Far1/MAPK interactions

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Contents 1 General Notes 2 Far1/Fus3 Interactions 2.1 Reaction Definition 3 Far1/Kss1 Interactions 3.1 Reaction Definition

General Notes

• Fus3, but not Kss1, phosphorylates Far1, and this phosphorylation correlates with Far1's ability to promote cell cycle arrest. Peter et al. 1993 PMID 8500168
  • Far1 is phosphorylated in response to pheromone treatment in kss1 mutants, but not in fus3 mutants.
  • kss1 mutants, but not in fus3 mutants, have a robust pheromone-dependent cell cycle arrest.
  • Fus3 purified from pheromone-treated cells is able to phosphorylate Far1 in vitro.

• 30min after exposure to 5 μM pheromone, Far1 is largely phosphorylated in WT and kss1 cells, but largely unphosphorylated in fus3 cells, suggesting that Fus3 and not Kss1 is responsible for Far1 phosphorylation in vivo. Breitkreutz et al. 2001 PMID 11525741

• Kss1-Myc phosphorylates Ste12-Flag at half the rate that Fus3-Myc does, and phosphorylates Far1 at one tenth the rate that Fus3-Myc does. This conclusion requires that the purified Kss1-Myc and Fus3-Myc are both maximally (or at least equivalently) activated. Breitkreutz et al. 2001 PMID 11525741
  • This explains why fus3 cells respond to pheromone and have the same transcriptional response, but fail to arrest, thus have a poor mating efficiency when compared to WT or kss1 cells.

• Far1's MAPK binding motif peptide induces a subtle rearrangement of residues in Fus3's docking groove upon binding. This rearrangement is not seen upon binding of Ste7 and Msg5's MAPK binding motif peptides. The authors solved the crystal structure of Fus3 unbound and bound to these peptides. Remenyi et al. 2005 PMID 16364919
  • The authors suspect that perhaps Kss1 isn't able to undergo this rearrangement of residues to allow for Far1 binding, which would explain the preference of Fus3 over Kss1 for phosphorylation of Far1.

• Deletion of Fus3 results in a defect in G1 arrest in response to pheromone (not all cells arrest), whereas deletion of Kss1 does not affect the ability of cells to enter G1 arrest in response to pheromone. Breitkreutz et al. 2001 PMID 11525741; Chekasova et al. 1999 PMID 10049917
  • This suggests that Fus3 but not Kss1 is able to phosphorylate Far1 efficiently enough to cause cell-cycle arrest.

Far1/Fus3 Interactions

• Fus3 binds a peptide with the MAPK consensus binding motif from Far1 (residues 72-83; KRGNIPKPLNLS) with a Kd = 7 ± 1 μM (measurements made by competition fluorescence polarizing assay). Remenyi et al. 2005 PMID 16364919

• Mutation of either Fus3's docking groove or Far1's docking site is sufficient to greatly reduce phosphorylation of Far1 by Fus3 in vitro. Remenyi et al. 2005 PMID16364919
  • Mutation of Far1's docking site results in the same cell-cycle arrest defect as deletion of Far1.

Reaction Definition

Since the measured Kd likely applies to unphosphorylated Fus3 binding to Far1, 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_Far1 = 7 μM / 4 = 1.75 μM.

Assumptions:

• Fus3 cannot bind to Far1 while Fus3 is bound to another target.

• This interaction follows the MAPK/target interaction properties.

• Fus3 and Kss1 interact with Far1 with different affinities.

• Fus3 does not bind phosphorylated Far1, and unphosphorylated Fus3 does not bind Far1

Far1(MAPK_site, T306~none) + Fus3(docking_site, T180~PO4, Y182~none) <-> 
   Far1(MAPK_site!1, T306~none).Fus3(docking_site!1, T180~PO4, Y182~none)

• Forward rate constant kon_Fus3pT_Far1

• Reverse rate constant koff_Fus3pT_Far1

Far1(MAPK_site, T306~none) + Fus3(docking_site, T180~none, Y182~PO4) <-> 
   Far1(MAPK_site!1, T306~none).Fus3(docking_site!1, T180~none, Y182~PO4)

