Dig1/Dig2/Ste12 phosphorylation

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Contents 1 General Notes 2 Dig1 Phosphorylation 2.1 Reaction Definition 3 Dig2 Phosphorylation 3.1 Reaction Definition 4 Ste12 Phosphorylation 4.1 Reaction Definition

General Notes

• Global transcriptional changes induced by saturating amounts of pheromone in kss1Δ cells and fus3Δ cells are very similar, suggesting that both have similar ability to relieve repression of Ste12 and activate transcription. Breitkreutz et al. 2001 PMID 11525741

Dig1 Phosphorylation

• Dig1 immunoprecipitates with Fus3-Myc in the absence and presence of pheromone. This is unaffected by deletion of Dig2. Tedford et al. 1997 PMID 9094309
  • Dig1 that is immunoprecipitated in the presence of pheromone has a lower electrophoretic mobility than Dig1 that is immunoprecipitated in the absence of pheromone. This decrease in mobility is eliminated by treatment with a phosphatase, suggesting the Dig1 is phosphorylated in response to pheromone, and phosphorylated Dig1 is immunoprecipitated efficiently by Fus3-Myc.

• Fus3 binds GST-Dig1(93-111) about 3-fold more weakly than it binds GST-Dig2(98-114) (which it binds with a Kd of 1 μM). Kusari et al. 2004 PMID 14734536
  • Kss1 binds both GST-Dig1(93-111) and GST-Dig2(98-114) about 17-fold more weakly than Fus3 binds these targets.

• Fus3 interacts with GST-Dig1(213-452) with about the same efficiency as Kss1. Bardwell et al. 1998 PMID 9744865
  • This interaction was later shown to be much weaker than the interaction between Kss1 and the putative MAPK docking site on Dig1 lying within residues 92-113, suggesting that it is not the primary means of interaction between the MAPKs and Dig1. Kusari et al. 2004 PMID 14734536

Reaction Definition

Fus3 binds Dig1 3-fold more weakly than it binds Dig2, which Fus3 binds with an affinity of ~1 μM. Since this Kd likely applies to unphosphorylated Fus3 binding to Dig1, we will adjust this number to account for the slightly higher affinity of phosphorylated Fus3 for its targets. According to the numbers measured by Zhou and Zhang (2002 PMID 11839761), unphosphorylated ERK2 has a ~2-fold weaker affinity for its targets than monophosphorylated ERK2, and doubly phosphorylated ERK2 has a ~2-fold weaker affinity for its targets than diphosphorylated ERK2 (see MAPK/target_interaction_properties). So we will assume an affinity of Kd_Fus3pTpY_Dig1 = 3 μM / 4 = 0.75 μM.

Since Kss1 binds Dig1 17-fold more weakly than Fus3 binds Dig1, Kd_Kss1pTpY_Dig1 = 0.75 μM * 17 ~ 13 μM.

Assumptions:

• The ability of Fus3 and Kss1 to bind and phosphorylate Dig1 is unaltered by whether Dig1 is bound to Ste12 or not, and by any other proteins that may be bound to Ste12.

• The interaction between Fus3/Kss1 and Dig1 follows the assumption outlined on the MAPK/target_interaction_properties page.

Dig1(MAPK_site, PO4_site~none) + Fus3(docking_site, T180~PO4, Y182~none) <->  
Dig1(MAPK_site!1, PO4_site~none).Fus3(docking_site!1, T180~PO4, Y182~none)

• Forward rate constant kon_Fus3pT_Dig1

• Reverse rate constant koff_Fus3pT_Dig1

Dig1(MAPK_site, PO4_site~none) + Fus3(docking_site, T180~none, Y182~PO4) <->  
Dig1(MAPK_site!1, PO4_site~none).Fus3(docking_site!1, T180~none, Y182~PO4)

• Forward rate constant kon_Fus3pY_Dig1

• Reverse rate constant koff_Fus3pY_Dig1

Dig1(MAPK_site, PO4_site~none) + Fus3(docking_site, T180~PO4, Y182~PO4) <-> 
Dig1(MAPK_site!1, PO4_site~none).Fus3(docking_site!1, T180~PO4, Y182~PO4)

• Forward rate constant kon_Fus3pTpY_Dig1

• Reverse rate constant koff_Fus3pTpY_Dig1

Dig1(MAPK_site, PO4_site~none) + Kss1(docking_site, T183~PO4, Y185~none) <-> 
Dig1(MAPK_site!1, PO4_site~none).Kss1(docking_site!1, T183~PO4, Y185~none)

• Forward rate constant kon_Kss1pT_Dig1

• Reverse rate constant koff_Kss1pT_Dig1

Dig1(MAPK_site, PO4_site~none) + Kss1(docking_site, T183~none, Y185~PO4) <-> 
Dig1(MAPK_site!1, PO4_site~none).Kss1(docking_site!1, T183~none, Y185~PO4)

