Figure 6.

hSpindly is farnesylated in vivo and farnesylation regulates its interaction with the RZZ complex. (A) HeLa cells transiently expressing GFP-WT hSpindly, GFP-C602A hSpindly (of CAAX motif), GFP-hSpindly-E, and GFP-hSpindly-F were metabolically labeled with alkynyl-farnesol (prenylation reporter) and HeLa cells expressing WT GFP-Spindly were either treated with FTI or DMSO. (top) Fluorescence detection of farnesylated immunoprecipitated GFP-tagged hSpindly protein as shown in gel. (bottom) Loading control is shown. WT Spindly, Spindly-E, and Spindly-F exhibit bands indicating farnesylation. Farnesylation of WT Spindly is inhibited in the presence of FTIs (lanes 3 and 4). As expected, C602A mutant of hSpindly is not farnesylated in vivo (lane 6). (B) Immunoprecipitation of hSpindly (70 kD) from mitotic HeLa cell lysates followed by Western blot against Zw10 (89 kD) or Rod (250 kD) RZZ complex subunits. I, input; IP, immunoprecipitated; FT, flowthrough. HeLa cells were treated with either DMSO or 10 µM of FTI-L744832 for 24 h before harvesting and arrested in mitosis with nocodazole treatment. Zw10 is indicated by an arrowhead (IP lanes), and an asterisk denotes a nonspecific band. Inhibition of farnesylation leads to loss of hSpindly and RZZ complex interaction. (C) GFP-Trap from mitotic HeLa cells with endogenous hSpindly knockdown and expressing RNAi resistant GFP-WT hSpindly (98.5 kD) or GFP-C602A hSpindly mutant (98.5 kD). GFP-Trap followed by Western blot against Zw10 (89 kD) or Rod (250 kD) subunits of the RZZ complex. Cells were incubated in nocodazole to accumulate in mitosis before harvesting. Cysteine-to-alanine mutant of GFP-hSpindly leads to loss of hSpindly and RZZ complex interaction.