• Forward rate constant kon_Fus3pY_Far1

• Reverse rate constant koff_Fus3pY_Far1

Far1(MAPK_site, T306~none) + Fus3(docking_site, T180~PO4, Y182~PO4) <-> 
   Far1(MAPK_site!1, T306~none).Fus3(docking_site!1, T180~PO4, Y182~PO4)

• Forward rate constant kon_Fus3pTpY_Far1

• Reverse rate constant koff_Fus3pTpY_Far1

Far1(MAPK_site!1, T306~none).Fus3(docking_site!1, T180~PO4, Y182~none) -> 
   Far1(MAPK_site, T306~PO4) + Fus3(docking_site, T180~PO4, Y182~none)

• Phosphorylation rate constant kcat_Fus3pT_Far1_PO4

Far1(MAPK_site!1, T306~none).Fus3(docking_site!1, T180~none, Y182~PO4) -> 
   Far1(MAPK_site, T306~PO4) + Fus3(docking_site, T180~none, Y182~PO4)

• Phosphorylation rate constant kcat_Fus3pY_Far1_PO4

Far1(MAPK_site!1, T306~none).Fus3(docking_site!1, T180~PO4, Y182~PO4) -> 
   Far1(MAPK_site, T306~PO4) + Fus3(docking_site, T180~PO4, Y182~PO4)

• Phosphorylation rate constant kcat_Fus3pTpY_Far1_PO4

Far1/Kss1 Interactions

• Kss1 binds a peptide with the MAPK consensus binding motif from Far1 (residues 72-83; KRGNIPKPLNLS) with a Kd = 46 ± 5 μM (measurements made by competition fluorescence polarizing assay). Remenyi et al. 2005 PMID 16364919

Reaction Definition

Since the measured Kd likely applies to unphosphorylated Kss1 binding to Far1, we will adjust this number to account for the slightly higher affinity of phosphorylated Kss1 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_Kss1pTpY_Far1 = 47 μM / 4 ~ 12 μM.

Assumptions:

• Kss1 cannot bind to Far1 while Kss1 is bound to another target.

• This interaction follows the MAPK/target interaction properties.

• Fus3 and Kss1 interact with Far1 with different affinities.

• Kss1 does not bind phosphorylated Far1, and unphosphorylated Kss1 does not bind Far1

Far1(MAPK_site, T306~none) + Kss1(docking_site, T183~PO4, Y185~none) <-> 
   Far1(MAPK_site!1, T306~none).Kss1(docking_site!1, T183~PO4, Y185~none)

• Forward rate constant kon_Kss1pT_Far1

• Reverse rate constant koff_Kss1pT_Far1

Far1(MAPK_site, T306~none) + Kss1(docking_site, T183~none, Y185~PO4) <-> 
   Far1(MAPK_site!1, T306~none).Kss1(docking_site!1, T183~none, Y185~PO4)

• Forward rate constant kon_Kss1pY_Far1

• Reverse rate constant koff_Kss1pY_Far1

Far1(MAPK_site, T306~none) + Kss1(docking_site, T183~PO4, Y185~PO4) <-> 
   Far1(MAPK_site!1, T306~none).Kss1(docking_site!1, T183~PO4, Y185~PO4)

• Forward rate constant kon_Kss1pTpY_Far1

• Reverse rate constant koff_Kss1pTpY_Far1

Far1(MAPK_site!1, T306~none).Kss1(docking_site!1, T183~PO4, Y185~none) -> 
   Far1(MAPK_site, T306~PO4) + Kss1(docking_site, T183~PO4, Y185~none)

• Phosphorylation rate constant kcat_Kss1pT_Far1_PO4

Far1(MAPK_site!1, T306~none).Kss1(docking_site!1, T183~none, Y185~PO4) -> 
   Far1(MAPK_site, T306~PO4) + Kss1(docking_site, T183~none, Y185~PO4)

• Phosphorylation rate constant kcat_Kss1pY_Far1_PO4

Far1(MAPK_site!1, T306~none).Kss1(docking_site!1, T183~PO4, Y185~PO4) -> 
   Far1(MAPK_site, T306~PO4) + Kss1(docking_site, T183~PO4, Y185~PO4)

• Phosphorylation rate constant kcat_Kss1pTpY_Far1_PO4