a–d, The morphology protein Wsh3 is required to recruit PP1Dis2 to cell tips19. Wsh3-recruited PP1 promotes the dephosphorylation of the DYRK family kinase Pom1 (ref. 42). Dephosphorylation of Pom1 promotes its association with the cell cortex. Subsequent auto-phosphorylation diminishes this newfound affinity for the cortex until the kinase loses its affinity for the cortex. Because Wsh3 is only found at cell tips, Pom1 only associates with the cortex at the tips42. We exploited this relationship to test the ability of the SKEVLF sequence in the PP1-docking consensus site of B56Par1 to function as a PP1Dis2-docking site in vivo. a, The indicated wsh3 alleles were cloned into the pINTL41PkN vector32 and integrated at the leu1 locus before introduction into wsh3.Δ background in which the endogenous wsh3+ gene had been deleted. b, Expression of the transgenes was repressed by the addition of thiamine to culture medium (uninduced) before filtration into thiamine-free medium induced expression of each allele 24 h later (induced). c, PP1Dis2.GFP was not enriched at cell tips in the absence of wsh3 induction. Induction of the wild-type or wsh3.I232SR234ET236L allele in which the SKEVLF motif of the B56Par1 PP1Dis2-docking site was substituted for the native IFRVTF motif of Wsh3 led to the recruitment of PP1Dis2.GFP to cell tips whereas expression of the PP1-docking-site mutant wsh3.F237A failed to do so. d, Pom1 recruitment to cell tips upon expression of both the wild-type and SKEVLF alleles, but not the PP1-docking-site alleles, indicated that the PP1Dis2 recruited to Wsh3 by the SKEVLF allele was functionally indistinguishable from that recruited by the wild-type molecule. e–g, Commitment to mitosis is promoted by the activation of Cdk–cyclin B. Cdk1–cyclin B activity is regulated by the level of phosphorylation on threonine 14 and tyrosine 15 within its ATP-binding site. Wee1 kinase phosphorylates these residues while Cdc25 removes the phosphate to trigger mitotic commitment43. The lack of Cdc25 phosphatase activity achieved by incubating cells harbouring the temperature-sensitive cdc25.22 mutation at temperatures above 34 °C kills cells because they are unable to remove the inhibitory phosphate from Cdk1. The cut12.s11 and cut12.s14 mutations enable cdc25.22 cells to divide at 34 °C (refs 20, 44). cut12.s11 is a G71V mutation at the start of a highly conserved bipartite PP1-docking site GILKTPGTLQIKKTVNF (the letters in bold indicate the residues that constitute the PP1-docking site). cut12.s14 is a F87L mutation at the end of the same docking motif20. e, Alignment of the conserved PP1-docking site of Cut12 homologues from the indicated fungi and the mutations generated in each of the indicated cut12 alleles. f, g, Mutation of the PP1-docking site of S. pombe Cut12 to abolish the function of either the GILK or the KxVxF motifs of Cut12 reduces PP1Dis2 association in immunoprecipitation assays (f) and suppresses the lethal mitotic commitment defect of cdc25.22 (g). Simultaneous mutation of both motifs abolishes PP1 association in immunoprecipitation assays and confers a higher level of suppression of cdc25.22 (g) that can enable cells to survive complete removal of cdc25+ coding sequences20. We asked whether the GLLR sequence found upstream of the B56Par1 KxVxF motif (Fig. 2a) would substitute for the GILK sequence of Cut12 in both the co-immunoprecipitation and cdc25.22 suppression assays; in both cases we found that it could. The association between PP1Dis2 and Cut12 in the cut12.I72LK74R mutant that converts the GILK motif to a GLLR was indistinguishable from wild type. Also, the G71V mutation of cut12.s11 suppressed cdc25.22 whereas cut12.I72LK74R did not (g). Thus, the function bestowed on Cut12 by the GILK sequence is fully maintained upon substitution with GLLR, indicating that GLLR is a functional variant of GILK. Error bars show s.d. Biological replicates: for b–d, n = 1; for f, n = 5; for g, n = 3.