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. Author manuscript; available in PMC: 2007 Aug 9.
Published in final edited form as: Cell. 2007 Feb 9;128(3):519–531. doi: 10.1016/j.cell.2006.12.032

Figure 2.

Figure 2

Multiple CDK sites flanking the Ste5 PM domain control Cln2/CDK inhibition
  1. Locations of potential CDK phosphorylation sites (SP or TP) in Ste5.
  2. Elimination of 8 N-terminal CDK sites in Ste5 (Ste5-8A) causes resistance to Cln2.
  3. Response to α factor was measured in ste5Δ ± PGAL1-CLN2 cells expressing Ste5 variants (WT or 8A) from the native STE5 promoter, or in ste20Δ ± PGAL1-CLN2 cells expressing Ste20 variants (WT or 13A) from the native STE20 promoter. Bars, mean ± SD (n = 8).
  4. CDK resistance caused by 8A mutations restores membrane signaling independent of Ste5-Gβγ interaction. Gβγ-independent signaling was activated by PGAL1-STE5-Q59L ± 8A in ste4Δ ste5Δ cells ± PGAL1-CLN2. Bars, mean ± SD (n = 7).
  5. The Ste5-8A mutant disrupts cell cycle periodicity of pheromone response. Cells (cdc15-2 or cdc15-2 STE5-8A) were synchronized in late M phase by arrest at 36°C, and then transferred to 25°C. At various times, response to brief treatment with α factor was monitored (see Experimental Procedures). Top, FUS1-lacZ induction (mean of 4 trials). Bottom, Fus3 activation (phospho-Fus3) was measured using phospho-specific antibodies (mean of 6 trials). Arrows mark the times of bud emergence (see Figures S1D and 5G).
  6. Ste5 phosphorylation sites were replaced with Ala residues either singly (1A) or in various combinations (2A, 3A, 4A, 8A). Response to α factor was tested in ste5Δ strains ± PGAL1-CLN2 (mean + SD, n = 8-16).