A novel role of farnesylation in targeting a mitotic checkpoint protein, human Spindly, to kinetochores.
Bottom Line: Inhibition of farnesylation using a farnesyl transferase inhibitor (FTI) abrogated hSpindly KT localization without affecting RZZ complex, CENP-E, and CENP-F KT localization.We showed that hSpindly is farnesylated in vivo and farnesylation is essential for its interaction with the RZZ complex and hence KT localization.Our data show a novel role of lipidation in targeting a checkpoint protein to KTs through protein-protein interaction.
Affiliation: Department of Oncology, University of Alberta, Edmonton, Alberta, Canada T6G 1Z2.Show MeSH
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Mentions: To investigate if hSpindly undergoes farnesylation in vivo, pEGFP-WT Spindly, pEGFP-C602A Spindly (of the CAAX motif), pEGFP-Spindly-E, and pEGFP-Spindly-F transfected HeLa cells were labeled with alkynyl-farnesol, a farnesyl group analogue (Charron et al., 2011), in the presence or absence of FTIs. Farnesylated proteins were detected by performing bioorthogonal ligation (click chemistry) with a fluorescent tag followed by in-gel fluorescence signal detection. Wild-type (WT) Spindly is farnesylated as indicated by the presence of a fluorescent band (Fig. 6 A, lane 5) and farnesylation is inhibited in the presence of FTIs (Fig. 6 A, lanes 3 and 4). Furthermore, Spindly-E and Spindly-F undergo farnesylation as expected because these constructs localize to KTs (Fig. 6 A, lanes 1 and 2). The hSpindly C602A mutant cannot localize to KTs and did not undergo farnesylation (Fig. 6 A, lane 6). These results provide an in vivo validation of hSpindly farnesylation.
Affiliation: Department of Oncology, University of Alberta, Edmonton, Alberta, Canada T6G 1Z2.