PMC

(A) Phosphorylation of a single substrate (Ste5^1–260) was monitored with and without the addition of the indicated docking sites, in cells expressing different P_GAL1-induced cyclins. Docking sites from Ste5, Ste20, and Exo84 preferentially enhance phosphorylation by Cln1/2-Cdc28, whereas an RxL-containing fragment from Fin1 converts the substrate into one that is preferred by Clb5 and Clb2
(B) Schematic comparison of different cyclin-Cdk complexes, with a general model for how cyclin-specific docking interactions can selectively enhance substrate phosphorylation by individual forms of cyclin-Cdk.

(A) Candidate Cln2 docking sites from seven Cdk substrates (see ) were inserted into a Ste20^Ste5PM chimera lacking its endogenous docking site. These derivatives were compared to chimeras containing the Ste5 or Ste20 docking sites inserted at the same position, or no docking site (none). Cln2 inhibition of pheromone response was assayed as in . Bars, mean ± SD (n = 5)
(B) The same candidate docking sites used in panel A were inserted at the end of a Ste5 fragment (Ste5^1–260) that lacks its endogenous docking site, and Cln2-driven phosphorylation was assayed. See for additional repetitions.

(A) Domain structures of Ste5 and Ste20. Red circles indicate Cdk sites [, ]; in Ste20, only the 13 confirmed sites (of 23 possible) are shown
(B) Phosphorylation of Ste5 and Ste20 fragments in synchronous cdc15-2 cultures, after release from M phase arrest. Full-length Ste20 is shown for comparison; its phosphorylation behavior was described previously [, ]. Cell cycle progression was monitored by anti-Clb2 immunoblot and by budding
(C) HA-tagged Ste5 or V5-tagged Ste20 fragments, expressed from native promoters, were monitored after galactose-induced expression of GST-tagged cyclins. Reduced electrophoretic mobility signifies phosphorylation, as confirmed by phosphatase treatment (data not shown). For comparison, V5-tagged Swe1 demonstrates activity for Clb2. The relative expression levels for GST-cyclins were highly reproducible; one representative example is shown. A variant of Cln3 lacking ten Cdk sites (Cln3^10A) was used to increase its stability. Results were similar in sic1Δ cells (data not shown), in which the Clb-Cdk inhibitor Sic1 is absent.

(A) The diagram indicates endpoints used for mapping the Cln2-binding region in Ste20, which is outlined in red
(B) Cells co-expressing GST fusions to Ste20 fragments and Cln2-myc₁₃ were lysed, and complexes were recovered using glutathione sepharose. Input (5%) and bound proteins were analyzed by anti-myc and anti-GST blots
(C) Starting with a Ste20^72–333 fragment, alanine substitutions were made at blocks of residues in the 72–118 region (see ; numbering starts at 2 because additional flanking mutations were used in other assays). Cln2 binding was tested as in panel B. Separately, the required Ste20 region was replaced by a Ste5^263–335 fragment (ii), in both wt and LLPP mutant forms, to test the ability of this Ste5 sequence to mediate Cln2 binding
(D) Ste20 and Ste5 docking sites show cyclin-specific binding. GST alone (−) or GST fusions (+) were used to co-precipitate myc₁₃-tagged cyclins (expressed from the CYC1 promoter) in yeast lysates. The GST fusions were to Ste20^1–333 (Ste20 motif) or to the Ste5^263–335-Ste20^120–333 chimera used in panel Cii (Ste5 motif). Cln3^10A showed varying levels of non-specific precipitation but no reproducible binding to either GST fusion
(E) Cln2-induced phosphorylation was assayed for V5-tagged forms of full-length Ste20 (1–939) or N-terminal fragments (80–590, 80–500), with or without mutations in the docking site (mut3) or the 13 confirmed Cdk sites (cdk*)
(F) The Cln2 docking site from Ste20 can drive phosphorylation of a heterologous substrate. Phosphorylation was analyzed using Ste5^1–283 (i), Ste5^1–260 (ii), and wt or mut3 versions of the Ste20 docking site (residues 80–115) fused to Ste5^1–260 (iii).

