Identification of a nuclear protein binding partner of phospholipase C

Abstract

The levels of nuclear phosphoinositides are governed, in part, by phospholipase C (PLC), including PLC-δ1 whose nuclear activity fluctuates with cell cycle. To understand the role this phospholipase plays in this compartment, a proteomics approach was used to identify nuclear binding partners. A PLC-δ1 affinity column was created to capture proteins extracted from purified nuclei. Specifically bound and eluted proteins were subjected to 2D electrophoresis, digested with trypsin, and the resulting peptides separated by microbore RP-HPLC and sequenced by MS/MS. Of the five proteins identified, Bub3 had the highest confidence score. Further testing showed Bub3 specifically and quantitatively co-immunoprecipitated with PLC-δ1 in extracts of C6 glioma cells, and was highly co-localized with PLC-δ1-eGFP in the nuclei of these cells synchronized to the G1/S boundary. Bub3, part of the anaphase promoting complex (APC), has previously been shown to associate with the kinetochore through Bub1. Previous work has also demonstrated that the yeast homolog, plc1, associates with this structure. Moreover, temperature sensitive PLC1 mutations cause mis-sorting of chromosomes; whereas, its deletion mutants require a functional BUB1/BUB3-dependent spindle checkpoint complex for viability. Taken together, our data suggest a link between PLC-δ1 and regulation of the kinetochore and APC in mammals.