title: The mitotic arrest deficient protein MAD2B interacts with the clathrin light chain A during mitosis.
abstract: BACKGROUND
  Although the mitotic arrest deficient protein MAD2B (MAD2L2) is thought to inhibit the anaphase promoting complex (APC) by binding to CDC20 and/or CDH1 (FZR1), its exact role in cell cycle control still remains to be established.
  
  
  RESULTS
  Using a yeast two-hybrid interaction trap we identified the human clathrin light chain A (CLTA) as a novel MAD2B binding protein. A direct interaction was established in mammalian cells via GST pull-down and endogenous co-immunoprecipitation during the G2/M phase of the cell cycle. Through subsequent confocal laser scanning microscopy we found that MAD2B and CLTA co-localize at the mitotic spindle. Clathrin forms a trimeric structure, i.e., the clathrin triskelion, consisting of three heavy chains (CLTC), each with an associated light chain. This clathrin structure has previously been shown to be required for the function of the mitotic spindle through stabilization of kinetochore fibers. Upon siRNA-mediated MAD2B depletion, we found that CLTA was no longer concentrated at the mitotic spindle but, instead, diffusely distributed throughout the cell. In addition, we found a marked increase in the percentage of misaligned chromosomes.
  
  
  CONCLUSIONS
  Previously, we identified MAD2B as an interactor of the renal cell carcinoma (RCC)-associated protein PRCC. In addition, we found that fusion of PRCC with the transcription factor TFE3 in t(X;1)(p11;q21)-positive RCCs results in an impairment of this interaction and a concomitant failure to shuttle MAD2B to the nucleus. Our current data show that MAD2B interacts with CLTA during the G2/M phase of the cell cycle and that depletion of MAD2B leads to a marked increase in the percentage of misaligned chromosomes and a redistribution of CLTA during mitosis.
authors.affiliation: Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen Centre for Molecular Life Sciences, Nijmegen, The Netherlands.
authors.forename: Klaas
authors.initials: K
authors.lastname: Medendorp
authors.forename: Lilian
authors.initials: L
authors.lastname: Vreede
authors.forename: Jan J M
authors.initials: JJ
authors.lastname: van Groningen
authors.forename: Lisette
authors.initials: L
authors.lastname: Hetterschijt
authors.forename: Linda
authors.initials: L
authors.lastname: Brugmans
authors.forename: Patrick A M
authors.initials: PA
authors.lastname: Jansen
authors.forename: Wilhelmina H
authors.initials: WH
authors.lastname: van den Hurk
authors.forename: Diederik R H
authors.initials: DR
authors.lastname: de Bruijn
authors.forename: Ad Geurts
authors.initials: AG
authors.lastname: van Kessel
mesh_terms: D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D033941:Clathrin Light Chains / Q000235:genetics / Q000378:metabolism*; D016195:G2 Phase; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D064246:Mad2 Proteins; D018613:Microscopy, Confocal; D008938:Mitosis; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism*; D014162:Transfection
chemical_list: D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D018797:Cell Cycle Proteins; D033941:Clathrin Light Chains; D008164:Luminescent Proteins; C120552:MAD2L2 protein, human; D064246:Mad2 Proteins; D009363:Neoplasm Proteins; C415042:PRCC protein, human; D011506:Proteins; D011993:Recombinant Fusion Proteins; C069203:TFE3 protein, human