(A) Locations of key Ste5 features and mutations. Residues 275–283 contain the putative Cln2 docking motif required for efficient phosphorylation of the N-terminal Cdk sites. Mutations used in later panels are indicated; see for details
(B) Phosphorylation of the Ste5 N-terminus (Ste5^1–337) requires both the Cdk sites and a distal LLPP motif between residues 275 and 283. Phosphorylation was triggered by galactose-induced expression of a P_GAL1-CLN2 construct (+) or a vector control (−)
(C) The role of the LLPP motif is independent of MAPKs (fus3Δ kss1Δ), the phosphatase Ptc1 (ptc1Δ), and the MAPK binding site (ND mutant). Results show the Ste5^1–337 fragment except as indicated otherwise. The fus3Δ kss1Δ strain (PPY1173) was tested in parallel with a congenic wild-type strain (PPY640). LLPP function also does not require the MAPK phosphorylation sites, but non-phosphorylatable mutations at these sites (4AV) mildly reduce the extent of Cln2-Cdc28 phosphorylation. Also see
(D) Cln2-driven phosphorylation of full-length Ste5 (V5-tagged) requires the LLPP motif. For the 8A lanes, a longer exposure (of the same blot) is shown to compensate for imperfect loading
(E) Mutation of the LLPP motif disrupts the ability of Cln2 to inhibit pheromone signaling. Pheromone-induced phosphorylation of Fus3 was monitored in ste5Δ fus3Δ kss1Δ strains (± P_GAL1-CLN2) harboring STE5 variants and wild-type FUS3 on plasmids
(F) The LLPP motif mediates regulation by Cln2 in the absence of Fus3-Ste5 binding (Ste5 ND mutant) and Fus3 kinase activity (fus3-K42R mutant). Strains (as in panel D) harbored plasmids with the indicated forms of STE5 and FUS3.

(A) The Ste20^Ste5PM chimeras. Ste20 residues 124–311, containing the membrane-binding BR domain, were replaced with three fragments from Ste5 that include its membrane-binding PM domain plus 7 or 8 flanking Cdk sites
(B) Pheromone signaling by Ste20^Ste5PM chimeras is inhibited by Cln2. Because Cln2-Cdk normally inhibits signaling via Ste5, these tests used cells with a non-phosphorylatable Ste5 variant (ste20Δ STE5-8A). Wild-type Ste20 (wt) or Ste20^Ste5PM chimeras (from panel A) were introduced on plasmids, and pheromone response was measured using a transcriptional reporter (FUS1-lacZ). Signaling by all three chimeras (#A, #B, #C) was inhibited by Cln2, but this was blocked by mutations in the Cdk phosphorylation sites (7A, 8A). Deletions of the Ste20 N-terminus, made in the #A chimera, show that residues 87–119 are required for regulation by Cln2. Bars, mean ± SD (n = 3)
(C) Sequences required for regulation by Cln2 were analyzed using a chimera similar to #A, containing only Ste20 residues 80–109 upstream of Ste5^1–85 (see ). Alanine mutations replaced eight blocks of residues (left) or individual residues in the SLDDPIQF motif (right). These were compared to an intact sequence (wt) and a chimera that lacks the sequence entirely (−). Signaling was assayed as in panel B. Bars, mean ± SD (n = 3)
(D) Docking sites from Ste20 (80–115) or Ste5 (257–330) were inserted at different positions (i–iv) into a variant of chimera #A that lacks residues 87–119 (see panel B). Insertions at position ii also removed residues 1–86. Signaling was assayed as above. Bars, mean ± SD (n = 3)
(E) Bud tip localization of Ste20^Ste5PM chimera in cycling cells is inhibited via the Cdk sites (7A) and the Cln2 docking site (mut3). Strain BY4741 harbored GFP-Ste20 plasmids. Representative images show unfixed cells (left); localization was quantified after formaldehyde fixation (right). Bars, mean ± SD (n = 3 experiments; >150 cells per allele per experiment)
(F) The growth function of Ste20 is inhibited in the Ste20^Ste5PM chimera, if Cdk and docking sites are intact. Serial (1/5x) dilutions of strain KBY211 harboring the indicated Ste20 plasmids were incubated at 25 or 36 C for 4 days. As a control, the ΔBR allele removes the BR domain in full-length Ste20 [].