Plk1 phosphorylates centromeric protein CENPB

A115 Plk1 is a serine-threonine kinase regulated in mitosis. Plk1 is over-expressed in many different patient cancers, and attenuation of Plk1 kinase activity inhibits tumor cell growth. Multiple substrates and interacting proteins of Plk1 have been reported. A high-content protein microarray was used to identify novel substrates of Plk1.
 Purified recombinant Plk1 was incubated with γ-32P-ATP on the Invitrogen ProtoArray v4 and signals were acquired with a phosphorimager. Several proteins on the array showed statistically significant increases in phosphorylation upon incubation with Plk1. One protein identified as a putative Plk1 substrate was CENPB, or centromeric protein B. CENPB is a component of the centromeric complex of the kinetochore. Its function has not been clearly defined, but may enhance centromere assembly by packaging repetitive DNA. Using recombinant CENPB and Plk1, phosphorylation was confirmed in solution kinase assays following detection by anti-phosphothreonine immunoblot analyses. In-gel tryptic digestion of the in vitro kinase reaction followed by ion trap mass spectrometry identified 5 phosphorylation sites, including Ser and Thr residues. One serine site matches the reported consensus phosphorylation sequence for Plk1. Interaction between these proteins was also observed in tumor cell lines by co-immunoprecipitation of exogenously expressed and tagged Plk1 and CENBP. Threonine phosphorylation of exogenously expressed CENPB was also observed only upon co-expression of Plk1 in cells. Immunofluoresence studies of HeLa and HEK293 cells demonstrated co-localization of endogenous Plk1 and CENPB at the centromeres during mitosis. Plk1 is known to be associated with several cellular structures, including centromeres, however interaction with CENPB has not previously been reported. These data suggest a novel regulation of CENPB by Plk1, implicating Plk1 phosphorylation of CENPB with mitotic checkpoint signaling and/or centrosome assembly.