Imported:YPM/Ste7/MAPK interactions

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Ste7/MAPK Interactions

Ste7/Fus3 Interactions

  • In vitro translated Ste7 interacts with in vitro translated Fus3 with a Kd ~ 5 nM. Bardwell et al. 1996 PMID 8668180
  • Ste7 interacts with Fus3 via its N-terminal 98 amino acids, a fragment which does not contain Ste7's catalytic domain. Bardwell et al. 1996 PMID 8668180
  • Amino acids 1-25 of Ste7 are sufficient to interact with Fus3. Bardwell et al. 2001 PMID 11134045
  • Ste7 coimmunoprecipitates with Fus3 both in the absence and presence of pheromone (when both Ste7 and Fus3 overexpressed). Bardwell et al. 1996 PMID 8668180
  • The estimated half-time of dissociation of Ste7:Fus3 is 2-3 minutes at 30°C. Bardwell et al. 1996 PMID 8668180
  • The Ste7:Fus3 complex is neither a Ste7:Fus3 enzyme:substrate nor a Fus3:Ste7 enzyme:substrate complex (i.e., it must dissociate in order for Ste7 to phosphorylate Fus3 and for Fus3 to phosphorylate Ste7). Bardwell et al. 1996 PMID 8668180
  • Purified GST-Fus3 (expressed in bacteria) and Ste7 interact with a Kd ~ 100 nM (measured by cosedimentation). Bardwell et al. 2001 PMID 11134045
    • The authors think that the Kd might be artificially weaker than previously the measured 5 nM (Bardwell et al. 1996 PMID 8668180) due to interference of the GST tag placed on Fus3, and/or a higher fraction of improperly folded Fus3 molecules produced by the bacterial expression system.
  • A mutation that decreases the Ste7/Fus3 interaction (Ste7 Δ2-19) causes decreased transcription when coexpressed with a constitutive Ste11 allele, both in cells that contain Ste5 and cell that lack Ste5. This suggests that the Ste7/Fus3 interaction is important for signaling on and off the scaffold. Bardwell et al. 2001 PMID 11134045
    • Further supporting this, in an in vitro assay peptides of the MAPK-docking site Ste7(2-22) were able in inhibit MEK1 (homologous to Ste7) phosphorylation of ERK2 (homologous to Fus3).
  • When coexpressed with a constitutive Ste11 allele, a Ste5 mutant that is unable to bind Ste7 (Ste5 V763A S861P) activates the same amount of transcription off the FUS1 gene as a Ste5-null mutant. Bardwell et al. 2001 PMID 11134045
    • This suggests that the Fus3 is not able to anchor Ste7 in place on Ste5 such that it is competent for phosphorylation by Ste11 on the scaffold. This suggests that the Ste7:Fus3 dimer might not be competent for binding to Ste5 such that both kinases are able to dock in their binding sites on Ste5.
  • Fus3 binds to GST-Ste7(1-98) with a Kd of 0.1 μM (via cosedimentation assay). Kusari et al. 2004 PMID 14734536
    • The authors believe that the value that they measured in a previous publication (Bardwell et al. 1996 PMID 8668180) may be incorrect.
  • Ste7 contains 2 MAPK consensus binding motifs. Fus3 binds a peptide with the first MAPK consensus binding motif from Ste7 (residues 7-19; LQRRNLKGLNLN) with a Kd = 0.08 ± 0.02 μM. Fus3 binds a peptide with the second MAPK consensus binding motif from Ste7 (residues 61-72; LRRGIKKKLTLD) with a Kd = 12 ± 1.5 μM. Measurements made by competition fluorescence polarizing assays. Remenyi et al. 2005 PMID 16364919
  • Mutation of either of the MAPK consensus binding sites had a moderate effect on pathway output (measured by Fus1-GFP), whereas simultaneous mutation of both MAPK consensus binding sites pretty much returned pathway output at background levels (similar to Ste7 deletion mutants). Bhattacharyya et al. 2006 PMID 16424299
    • This shows that the Ste7/Fus3 interaction is required for mating signal propagation.
  • Feedback phosphorylation of Ste7 by Fus3 may inhibit binding of hyperphosphorylated Ste7 to Ste5. Mutating the feedback phosphorylation sites on Ste7 to glutamates to mimic phosphorylation yielded a mutant Ste7 with reduced binding to Ste5. Maleri et al. 2004 PMID 15456892
  • Using FCS to estimate concentrations of Fus3 and Ste7, and FCCS to estimate the concentration of Fus3:Ste7, the apparent affinity of interaction of Fus3 and Ste7 is 174 nM. Maeder et al. 2007 PMID 17952059
    • This Kd represents the overall affinity of interaction via direct interaction and indirect interaction.
    • This apparent affinity was not affected by pheromone treatment.
  • Using FCS to estimate concentrations of Fus3 and Ste7, and FCCS to estimate the concentration of Fus3:Ste7, the apparent affinity of interaction of Fus3 and Ste7 is 111 nM two hours after pheromone treatment, and the interaction cannot be detected prior to pheromone treatment. Slaughter et al. 2007 PMID 18077328
    • This Kd represents the overall affinity of interaction via direct interaction and indirect interaction.
    • It is not clear why the interaction between Ste7 and Fus3 was not detected prior to pheromone treatment, as this interaction was not found to be pheromone regulated by other researchers (see above).

