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Molecular and Cellular Biology, August 2005, p. 6660-6672, Vol. 25, No. 15
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.15.6660-6672.2005

Sil Phosphorylation in a Pin1 Binding Domain Affects the Duration of the Spindle Checkpoint

Stefano Campaner,^1, Philipp Kaldis,² Shai Izraeli,^1, and Ilan R. Kirsch^1*

Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892,¹ Mouse Cancer Genetics Program, CCR, NCI, Frederick, Maryland 21702²

Received 14 December 2004/ Returned for modification 25 January 2005/ Accepted 6 May 2005

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.

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* Corresponding author. Present address: Research Oncology, Amgen, 1201 Amgen Court West, AW1-J4144, Seattle, WA 98119-3105. Phone: (206) 265-7316. Fax: (206) 216-5930. E-mail: lkirsch{at}amgen.com.

Supplemental material for this article may be found at http://mcb.asm.org/.

Present address: Department of Experimental Oncology, European Institute of Oncology, Milan, Italy.

Present address: Department of Pediatric Hematology-Oncology, The Chaim Sheba Medical Center, Tel-Aviv University, Tel-Hashomer, 52621 Israel.

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Molecular and Cellular Biology, August 2005, p. 6660-6672, Vol. 25, No. 15
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.15.6660-6672.2005

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