• Forward rate constant kon_Kss1pY_Dig1

• Reverse rate constant koff_Kss1pY_Dig1

Dig1(MAPK_site, PO4_site~none) + Kss1(docking_site, T183~PO4, Y185~PO4) <-> 
Dig1(MAPK_site!1, PO4_site~none).Kss1(docking_site!1, T183~PO4, Y185~PO4)

• Forward rate constant kon_Kss1pTpY_Dig1

• Reverse rate constant koff_Kss1pTpY_Dig1

Dig1(MAPK_site!1, PO4_site~none).Fus3(docking_site!1, T180~PO4, Y182~none) ->  
Dig1(MAPK_site, PO4_site~PO4) + Fus3(docking_site, T180~PO4, Y182~none)

• Phosphorylation rate constant kcat_Fus3pT_Dig1_PO4

Dig1(MAPK_site!1, PO4_site~none).Fus3(docking_site!1, T180~none, Y182~PO4) -> 
Dig1(MAPK_site, PO4_site~PO4) + Fus3(docking_site, T180~none, Y182~PO4)

• Phosphorylation rate constant kcat_Fus3pY_Dig1_PO4

Dig1(MAPK_site!1, PO4_site~none).Fus3(docking_site!1, T180~PO4, Y182~PO4) -> 
Dig1(MAPK_site, PO4_site~PO4) + Fus3(docking_site, T180~PO4, Y182~PO4)

• Phosphorylation rate constant kcat_Fus3pTpY_Dig1_PO4

Dig1(MAPK_site!1, PO4_site~none).Kss1(docking_site!1, T183~PO4, Y185~none) -> 
Dig1(MAPK_site, PO4_site~PO4) + Kss1(docking_site, T183~PO4, Y185~none)

• Phosphorylation rate constant kcat_Kss1pT_Dig1_PO4

Dig1(MAPK_site!1, PO4_site~none).Kss1(docking_site!1, T183~none, Y185~PO4) -> 
Dig1(MAPK_site, PO4_site~PO4) + Kss1(docking_site, T183~none, Y185~PO4)

• Phosphorylation rate constant kcat_Kss1pY_Dig1_PO4

Dig1(MAPK_site!1, PO4_site~none).Kss1(docking_site!1, T183~PO4, Y185~PO4) -> 
Dig1(MAPK_site, PO4_site~PO4) + Kss1(docking_site, T183~PO4, Y185~PO4)

• Phosphorylation rate constant kcat_Kss1pTpY_Dig1_PO4

Dig2 Phosphorylation

• Dig2 immunoprecipitates with Fus3-Myc in the absence and presence of pheromone. This is unaffected by deletion of Dig1. Tedford et al. 1997 PMID 9094309
  • Dig2 that is immunoprecipitated in the presence of pheromone has a lower electrophoretic mobility than Dig2 that is immunoprecipitated in the absence of pheromone. This decrease in mobility is eliminated by treatment with a phosphatase, suggesting the Dig2 is phosphorylated in response to pheromone, and phosphorylated Dig2 is immunoprecipitated efficiently by Fus3-Myc.

• Fus3 binds GST-Dig2(98-114) (which contains a MAPK docking site) with a Kd of 1 μM (by cosedimentation assay). Kusari et al. 2004 PMID 14734536
  • Kss1 binds both GST-Dig1(93-111) and GST-Dig2(98-114) about 17-fold more weakly than Fus3 binds these targets.

Reaction Definition

Fus3 binds Dig1 with an affinity of ~1 μM. Since this Kd likely applies to unphosphorylated Fus3 binding to Dig2, we will adjust this number to account for the slightly higher affinity of phosphorylated Fus3 for its targets. According to the numbers measured by Zhou and Zhang (2002 PMID 11839761), unphosphorylated ERK2 has a ~2-fold weaker affinity for its targets than monophosphorylated ERK2, and doubly phosphorylated ERK2 has a ~2-fold weaker affinity for its targets than diphosphorylated ERK2 (see MAPK/target_interaction_properties). So we will assume an affinity of Kd_Fus3pTpY_Dig2 = 1 μM / 4 = 0.25 μM.

Since Kss1 binds Dig2 ~17-fold more weakly than Fus3 binds Dig2, Kd_Kss1pTpY_Dig2 = 0.25 μM * 17 ~ 4.3 μM.

Assumptions:

• The ability of Fus3 and Kss1 to bind and phosphorylate Dig2 is unaltered by whether Dig2 is bound to Ste12 or not, and by any other proteins that may be bound to Ste12.

• The interaction between Fus3/Kss1 and Dig2 follows the assumption outlined on the MAPK/target_interaction_properties page.

