Figure 5.

Phosphorylation of CENP-50 by PLK1 is essential for recovery from spindle damage. (Aa) Low-mobility bands were detected by Western blot analysis with anti-CENP-50 antibody. The amounts of the low-mobility bands (P) were increased in cells enriched in M phase by nocodazole treatment for 12 h (WT, −λ-protein phosphatase [PPase], +nocodazole [Noc]) compared with those in asynchronous cells (WT, −λ-PPase, −Noc). These low-mobility bands were abolished by treatment of the cell lysates with λ-PPase (+λ-PPase), indicating that CENP-50 is phosphorylated. The arrow indicates the unphosphorylated form of CENP-50. (b) Western blot analysis of WT cells (WT) and 62.63A/17-15 cells (62.63A) in the presence of tetracycline, in which mutant CENP-50 cDNA (with substitution of two potential serine/threonine phosphorylation sites with alanine) was transfected into 17-15 cells (CENP-50–conditional KO cells). The amounts of the low-mobility CENP-50 bands were reduced in 62.63A/17-15 cells. Arrowhead and arrow indicate low- and high-mobility bands, respectively. Arrowhead with asterisk indicates hyperphosphorylation caused by M phase arrest with nocodazole treatment. Anti-tubulin antibody was used as a loading control. (c) CENP-50 amino acid sequences of human, mouse, and chicken. S62 and T63 of chicken CENP-50 corresponds S77 and T78 of human sequence. (B) Spindle damage recovery assay as described in Figure 4 (A) for 17-15 cells (CENP-50–conditional KO cells),WT/17-15 cells, in which WT CENP-50 cDNA was transfected into 17-15 cells, and 62.63A/17-15 cells. +tet indicates addition of tetracycline to the culture medium. (C) Western blot analysis of WT cells (WT) and 62.63A/17-15 cells (62.63A) in the presence of tetracycline. Anti-CENP-50, anti-CENP-O, anti-CENP-P, anti CENP-Q, and anti-CENP-R antibodies were used. Anti-tubulin antibody was used as a loading control. Arrows indicate each protein. Arrowheads indicate phosphorylated bands. Phosphorylated bands denoted by arrowhead with asterisk were reduced in 62.63A/17-15 cells. (D) Immunofluorescence analysis of 62.63A/17-15 cells with anti-CENP-50, anti-CENP-O, anti-CENP-P, anti-CENP-Q, and anti-CENP-R antibodies. Arrowheads indicate mitotic cells. (E) (a) Western blot analysis of cells expressing CENP-50 cDNA mutant 16×A or cDNA 16×A + 62.63A with anti-CENP-50 antibodies. Western blot data shows that mobility of CENP-50 with 16×A or 16×A + 62.63A is faster than that of phosphorylated CENP-50. (b) Results of spindle damage recovery assay for cells expressing CENP-50 mutant with 16×A or 16×A + 62.63A are also shown.