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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: We showed that hSpindly is farnesylated in vivo and farnesylation is essential for its interaction with the RZZ complex and hence KT localization.FTI treatment and hSpindly knockdown displayed the same mitotic phenotypes, indicating that hSpindly is a key FTI target in mitosis.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.

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hSpindly farnesylation motif can be substituted with CENP-E or CENP-F farnesylation motif but not a geranylgeranylation motif. (A) A schematic diagram of hSpindly depicting substitution of its farnesylation motif with CENP-E and CENP-F, referred to as Spindly-E and Spindly-F, respectively. The last amino acid of Spindly is changed such that it is a geranylgeranylation motif instead of farnesylation motif referred to as Spindly-GG1 and Spindly-GG2. The KT localization of each construct is shown (+, KT localization; −, non-KT localization). Amino acid numbers are indicated. (B) HeLa cells were transiently transfected with EGFP-hSpindly fusion constructs (shown in A) and KT localization ability of each construct was analyzed using fluorescence microscopy. Representative images show that the farnesylation motif of Spindly can be replaced with the farnesylation motif of known farnesylated proteins CENP-E and CENP-F, but a geranylgeranylation motif cannot target hSpindly to KTs. Bar, 10 µm.
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fig5: hSpindly farnesylation motif can be substituted with CENP-E or CENP-F farnesylation motif but not a geranylgeranylation motif. (A) A schematic diagram of hSpindly depicting substitution of its farnesylation motif with CENP-E and CENP-F, referred to as Spindly-E and Spindly-F, respectively. The last amino acid of Spindly is changed such that it is a geranylgeranylation motif instead of farnesylation motif referred to as Spindly-GG1 and Spindly-GG2. The KT localization of each construct is shown (+, KT localization; −, non-KT localization). Amino acid numbers are indicated. (B) HeLa cells were transiently transfected with EGFP-hSpindly fusion constructs (shown in A) and KT localization ability of each construct was analyzed using fluorescence microscopy. Representative images show that the farnesylation motif of Spindly can be replaced with the farnesylation motif of known farnesylated proteins CENP-E and CENP-F, but a geranylgeranylation motif cannot target hSpindly to KTs. Bar, 10 µm.

Mentions: Because the C terminus of hSpindly is required for KT localization through the CAAX farnesylation motif, we hypothesized that any farnesylation motif on the C terminus of hSpindly regardless of the specific residues is sufficient for it to undergo farnesylation and target it to KTs. We replaced the CPQQ residues of hSpindly with the CKTQ (CENP-E) and CKVQ (CENP-F) farnesylation motifs, referred to as Spindly-E and Spindly-F, respectively. Both Spindly-E and -F localized to KTs during prometaphase, supporting our hypothesis that the exact amino acid sequence is not important and the ability to be farnesylated is sufficient for hSpindly KT localization (Fig. 5, A and B; and Fig. S1 G).


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 farnesylation motif can be substituted with CENP-E or CENP-F farnesylation motif but not a geranylgeranylation motif. (A) A schematic diagram of hSpindly depicting substitution of its farnesylation motif with CENP-E and CENP-F, referred to as Spindly-E and Spindly-F, respectively. The last amino acid of Spindly is changed such that it is a geranylgeranylation motif instead of farnesylation motif referred to as Spindly-GG1 and Spindly-GG2. The KT localization of each construct is shown (+, KT localization; −, non-KT localization). Amino acid numbers are indicated. (B) HeLa cells were transiently transfected with EGFP-hSpindly fusion constructs (shown in A) and KT localization ability of each construct was analyzed using fluorescence microscopy. Representative images show that the farnesylation motif of Spindly can be replaced with the farnesylation motif of known farnesylated proteins CENP-E and CENP-F, but a geranylgeranylation motif cannot target hSpindly to KTs. Bar, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4384735&req=5

fig5: hSpindly farnesylation motif can be substituted with CENP-E or CENP-F farnesylation motif but not a geranylgeranylation motif. (A) A schematic diagram of hSpindly depicting substitution of its farnesylation motif with CENP-E and CENP-F, referred to as Spindly-E and Spindly-F, respectively. The last amino acid of Spindly is changed such that it is a geranylgeranylation motif instead of farnesylation motif referred to as Spindly-GG1 and Spindly-GG2. The KT localization of each construct is shown (+, KT localization; −, non-KT localization). Amino acid numbers are indicated. (B) HeLa cells were transiently transfected with EGFP-hSpindly fusion constructs (shown in A) and KT localization ability of each construct was analyzed using fluorescence microscopy. Representative images show that the farnesylation motif of Spindly can be replaced with the farnesylation motif of known farnesylated proteins CENP-E and CENP-F, but a geranylgeranylation motif cannot target hSpindly to KTs. Bar, 10 µm.
Mentions: Because the C terminus of hSpindly is required for KT localization through the CAAX farnesylation motif, we hypothesized that any farnesylation motif on the C terminus of hSpindly regardless of the specific residues is sufficient for it to undergo farnesylation and target it to KTs. We replaced the CPQQ residues of hSpindly with the CKTQ (CENP-E) and CKVQ (CENP-F) farnesylation motifs, referred to as Spindly-E and Spindly-F, respectively. Both Spindly-E and -F localized to KTs during prometaphase, supporting our hypothesis that the exact amino acid sequence is not important and the ability to be farnesylated is sufficient for hSpindly KT localization (Fig. 5, A and B; and Fig. S1 G).

Bottom Line: We showed that hSpindly is farnesylated in vivo and farnesylation is essential for its interaction with the RZZ complex and hence KT localization.FTI treatment and hSpindly knockdown displayed the same mitotic phenotypes, indicating that hSpindly is a key FTI target in mitosis.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