Study On The Effect Of MERIT40-Tankyrase1 Interaction On Spindle Assembly

Accurate segregation of the replicated chromosome to daughter cells during cell division in higher eukaryotes cells through mitosis that tightly related to assembly and function of bipolar spindle is critical for genome stability.It is regulated by various post-translational modifications,including phosphorylation,ubiquitination,ADP-ribosylation and so forth.Ubiquitination is an important post-translational modification that tightly regulates proteins location,translocation,interaction and participate in many biological processes,including mitosis.Similar to the other post-translational modifications,ubiquitination is a dynamic and reversible process in cell.Ubiquitin or ubiquitin chain can be hydrolyed under the catalysis of deubquitinase(DUBs),thereby regulates the ubiquitination level of target proteins.BRISC is a K63 specific deubquitinase containing MERIT40,ABRO1,BRCC45 and BRCC36,and is involved in multiple biological cellular processes,such as IFNs mediated antiviral immune regulation,inflammatory reaction,and spindle assembly.However,little is known about the role and mechanism of BRISC in the regulation of spindle assembly.Through immunofluorescence,immunoimpricipitation,Pull Down,as well as western blot,we found that(i)Tankyrase1,MERIT40,BRCC36 colocalize to spindle pole;(ii)RXXPEG motif is required for MERIT40 localization in spindle pole and it’s interaction with Tankyrase1;(iii)the ARC V subdomain of Tankyrase1 is required for interacting with MERI40 and BRCC36;(iv)the RXXPEG motif mutant R28 A of MERIT40 displays defects in mitosis;(v)during mitosis,Tankyrase1 can be modified by K63-linked ubiquitin chain,the level of which is negatively regulated by MERTI40 complex.Mutation of the RXXPEG motif mitigates or even abolishes the interaction between MERIT40 and Tankyrase1,leading to significant K63-linked ubiquitination of affect the catalytic activity of Tankyrase1 and ultimately lead to dysfunction in spindle assembly.