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Involvement of C-terminal truncation mutation of kinesin-5 in resistance to kinesin-5 inhibitor.

ABSTRACT: Cultured cells easily develop resistance to kinesin-5 inhibitors (K5Is) often by overexpressing a related motor protein, kinesin-12/KIF15, or by acquiring mutations in the N-terminal motor domain of kinesin-5/KIF11 itself. We aimed to identify novel mechanisms responsible for resistance to S-trityl L-cysteine (STLC), one of the K5Is, using human osteosarcoma cell lines. Among six lines examined, U-2OS and HOS survived chronic STLC treatment and gave rise to resistant cells with IC50s at least 10-fold higher than those of the respective parental lines. Depletion of KIF15 largely eliminated the acquired K5I resistance in both cases, consistent with the proposed notion that KIF15 is indispensable for it. In contrast to the KIF11-independent property of the cells derived from HOS, those derived from U-2OS still required KIF11 for their growth and, intriguingly, expressed a C-terminal truncated variant of KIF11 resulting from a frame shift mutation (S1017fs). All of the isolated clones harbored the same mutation, suggesting its clonal expansion in the cell population due to the growth advantage during chronic STLC treatment. Transgenic expression of KIF11S1017fs in the parental U-2OS cells, as well as in HeLa cells, conferred a moderate but reproducible STLC resistance, probably owing to STLC-resistant localization of the mutant KIF11 on mitotic spindle. Our observations indicate that both KIF15 and the C-terminal-truncated KIF11 contributes to the STLC resistance of the U-2OS derived cells.

SUBMITTER: Saeki E

PROVIDER: S-EPMC6296710 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Involvement of C-terminal truncation mutation of kinesin-5 in resistance to kinesin-5 inhibitor.

Saeki Eri E Yasuhira Shinji S Shibazaki Masahiko M Tada Hiroshi H Doita Minoru M Masuda Tomoyuki T Maesawa Chihaya C

PloS one 20181217 12

Cultured cells easily develop resistance to kinesin-5 inhibitors (K5Is) often by overexpressing a related motor protein, kinesin-12/KIF15, or by acquiring mutations in the N-terminal motor domain of kinesin-5/KIF11 itself. We aimed to identify novel mechanisms responsible for resistance to S-trityl L-cysteine (STLC), one of the K5Is, using human osteosarcoma cell lines. Among six lines examined, U-2OS and HOS survived chronic STLC treatment and gave rise to resistant cells with IC50s at least 10 ...[more]

PMID: 30557316

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Project description:BACKGROUND: The amyloid-? (A?) peptide is the primary component of the extracellular senile plaques characteristic of Alzheimer's disease (AD). The metals hypothesis implicates redox-active copper ions in the pathogenesis of AD and the Cu(2+) coordination of various A? peptides has been widely studied. A number of disease-associated modifications involving the first 3 residues are known, including isomerisation, mutation, truncation and cyclisation, but are yet to be characterised in detail. In particular, A? in plaques contain a significant amount of truncated pyroglutamate species, which appear to correlate with disease progression. METHODOLOGY/PRINCIPAL FINDINGS: We previously characterised three Cu(2+)/A?1-16 coordination modes in the physiological pH range that involve the first two residues. Based upon our finding that the carbonyl of Ala2 is a Cu(2+) ligand, here we speculate on a hypothetical Cu(2+)-mediated intramolecular cleavage mechanism as a source of truncations beginning at residue 3. Using EPR spectroscopy and site-specific isotopic labelling, we have also examined four A? peptides with biologically relevant N-terminal modifications, A?1[isoAsp]-16, A?1-16(A2V), A?3-16 and A?3[pE]-16. The recessive A2V mutation preserved the first coordination sphere of Cu(2+)/A?, but altered the outer coordination sphere. Isomerisation of Asp1 produced a single dominant species involving a stable 5-membered Cu(2+) chelate at the amino terminus. The A?3-16 and A?3[pE]-16 peptides both exhibited an equilibrium between two Cu(2+) coordination modes between pH 6-9 with nominally the same first coordination sphere, but with a dramatically different pH dependence arising from differences in H-bonding interactions at the N-terminus. CONCLUSIONS/SIGNIFICANCE: N-terminal modifications significantly influence the Cu(2+) coordination of A?, which may be critical for alterations in aggregation propensity, redox-activity, resistance to degradation and the generation of the A?3-× (×?=?40/42) precursor of disease-associated A?3[pE]-x species.

Dataset Information

Involvement of C-terminal truncation mutation of kinesin-5 in resistance to kinesin-5 inhibitor.

Publications

Involvement of C-terminal truncation mutation of kinesin-5 in resistance to kinesin-5 inhibitor.

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