Two major alternative splice variants of beta-TrCP1 interact with CENP-W with different binding preferences.

Beta-transducin repeat containing protein 1 (β-TrCP1) is a versatile F-box protein that is responsible for substrate recognition of SCFβ-TrCP1 ubiquitin ligase. In human cells, two major alternatively spliced isoforms (b and f) of β-TrCP1 were found. Recently, we identified that CENP-W interacts with the β-TrCP1 and regulates the cellular distribution of β-TrCP1. In this study, we examined whether CENP-W, a new kinetochore component, may differentially regulate the two major isoforms of human β-TrCP1 (b and f), especially in the cytoplasmic-nuclear shuttling of β-TrCP1. An in vivo binding assay was performed to examine whether CENP-W binds differently to the two isoforms of β-TrCP1. EGFP-conjugated β-TrCP1 isoforms were co-transfected with NLS-defective mutant CENP-W and their cellular distribution were observed using a fluorescence microscopy. Although CENP-W interacts with both b and f isoforms, it has a greater affinity for the b isoform rather than f isoform. Moreover, CENP-W effectively regulates the nuclear-cytoplasmic shuttling of these two β-TrCP1 isoforms, but with a slight preference towards the b isoform. The Elongin C-binding motif existing in the b isoform may be involved in their specific association. CENP-W showed a higher affinity toward the β-TrCP1 b isoform, and translocated isoform b more efficiently than isoform f, which may allow a fine regulation of of β-TrCP1 in the cells.