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A novel role of farnesylation in targeting a mitotic checkpoint protein, human Spindly, to kinetochores.

Moudgil DK, Westcott N, Famulski JK, Patel K, Macdonald D, Hang H, Chan GK - J. Cell Biol. (2015)

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.

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Affiliation: Department of Oncology, University of Alberta, Edmonton, Alberta, Canada T6G 1Z2.

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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.
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fig6: 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.

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.


A novel role of farnesylation in targeting a mitotic checkpoint protein, human Spindly, to kinetochores.

Moudgil DK, Westcott N, Famulski JK, Patel K, Macdonald D, Hang H, Chan GK - J. Cell Biol. (2015)

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.
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fig6: 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.
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.

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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oncology, University of Alberta, Edmonton, Alberta, Canada T6G 1Z2.

Show MeSH
Related in: MedlinePlus