Sil phosphorylation in a Pin1 binding domain affects the duration of the spindle checkpoint.
SIL is an immediate-early gene that is essential for embryonic development and is implicated in T-cell leukemia-associated translocations. We now show that the Sil protein is hyperphosphorylated during mitosis or in cells blocked at prometaphase by microtubule inhibitors. Cell cycle-dependent phosphorylation of Sil is required for its interaction with Pin1, a regulator of mitosis. Point mutation of the seven (S/T)P sites between amino acids 567 and 760 reduces mitotic phosphorylation of Sil, Pin1 binding, and spindle checkpoint duration. When a phosphorylation site mutant Sil is stably expressed, the duration of the spindle checkpoint is shortened in cells challenged with taxol or nocodazole, and the cells revert to a G2-like state. This event is associated with the downregulation of the kinase activity of the Cdc2/cyclin B1 complex and the dephosphorylation of the threonine 161 on the Cdc2 subunit. Sil downregulation by plasmid-mediated RNA interference limited the ability of cells to activate the spindle checkpoint and correlated with a reduction of Cdc2/cyclin B1 activity and phosphorylation on T161 on the Cdc2 subunit. These data suggest that a critical region of Sil is required to mediate the presentation of Cdc2 activity during spindle checkpoint arrest.
Campaner
Stefano
S
Research Oncology, Amgen, 1201 Amgen Court West, AW1-J4144, Seattle, WA 98119-3105, USA.
Kaldis
Philipp
P
Izraeli
Shai
S
Kirsch
Ilan R
IR
D001665:Binding Sites; D002453:Cell Cycle / Q000502:physiology*; D004247:DNA / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008938:Mitosis / Q000502:physiology; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D015514:Oncogene Proteins, Fusion / Q000378:metabolism*; D019696:Peptidylprolyl Isomerase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D008941:Spindle Apparatus / Q000378:metabolism*; D013997:Time Factors
D047908:Intracellular Signaling Peptides and Proteins; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D015514:Oncogene Proteins, Fusion; C071236:STIL protein, human; D004247:DNA; C000606846:PIN1 protein, human; D019696:Peptidylprolyl Isomerase