Sgt1 is required for human kinetochore assembly

Steensgaard, Peter; Garré, Massimiliano; Muradore, Ivan; Transidico, Pietro; Nigg, Erich A.; Kitagawa, Katsumi; Earnshaw, William C.; Faretta, Mario; Musacchio, Andrea

doi:10.1038/sj.embor.7400154

Cited by 74 publications

(94 citation statements)

References 32 publications

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“…Human SGT1 (SUGT1) can rescue the sgt1-null mutant (Kitagawa et al, 1999), a result that suggests that the function of SGT1 is conserved in humans and yeast. This hypothesis is also supported by the finding that CENP-I, CENP-F, and HEC1, but not CENP-C, are absent from kinetochores in SGT1-depleted human cells (Steensgaard et al, 2004). However, the role of HSP90 at human kinetochores has not been explored.…”

Section: Introductionsupporting

confidence: 68%

17-AAG, an Hsp90 inhibitor, causes kinetochore defects: a novel mechanism by which 17-AAG inhibits cell proliferation

et al. 2006

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The Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG), which is currently in clinical trials, is thought to exert antitumor activity by simultaneously targeting several oncogenic signaling pathways. Here we report a novel mechanism by which 17-AAG inhibits cell proliferation, and we provide the first evidence that HSP90 is required for the assembly of kinetochore protein complexes in humans. 17-AAG caused delocalization of several kinetochore proteins including CENP-I and CENP-H but excluding CENP-B and CENP-C. Consistently, 17-AAG induced a mitotic arrest that depends on the spindle checkpoint and induced misalignment of chromosomes and aneuploidy. We found that HSP90 associates with SGT1 (suppressor of G2 allele of skp1; SUGT1) in human cells and that depletion of SGT1 sensitizes HeLa cells to 17-AAG. Overexpression of SGT1 restored the localization of specific kinetochore proteins and chromosome alignment in cells treated with 17-AAG. Biochemical and genetic results suggest that HSP90, through its interaction with SGT1 (SUGT1), is required for kinetochore assembly. Furthermore, timecourse experiments revealed that transient treatment with 17-AAG between late S and G2/M phases causes substantial delocalization of CENP-H and CENP-I, a finding that strongly suggests that HSP90 participates in kinetochore assembly in a cell cycle-dependent manner.

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Section: Introductionsupporting

confidence: 68%

17-AAG, an Hsp90 inhibitor, causes kinetochore defects: a novel mechanism by which 17-AAG inhibits cell proliferation

et al. 2006

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“…Anti-GFP antibody (a gift from Dr J Neefjes) was used 1:500. Mad1 antibody (a gift from Dr Musacchio) (Steensgaard et al, 2004) was used 1:10. a-tubulin antibody (Sigma) was used 1:750. The p150glued, EB1 antibodies (Transduction Laboratories) and anti-dynein intermediate chain (Sigma) were used 1:200.…”

Section: Antibodiesmentioning

confidence: 99%

CLIP-170 facilitates the formation of kinetochore–microtubule attachments

Tanenbaum

Galjart

Vugt³

et al. 2005

EMBO J

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CLIP-170 is a microtubule 'plus end tracking' protein involved in several microtubule-dependent processes in interphase. At the onset of mitosis, CLIP-170 localizes to kinetochores, but at metaphase, it is no longer detectable at kinetochores. Although RNA interference (RNAi) experiments have suggested an essential role for CLIP-170 during mitosis, the molecular function of CLIP-170 in mitosis has not yet been revealed. Here, we used a combination of high-resolution microscopy and RNAi-mediated depletion to study the function of CLIP-170 in mitosis. We found that CLIP-170 dynamically localizes to the outer most part of unattached kinetochores and to the ends of growing microtubules. In addition, we provide evidence that a pool of CLIP-170 is transported along kinetochoremicrotubules by the dynein/dynactin complex. Interference with CLIP-170 expression results in defective chromosome congression and diminished kinetochoremicrotubule attachments, but does not detectibly affect microtubule dynamics or kinetochore-microtubule stability. Taken together, our results indicate that CLIP-170 facilitates the formation of kinetochore-microtubule attachments, possibly through direct capture of microtubules at the kinetochore.

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“…Like HSP90, both SGT1 and RAR1 are required for the stabilization of many R proteins (Tornero et al, 2002;Bieri et al, 2004;Azevedo et al, 2006). Similarly, both HSP90 and SGT1 are required for the stabilization and assembly of the yeast CBF3 (for centromere binding factor3) and the human kinetochore complexes (Bansal et al, 2004;Lingelbach and Kaplan, 2004;Steensgaard et al, 2004;Niikura et al, 2006). As yeast does not contain any RAR1 homolog, CBF3 complex assembly is a RAR1-independent process.…”

Section: Introductionmentioning

confidence: 99%

Structural and Functional Analysis of SGT1 Reveals That Its Interaction with HSP90 Is Required for the Accumulation of Rx, an R Protein Involved in Plant Immunity

et al. 2007

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SGT1 (for suppressor of G2 allele of skp1) and RAR1 (for required for Mla12 resistance) are highly conserved eukaryotic proteins that interact with the molecular chaperone HSP90 (for heat shock protein90). In plants, SGT1, RAR1, and HSP90 are essential for disease resistance triggered by a number of resistance (R) proteins. Here, we present structural and functional characterization of plant SGT1 proteins. Random mutagenesis of Arabidopsis thaliana SGT1b revealed that its CS (for CHORD-SGT1) and SGS (for SGT1 specific) domains are essential for disease resistance. NMR-based interaction surface mapping and mutational analyses of the CS domain showed that the CHORD II domain of RAR1 and the N-terminal domain of HSP90 interact with opposite sides of the CS domain. Functional analysis of the CS mutations indicated that the interaction between SGT1 and HSP90 is required for the accumulation of Rx, a potato (Solanum tuberosum) R protein.Biochemical reconstitution experiments suggest that RAR1 may function to enhance the SGT1-HSP90 interaction by promoting ternary complex formation.

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Sgt1 is required for human kinetochore assembly

Cited by 74 publications

References 32 publications

17-AAG, an Hsp90 inhibitor, causes kinetochore defects: a novel mechanism by which 17-AAG inhibits cell proliferation

17-AAG, an Hsp90 inhibitor, causes kinetochore defects: a novel mechanism by which 17-AAG inhibits cell proliferation

CLIP-170 facilitates the formation of kinetochore–microtubule attachments

Structural and Functional Analysis of SGT1 Reveals That Its Interaction with HSP90 Is Required for the Accumulation of Rx, an R Protein Involved in Plant Immunity

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