Dig2(MAPK_site, PO4_site~none) + Fus3(docking_site, T180~PO4, Y182~none) <-> 
Dig2(MAPK_site!1, PO4_site~none).Fus3(docking_site!1, T180~PO4, Y182~none)

• Forward rate constant kon_Fus3pT_Dig2

• Reverse rate constant koff_Fus3pT_Dig2

Dig2(MAPK_site, PO4_site~none) + Fus3(docking_site, T180~none, Y182~PO4) <-> 
Dig2(MAPK_site!1, PO4_site~none).Fus3(docking_site!1, T180~none, Y182~PO4)

• Forward rate constant kon_Fus3pY_Dig2

• Reverse rate constant koff_Fus3pY_Dig2

Dig2(MAPK_site, PO4_site~none) + Fus3(docking_site, T180~PO4, Y182~PO4) <-> 
Dig2(MAPK_site!1, PO4_site~none).Fus3(docking_site!1, T180~PO4, Y182~PO4)

• Forward rate constant kon_Fus3pTpY_Dig2

• Reverse rate constant koff_Fus3pTpY_Dig2

Dig2(MAPK_site, PO4_site~none) + Kss1(docking_site, T183~PO4, Y185~none) <-> 
Dig2(MAPK_site!1, PO4_site~none).Kss1(docking_site!1, T183~PO4, Y185~none)

• Forward rate constant kon_Kss1pT_Dig2

• Reverse rate constant koff_Kss1pT_Dig2

Dig2(MAPK_site, PO4_site~none) + Kss1(docking_site, T183~none, Y185~PO4) <-> 
Dig2(MAPK_site!1, PO4_site~none).Kss1(docking_site!1, T183~none, Y185~PO4)

• Forward rate constant kon_Kss1pY_Dig2

• Reverse rate constant koff_Kss1pY_Dig2

Dig2(MAPK_site, PO4_site~none) + Kss1(docking_site, T183~PO4, Y185~PO4) <-> 
Dig2(MAPK_site!1, PO4_site~none).Kss1(docking_site!1, T183~PO4, Y185~PO4)

• Forward rate constant kon_Kss1pTpY_Dig2

• Reverse rate constant koff_Kss1pTpY_Dig2

Dig2(MAPK_site!1, PO4_site~none).Fus3(docking_site!1, T180~PO4, Y182~none) -> 
Dig2(MAPK_site, PO4_site~PO4) + Fus3(docking_site, T180~PO4, Y182~none)

• Phosphorylation rate constant kcat_Fus3pT_Dig2_PO4

Dig2(MAPK_site!1, PO4_site~none).Fus3(docking_site!1, T180~none, Y182~PO4) -> 
Dig2(MAPK_site, PO4_site~PO4) + Fus3(docking_site, T180~none, Y182~PO4)

• Phosphorylation rate constant kcat_Fus3pY_Dig2_PO4

Dig2(MAPK_site!1, PO4_site~none).Fus3(docking_site!1, T180~PO4, Y182~PO4) -> 
Dig2(MAPK_site, PO4_site~PO4) + Fus3(docking_site, T180~PO4, Y182~PO4)

• Phosphorylation rate constant kcat_Fus3pTpY_Dig2_PO4

Dig2(MAPK_site!1, PO4_site~none).Kss1(docking_site!1, T183~PO4, Y185~none) -> 
Dig2(MAPK_site, PO4_site~PO4) + Kss1(docking_site, T183~PO4, Y185~none)

• Phosphorylation rate constant kcat_Kss1pT_Dig2_PO4

Dig2(MAPK_site!1, PO4_site~none).Kss1(docking_site!1, T183~none, Y185~PO4) -> 
Dig2(MAPK_site, PO4_site~PO4) + Kss1(docking_site, T183~none, Y185~PO4)

• Phosphorylation rate constant kcat_Kss1pY_Dig2_PO4

Dig2(MAPK_site!1, PO4_site~none).Kss1(docking_site!1, T183~PO4, Y185~PO4) -> 
Dig2(MAPK_site, PO4_site~PO4) + Kss1(docking_site, T183~PO4, Y185~PO4)

• Phosphorylation rate constant kcat_Kss1pTpY_Dig2_PO4

Ste12 Phosphorylation

• Ste12 is phosphorylated in response to pheromone exposure. Ste12 phosphorylation can be detected 2.5 min after pheromone exposure, and is at its maximal level after 5 min. Song et al. 1991 PMID 2026326

• Ste12 is constitutively phosphorylated on serine and threonine residues, but not on tyrosine residues. This distribution is unchanged by pheromone treatment. Hung et al. 1997 PMID 9151949

• In Dig1Δ Dig2Δ cells, treatment with pheromone does not affect the expression of FUS1-LacZ. Bardwell et al. 1998 PMID 9860980; Tedford et al. 1997 PMID 9094309
  • This suggests that either the basal activity of the MAPKs is high enough to keep Ste12 phosphorylated in Dig1Δ Dig2Δ cells in the absence of pheromone, or phosphorylation of Ste12 does not affect the expression of FUS1-LacZ.

• Expression of mating genes in Dig1Δ Dig2Δ cells is similar to the expression found in WT cells exposed to 50 nM pheromone for 30 minutes. Roberts et al. 2000 PMID 10657304
  • This suggests that derepression of Ste12 is sufficient for pheromone-induced levels of gene expression.

Reaction Definition

Since in Dig1Δ Dig2Δ cells, treatment with pheromone does not affect the expression of FUS1-LacZ, we will assume that phosphorylation of Ste12 in response to pheromone does not affect Ste12's activity as a transcription factor.