Ste7/Kss1 Interactions

  • In vitro translated Ste7 interacts with in vitro translated Kss1 with a Kd ~ 5 nM. Bardwell et al. 1996 PMID 8668180
    • The estimated half-time of dissociation of Ste7:Kss1 is 2-3 minutes at 30°C.
  • The N-terminal third of Ste7 interacts with Kss1, a fragment which does not contain Ste7's catalytic domain. This interaction does not require Kss1's phosphoacceptor loop, which is the site where Kss1 is phosphorylated by Ste7. Bardwell et al. 1996 PMID 8668180
  • Ste7 coimmunoprecipitates with Kss1 both in the absence and presence of pheromone (when both Ste7 and Fus3 overexpressed). Bardwell et al. 1996 PMID 8668180
  • The Ste7:Kss1 complex is neither a Ste7:Kss1 enzyme:substrate nor a Kss1:Ste7 enzyme:substrate complex (ie it must dissociate in order for Ste7 to phosphorylate Kss1 and for Kss1 to phosphorylate Ste7). Bardwell et al. 1996 PMID 8668180
  • Kss1 binds to GST-Ste7(1-98) with a Kd of 0.1 μM (via cosedimentation assay). Kusari et al. 2004 PMID 14734536
    • The authors believe that the value that they measured in a previous publication (Bardwell et al. 1996 PMID 8668180) may be incorrect.
  • Ste7 contains 2 MAPK consensus binding motifs. Kss1 binds a peptide with the first MAPK consensus binding motif from Ste7 (residues 7-19; LQRRNLKGLNLN) with a Kd = 0.1 ± 0.05 μM. Kss1 binds a peptide with the second MAPK consensus binding motif from Ste7 (residues 61-72; LRRGIKKKLTLD) with a Kd = 9 ± 1.5 μM. Measurements made by competition fluorescence polarizing assays. Remenyi et al. 2005 PMID 16364919
  • Mutation of either of Ste7's consensus MAPK binding motifs has little effect on in vitro phosphorylation of Kss1 by a constitutive Ste7 allele (S359E T363E), but mutation of both eliminates Kss1 phosphorlyation. Remenyi et al. 2005 PMID 16364919
    • This suggests that stable binding to Ste7 is required for Kss1's phosphorylation.
  • Using FCS to estimate concentrations of Kss1 and Ste7, and FCCS to estimate the concentration of Kss1:Ste7, the apparent affinity of interaction of Kss1 and Ste7 is 32 nM in the absence of pheromone, and 61 nM two hours after pheromone treatment. Slaughter et al. 2007 PMID 18077328
    • This Kd represents the overall affinity of interaction via direct interaction and indirect interaction.
    • It is not clear whether the difference in affinity measured before/after pheromone treatment is statistically significant. Assuming this variation is due to experimental error, the average affinity is 47 nM.

Reaction Definition

Ste7 and Fus3 interact with an average affinity Kd_Ste7_Fus3 = 0.12 μM (ignoring the Kd of 5nM that was later discarded by the group that made the measurement), and Ste7 and Kss1 interact with an average affinity Kd_Ste7_Kss1 = 0.08 μM (again ignoring the discarded 5nM measurement). Since Ste7 interacts with Kss1 and Fus3 with near equal affinity, we will use a single association and dissociation constants Kd_Ste7_MAPK = 0.1 μM for both MAPKs (see MAPK/target_interaction_properties). Because the two MAPK consensus binding sites on Ste7 appear to be redundant, we will only model one binding site on Ste7.

Assumptions:

  • Binding affinity is independent of phosphorylation state of either Ste7 or the MAPK.

<modelRxnFull><modelRxnRule>

Ste7(Ste5_site, MAPK_site) + Fus3(docking_site) <-> Ste7(Ste5_site, MAPK_site!1).Fus3(docking_site!1)

</modelRxnRule> <modelRxnRule>

Ste7(Ste5_site, MAPK_site) + Kss1(docking_site) <-> Ste7(Ste5_site, MAPK_site!1).Kss1(docking_site!1)

</modelRxnRule>

  • Forward rate constant <modelRxnParam>kon_Ste7_MAPK</modelRxnParam>
  • Reverse rate constant <modelRxnParam>koff_Ste7_MAPK</modelRxnParam></modelRxnFull>