(A) Alignment of human (Skb1Hs), S. pombe (Skb1Sp), and C. elegans (Skb1Ce) Skb1 homologs. Alignments were generated by using the lasergene megalign program (DNAstar, Madison, WI). Identical amino acid residues are indicated by black boxes. (B) Southern blot of BamHI- (Left) and EcoRI- (Right) digested human genomic DNA by using the SKB1Hs cDNA as a probe.

Skb1 and Shk1 associate with Cdc2 and with each other in fission yeast. (A) S. pombe strain CHP428 was transformed with plasmids for expression of GST (pAAUGST), GST-Skb1 (pAAUGSTSkb1), or GST-Shk1 (pAAUGSTShk1) fusion proteins. Transformants were grown to about 10⁷ cells per ml in EMM, then lysed and GST complexes precipitated as described in Methods. Complexes were resolved by SDS/PAGE and transferred to nitrocellulose. Immunoblotting was performed by using anti-Cdc2 PSTAIRE antibody (Top). Membranes were then stripped and reprobed with anti-GST antibody (Bottom). Cdc2 protein was associated with GST-Skb1 and GST-Shk1, but not with GST. (B) Shk1 and Skb1 proteins associate in S. pombe cells. Lysates were prepared from CHP428 cells overexpressing skb1 and either GST or GST-Shk1ΔN, as described for A. GST and GST-Shk1ΔN complexes were resolved by SDS/PAGE and subjected to immunoblot analysis by using anti-Skb1 antibody (Top). Blots were subsequently stripped and probed with anti-GST antibody (Bottom). Skb1 protein was associated with GST-Shk1ΔN, but not with GST.

Skb1Hs functions as a mitotic inhibitor in S. pombe. (A) Cell division size data for wild-type and skb1Δ cells transformed with pREP1Skb1Hs, for expression of SKB1Hs from the S. pombe nmt1 promoter, or the control plasmid pREP1. SKB1Hs expression causes wild-type and skb1Δ cells to divide at a substantially greater length. (B) SKB1Hs expression exacerbates the t.s. growth defect of a cdc25–22 skb1Δ mutant (Bottom), but does not inhibit the growth of wild-type S. pombe cells (Top). (C) SKB1Hs expression is inhibitory to the growth of cdc2–3w cdc25Δ mutant (Bottom) but not the cdc2–3w mutant (Top). (D) Cdc2 associates with GST-Skb1Hs protein in fission yeast. Cells expressing either GST or GST-Skb1Hs were grown to log phase, lysed, and GST complexes isolated and subjected to immunoblot analysis with anti-Cdc2 and anti-GST antibody, as described in Fig. 3.

Genetic evidence that Shk1 has a mitotic inhibitory function. (A) Exacerbation of the cdc25–22 t.s. growth defect caused by skb1 overexpression is Shk1-dependent. PAAUCM- and pAAUCMSkb1-transformed cdc25–22 (Top) and cdc25–22 nmt-shk1 (Bottom) cells were streaked onto EMM containing 15 μM thiamine and incubated at 33°C for 3 days. (B) Overexpression of shk1 exacerbates the cdc25–22 t.s. growth defect. The cdc25–22 mutant was transformed with a plasmid for overexpression of shk1 (pAAUCMShk1) or with a control plasmid (pAAUCM). Transformants were streaked onto EMM plates and assayed for growth at 30°C (Top) and 33°C (Bottom). Overexpression of shk1 retarded the growth of the cdc25–22 mutant at 30°C and completely inhibited growth at 33°C. (C) Cooverexpression of skb1 and shk1 is only slightly inhibitory to growth of wild-type (CHP428) S. pombe cells. (D) Cooverexpression of skb1 and shk1 more severely exacerbates the cdc25–22 t.s. growth defect than overexpression of either gene alone. The cdc25–22 mutant was transformed with pREP1Skb1 (Skb1) and/or pAAUCMShk1 (Shk1) or with the control plasmids pREP1 and/or pAAUCM. Primary transformants were streaked onto EMM plates and incubated at 30°C. Cooverexpression of skb1 and shk1 completely inhibited growth of the cdc25–22 mutant at 30°C, whereas overexpression of either gene alone only retarded growth.

Genetic interactions of skb1 with genes encoding key mitotic regulators in S. pombe. (A) The skb1 null mutation partially suppresses the t.s. growth defect of the cdc25–22 mutant. cdc25–22 and cdc25–22 skb1Δ S. pombe strains were streaked on YEA plates and incubated at either 36°C (Top) or 33°C (Bottom). The cdc25–22 skb1Δ mutant strain produced visible colonies at 36°C, whereas the cdc25–22 strain did not. At 33°C, the cdc25–22 skb1Δ strain grew significantly faster than the cdc25–22 mutant. (B) Overexpression of skb1 exacerbates the cdc25–22 t.s. growth defect. The cdc25–22 mutant was transformed with a plasmid for overexpression of skb1 (pREP1Skb1) or with a control plasmid (pREP1). Transformants were streaked onto EMM plates and were assayed for growth at 30°C (Top) or 33°C (Bottom). Overexpression of skb1 retarded the growth of the cdc25–22 mutant at 30°C and completely inhibited growth at 33°C. (C) cdc25–22 pREP1- and pREP1Skb1-transformed cells were grown at 26°C in liquid EMM to a density of about 10⁶ cells per ml then shifted to either 33°C or 36°C and incubated for 5 hr before 4′,6-diamidino-2-phenylindole (DAPI) staining and microscopic analysis to determine the percentage of binucleated and septated cells in each culture. (D) skb1 overexpression is inhibitory to the growth of cdc2–3w cdc25Δ, but not cdc2–3w cells. pREP1- and pREP1Skb1-transformed cdc2–3w cells (Top) and cdc2–3w cdc25Δ cells (Bottom) were assayed for growth by being streaked on EMM plates and incubated at 30°C. (E) pREP1- and pREP1Skb1-transformed wild-type cells (CHP428) were streaked onto EMM and grown at either 30°C (Top) or 36°C (Bottom). (F) skb1 overexpression inhibits growth of the wee1Δ mutant at 36°C. pREP1- and pREP1Skb1-transformed wee1Δ cells were streaked onto EMM and grown at either 30°C (Top) or 36°C (Bottom).