ABSTRACT
Human repp86 is a nuclear protein that is expressed in a tightly limited period of time during the cell cycle and plays an essential role in its progression. Manipulation of repp86 expression by reduction of endogenous repp86 or overexpression of exogenous repp86 results in cell cycle arrest. We found that repp86 interacts with human Siah2, which is a known mediator for proteasomal degradation. Siah2 failed to interact with repp86 lacking the first 67 N-terminal amino acids. Overexpression of Siah2 reduced endogenous and exogenous repp86 at the protein level without affecting its mRNA, as shown by cotransfection and RT-PCR experiments. Furthermore, MG-132--a specific inhibitor of the proteasome--blocked the degradation of repp86 in Siah2 overexpressing cells. Moreover, transiently transfected Siah2 abrogated the mitotic arrest in repp86 overexpressing cells. Our data show that Siah2 is an important mediator of repp86 protein degradation.
Subject(s)
Nuclear Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Blotting, Western , Cell Cycle/genetics , Cell Cycle/physiology , Cell Line , Cell Line, Tumor , Cysteine Proteinase Inhibitors/pharmacology , Endonucleases , Gene Expression Regulation/drug effects , HeLa Cells , Humans , Leupeptins/pharmacology , Nuclear Proteins/genetics , Protein Binding , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Saccharomyces cerevisiae/genetics , Transfection , Two-Hybrid System Techniques , Ubiquitin-Protein Ligases/geneticsABSTRACT
Human EML4 (EMAP-like protein 4) is a novel microtubule-associated WD-repeat protein of 120 kDa molecular weight, which is classified as belonging to the conserved family of EMAP-like proteins. Cosedimentation assays demonstrated that EML4 associates with in vitro polymerized microtubules. Correspondingly, immunofluorescence stainings and transient expression of EGFP-labeled EML4 revealed a complete colocalization of EML4 with the interphase microtubule array of HeLa cells. We present evidence that the amino-terminal portion of EML4 (amino acids 1-249) is essential for the association with microtubules. Immunoprecipitation experiments revealed that EML4 is hyperphosphorylated on serine/threonine residues during mitosis. In addition, immunofluorescence stainings demonstrated that hyperphosphorylated EML4 is associated with the mitotic spindle, suggesting that the function of EML4 is regulated by phosphorylation. siRNA-mediated knockdown of EML4 in HeLa cells led to a significant decrease in the number of cells. In no case mitotic figures could be observed in EML4 negative HeLa cells. Additionally, we observed a significant reduction of the proliferation rate and the uptake of radioactive [3H]-thymidine as a result of EML4 silencing. Most importantly, EML4 negative cells showed a completely modified microtubule network, indicating that EML4 is necessary for correct microtubule formation.
Subject(s)
Cell Cycle Proteins/physiology , Microtubule-Associated Proteins/physiology , Microtubules/physiology , Serine Endopeptidases/physiology , Animals , Cell Cycle , Cell Cycle Proteins/genetics , Cell Line , Cell Line, Tumor , Cell Survival , Green Fluorescent Proteins/genetics , HeLa Cells , Humans , Immunoprecipitation , Mice , Mice, Inbred BALB C , Microtubule-Associated Proteins/genetics , Microtubules/ultrastructure , Phosphorylation , RNA Interference , RNA, Small Interfering/genetics , Serine Endopeptidases/genetics , TransfectionABSTRACT
PURPOSE: Given the well-known challenges of neuroblastoma prognosis, we investigated whether the expression of restrictedly expressed proliferation-associated protein of 86 kDa theoretical molecular mass (repp86), a proliferation-associated protein expressed in S, G2, and M phases of the cell cycle, correlates with the clinical outcome in patients with neuroblastoma. PATIENTS AND METHODS: 161 children with different stages of neuroblastoma were studied; the median follow-up time was 72.8 months. The patients were staged according to the International Neuroblastoma Staging System, and histologic grading of the tumors was performed according to the criteria of Hughes and those of the International Neuroblastoma Pathology Classification. The MYCN gene copy number was determined by Southern blot analysis or fluorescence in situ-hybridization, and repp86 expression was assessed immunohistochemically by means of monoclonal antibody Ki-S2 on paraffin sections from archival tumor samples. RESULTS: A repp86 labeling index (RI) of more than 10% positive tumor cells significantly predicted a shortened disease-free interval and an increased tumor mortality (both P <.0001). Moreover, the RI allowed the identification of patients with favorable and adverse prognosis in subsets defined by stage, grade, age, and MYCN status. In a multivariate analysis, the RI emerged as the most important predictor of event-free and disease-specific survival with hazard ratios of 11.7 and 10.5, respectively (both P <.0001). CONCLUSION: It seems that repp86 expression is closely associated with the biologic behavior of neuroblastoma. Assessment of the RI might, therefore, considerably refine prognostic models.
Subject(s)
Cell Cycle Proteins/biosynthesis , Gene Expression Regulation, Neoplastic , Neuroblastoma/genetics , Neuroblastoma/pathology , Adolescent , Blotting, Southern , Child , Child, Preschool , Disease-Free Survival , Female , Humans , Immunohistochemistry , In Situ Hybridization, Fluorescence , Infant , Infant, Newborn , Male , N-Myc Proto-Oncogene Protein , Nuclear Proteins/biosynthesis , Oncogene Proteins/biosynthesis , Prognosis , Retrospective Studies , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Human repp86 becomes detectable in the nucleoplasm of cycling cells at the G(1)-S boundary, condenses at the centrosomes with the onset of mitosis, during which it progressively locates to the mitotic spindle and to the midbody, and vanishes at the completion of cytokinesis. The repp86 cDNA was cloned and sequenced. Full-length repp86 and its COOH-terminal domain cosediment with polymerized microtubules, linking repp86 to the family of microtubule-associated proteins. During prophase and metaphase, repp86 interacts on the mitotic spindle with the putative motor protein Hklp2. Thus, repp86 may function in targeting Hklp2 to the microtubule minus ends, its activity being regulated by phosphorylation of serine/threonine residues. Exogenous overexpression of repp86 provokes accumulation of cells in G(2)-M phase and subsequent polyploidization, suggesting that excess repp86 may interfere with correct nuclear division.
