1. Specificity of MAP Kinase Signaling in Yeast Differentiation Involves Transient versus Sustained MAPK Activation 
Walid Sabbagh, Laura J Flatauer, A.Jane Bardwell, Lee Bardwell 
Molecular Cell 
Volume 8, Issue 3, Pages (September 2001)
DOI: /S (01)

2. Figure 1
Model of Signal-Response Specificity in Mating versus Filamentous Invasive Growth
In haploid yeast cells, mating is initiated by the binding of pheromone to a G protein-coupled receptor. Gβγ (shown) then binds to the scaffold/adaptor protein Ste5. Ste5 binds to all three tiers of the MAPK cascade and promotes transmission of the mating signal via Ste20 (not shown) to Ste11 (MEKK) and thence to Ste7 (MEK). The pheromone receptor, the G protein, and Ste5 are all mating-pathway-specific components. In filamentous invasive growth, unknown filamentation-pathway-specific components couple to the Ste20-Ste11-Ste7-Kss1 MAPK cascade. Nutrient limitation signals initiate filamentation (Cullen and Sprague, 2000) but if or how these couple to the MAPK cascade is unclear.
(A) In the model (Madhani and Fink, 1998; Madhani et al., 1997), Fus3 is presumed to be the MAPK for mating; Fus3, but not Kss1, is activated by the pheromone response pathway. Activation of Fus3 stimulates transcription of mating genes, driven by pheromone response elements (PREs) as well as by G1 arrest (data not shown). Phosphorylation of Kss1 by the invasive growth pathway stimulates the transcription of genes driven by filamentation response elements (FREs; Madhani and Fink, 1997).
(B) The model suggests that in the absence of Fus3, Kss1 gains illicit access to the mating pathway and is thereby activated by pheromone stimulation. Mating proceeds, albeit at reduced efficiency. However, filamentation-specific genes (driven by FREs) are now erroneously induced by mating pheromone
Molecular Cell 2001 8, DOI: ( /S (01) )

3. Figure 2
Kss1 Is Phosphorylated and Activated during Normal Pheromone Response
(A) Midlog phase cultures of strains of the indicated genotypes were treated (or not) for 15 min with 150 nM α factor mating pheromone (phm), and MAPK phosphorylation levels were determined by immunoblotting with a phosphorylation-state specific antibody (αpTEpY). Total MAPK protein levels were determined by reprobing with anti-Kss1 (αKss1) or anti-Fus3 (αFus3) antisera. 7Δ indicates strain JCY107 (ste7Δ) and ΔΔ indicates strain JCY130 (kss1Δ fus3Δ).
(B) MAPK phosphorylation in wild-type strain stimulated for 15 min with the indicated concentration of pheromone.
(C) MAPK phosphorylation in wild-type strain treated with 150 nM pheromone for the indicated time.
(D) MAPK phosphorylation in wild-type MATa and MATα strains and at the indicated time (in minutes) after their mixing.
(E) Kinase activity of Kss1 protein. Strains JCY110 (kss1Δ; lanes 1, 2, and 5) or JCY 130 (kss1Δ fus3Δ; lanes 3, 4, and 6) carrying epitope-tagged wild-type (wt) Kss1 (lanes 1–4), catalytically inactive (ci) Kss1Y24F (lane 5), or an empty vector (lane 6) were treated (or not) for 15 min with 150 nM pheromone and lysed, and immunoprecipitation-kinase assays were performed using recombinant GST-Ste71–172 as a substrate (top panel). The amount of Kss1 immunoprecipitated is shown in the middle panel. The bottom panel shows the quantitation (by phosphoimager) of substrate phosphorylation (average of three experiments). Results are normalized to wt, no pheromone = 1. Standard error bars are shown
Molecular Cell 2001 8, DOI: ( /S (01) )

4. Figure 3
Kss1 Phosphorylation Is Required for Full Pheromone-Induced Gene Expression
Strains JCY100 (wt) or JCY 110 (kss1Δ) carrying an empty vector (ev) or expressing from plasmid YCpU either wild-type KSS1 or an unphosphorylatable mutant, kss1(AEF), were treated for 2 hr with the indicated concentration of pheromone or mixed with an equal number of α cells for 2 hr, and expression of the FUS1-lacZ reporter gene was determined. The activity obtained from the α cell-mixing experiments was doubled to compensate for the dilution by the α cells, which did not contain the reporter. Results are normalized to WT + [ev], no pheromone = 1. Standard error bars are shown
Molecular Cell 2001 8, DOI: ( /S (01) )

