Figure 2. A distal docking motif promotes Cln2-Cdc28 phosphorylation of Ste5.

(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 Figure S1A 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 Figure S1B,C

(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.