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. 2009 Feb 19;28(7):902–914. doi: 10.1038/emboj.2009.38

Figure 8.

Figure 8

Phosphorylation of rLIC1 on S207 is important for resolution of the spindle assembly checkpoint. (A) Endogenous hLIC1 in interphase cells (I) undergoes a gel shift during mitosis (M, left). Both the mitotic form of LIC1 (above) and the interphase form (below) co-immunoprecipitate with dynein IC-1. (B) Myc-tagged rLIC1 also undergoes a mitotic shift and is reduced to the lower form with lambda protein phosphatase treatment (λPP). (C) The mitotic shift of myc–rLIC1 can be partially suppressed by a S207A mutation and restored with a S207E mutation (three left lanes). All myc–rLIC1 mutants co-immunoprecipitate with dynein IC-1 antibodies (centre) but not control IgG (right). S, A, E=S207, S207A, S207E, respectively. (D) Top: Myc–rLIC1 mutants (S, A, E) immunoprecipitated from interphase lysates undergo phosphorylation on incubation with cdk1–cyclin B. Arrows indicate the small difference in upward migration (see text) among the S, A and E mutants. Bottom: Observed masses (italics) and single letter amino-acid sequences (in parentheses with N-terminal and C-terminal residue numbers indicated and phosphorylated residues underlined) from MALDI-QIT-TOF mass spectrometric analysis of the three phosphopeptides obtained from a tryptic digest of the phosphorylated S207 band (indicated by dotted lines). (E) Mitotic index of HeLa cells treated with hLIC1 siRNA and overexpressing myc–rLIC1 or point mutants (for S207, S207A and S207E, n=approximately 1500 cells each from three experiments; for LIC1 knockdown alone, approximately 500 cells were counted from three experiments). OX'd, overexpressed. Mitotic index of control (GFP siRNA treated) Hela cells was approximately 5%. (F) Average interkinetochore distance in LIC1 siRNA treatment (first bar) followed by rescue with myc–rLIC1 constructs. Bars, average interkinetochore distance for 30 kinetochore pairs (five cells) per experiment for each construct, total of three experiments. (G) All three myc–rLIC1 constructs localise to kinetochores (prometaphase) and spindle poles (metaphase). Scale bar=2 μM.