flow of regulatory information, thereby coordinating the cell cycle progression. We provided new clue for scaffold proteins mediating the structural and functional organization of the MAPK signaling pathway. We also gave evidence in support of regulation of the G2/M transition and mitosis by MAPK signaling pathway.