5. Figure 4
Fus3 Prevents a Leak from the Mating Pathway from Activating Filamentation-Specific Gene Expression
Data are normalized as indicated. Standard error bars are shown.
(A) Expression of FRE-driven reporter in wild-type (JCY100) and fus3Δ (JCY120) strains, following growth on plates for 24 hr.
(B) Expression of PRE-driven reporter (FUS1-lacZ; diamonds) and FRE-driven reporter (FRE-lacZ; circles) in strains JCY100 (wt; open symbols) and JCY120 (fus3Δ; filled symbols), following treatment for indicated time with 250 nM pheromone.
(C) Effect of initiating physiological mating. Strains JCY100 (wt) and JCY120 (fus3Δ) carrying the indicated reporters were mixed with an equal number of α cells. After 2 hr, cells were harvested and reporter gene expression was determined.
(D) Effect of manipulating Ste5 activity. Expression of the indicated reporter was determined in strains JCY100 (KSS1+ FUS3+) and JCY120 (KSS1+ fus3Δ) carrying an empty vector (−) or expressing from a multicopy plasmid STE5T52M (STE5Hyp). Expression of the FRE-driven reporter was also determined in strains LFY 105 (KSS1+ FUS3+ ste5Δ) and LFY 125 (KSS1+ fus3Δ ste5Δ)
Molecular Cell 2001 8, DOI: ( /S (01) )

6. Figure 5
MAPK Catalytic Activity Is Required to Transmit and Block Signal Crossover
Data are normalized as indicated. Standard error bars are shown.
(A) Kss1 phosphorylation and catalytic activity are required to transmit the mating → filamentation leak. Expression of FRE-driven reporter in strain JCY130 (kss1Δ fus3Δ), carrying either an empty vector (ev), or expressing from plasmid YCpU either wild-type KSS1, or a catalytically-inactive kss1 mutant (Y24F), or an unphosphorylatable kss1 mutant (AEF).
(B) Catalytic activity of Fus3 is required to prevent hyper-invasive growth. Strains JCY100 (WT), JCY107 (ste7Δ, indicated by 7Δ) and JCY120 (fus3Δ, indicated by f3Δ), carrying either an empty vector (ev), or overexpressing from a multicopy plasmid either wild-type FUS3, or catalytically-inactive (K42R) or unphosphorylatable (AEF) derivatives, were streaked onto plates selective for plasmid maintenance. After 1 day growth, plates were replica plated onto YPD plates, grown for 24 hr at 30°C, scored for total growth, then scored for invasive growth by washing under a gentle stream of water to remove non-adherent cells (Roberts and Fink, 1994).
(C) Fus3 phosphorylation and catalytic activity are required to block the mating → filamentation leak. Expression of FRE-driven reporter in strain JCY100 (wt) or JCY120 (fus3Δ) carrying either an empty vector (ev) or overexpressing from plasmid YEpU either wild-type FUS3, a catalytically-inactive fus3 mutant (K42R), or an unphosphorylatable fus3 mutant (AEF)
Molecular Cell 2001 8, DOI: ( /S (01) )

7. Figure 6
Active Fus3 Limits the Magnitude and Duration of Kss1 Phosphorylation
(A) Basal phosphorylation of Kss1. Strains of the indicated genotypes (see Figure 2) were grown on plates for 2 days, and MAPK phosphorylation levels were determined by immunoblotting with a phosphorylation-state-specific antibody (αpTEpY). Total MAPK protein levels were determined by reprobing with anti-Kss1 (αKss1) or anti-Fus3 (αFus3) antisera.
(B) Overproduced active Fus3 inhibits Kss1 phosphorylation. MAPK phosphorylation levels were determined in strains JCY110 (kss1Δ; lane 1) or JCY 120 (fus3Δ; lanes 2-7) carrying either an empty vector (ev) or overexpressing from plasmid YEpU either wild-type FUS3 or a catalytically-inactive mutant (K42R).
(C) MAPK phosphorylation in wild-type and fus3Δ strain treated with 250 nM pheromone for the indicated time (upper panel). Total Kss1 and Fus3 protein levels, determined by reprobing with a mixture of anti-Kss1 and anti-Fus3 antisera (lower panel)
Molecular Cell 2001 8, DOI: ( /S (01) )

8. Figure 7 Model of Signal Identity in Mating versus Invasive Growth
Stimulation of the mating pheromone response pathway leads to the phosphorylation and activation of both Kss1 and Fus3. Both MAPKs participate in the induction of mating genes. Active Fus3 potentiates a feedback mechanism that limits the magnitude and duration of Kss1 activation, thereby plugging a Kss1-mediated leak that would otherwise result in inappropriate hyperactivation of FRE-driven transcription. Active Fus3 also limits its own phosphorylation (not shown), presumably by the same mechanism. Finally, active Fus3 also inhibits filamentation by a Kss1-independent mechanism (data not shown)
Molecular Cell 2001 8, DOI: ( /S (01) )