Ste5/MAPK cascade interactions

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General Comments

  • Tethering of kinases to Ste5 is sufficient to restore mating in Ste5 kinase-binding mutants (unable to bind either Ste11 or Ste7). Park et al. 2003 PMID 12511654
  • Fus3 binds in a similar manner (though opposite orientation) to a Ste5 peptide (residues 287-316) and a Far1 peptide (see Far1/MAPK_interactions for more details). This may explain why Fus3 both binds Ste5 and more strongly and phosphorylates Far1 more efficiently than does Kss1. Bhattacharyya et al. 2006 PMID 16424299

Ste5 interactions with Ste11

  • Ste5 binding to Ste11 is sufficiently tight to withstand density gradient sedimentation. Choi et al. 1994 PMID 8062390
  • Ste11-myc copurifies with GST-Ste5 in the presence and absence of pheromone. A catalytically inactive Ste11 mutant (R444 mutation) also copurifies with Ste5. The copurification is not diminished in ste7Δ fus3Δ cells. Choi et al. 1994 PMID 8062390
  • Mutation of the phosphorylation sites in the Ste11 amino terminal domain (residues 1-424) to make Ste11(S302A S306A T307A) and Ste11(S302D S306D T307D) yields proteins that still exhibit wild-type interactions with Ste5 and Ste50 by yeast two-hybrid, suggesting that phosphorylation of Ste11 does not modulate its binding to Ste5 or Ste50. van Drogen et al. 2000 PMID 10837245
  • Nuclear accumulation of Ste5-GST (which results in nearly complete oligomerization of the Ste5 population) is increased 7-fold in ste11Δ cells, whereas nuclear accumulation of Ste5-Myc9 (which is mainly monomeric) is unchanged in ste11Δ cells. This suggests that Ste11 may act as a cytoplasmic anchor for Ste5 oligomers. Wang and Elion. 2003 PMID 12808050
    • This was further supported by evidence that more HA3-Ste5-GST co-IPs with Ste11-Myc than HA3-Ste5, implying that Ste5 oligomers might have a higher affinity for Ste11. This conclusion is somewhat clouded by the differences in the amounts of Ste11 and Ste5 in the strains used (see figure 8A).
  • Ste11 likely has a dissociation half-time from Ste5 that is faster than 15 min. Flatauer et al. 2005 PMID 15713635
    • Mutation of Ste5 (V763A S861P) such that it no longer binds Ste7 only slightly decreases Kss1 phosphorylation 15 min after pheromone exposure, suggesting that either Ste11 bound to Ste5 can phosphorylate Ste7 in solution, or activated Ste11 is able to dissociate from Ste5 in less than 15 min to phosphorylate Ste7 and propagate the signal.
  • Mutation of Ste5 to prevent it's association with Ste11 eliminates mating, demonstrating that this interaction is necessary for signaling. Inyoue et al. 1997 PMID 9335587; Park et al. 2003 PMID 12511654
  • Using FCS to estimate concentrations of Ste11 and Ste5, and FCCS to estimate the concentration of Ste11:Ste5, the apparent affinity of interaction of Ste11 and Ste5 is 89 nM (averaging the 54 nM affinity of the Ste11-GFP/Ste5-RFP interaction and the 124 nM affinity of the Ste11-RFP/Ste5-GFP interaction). Maeder et al. 2007 PMID 17952059
    • This Kd represents the overall affinity of interaction via direct interaction and indirect interaction (perhaps via a Ste50-Cdc42-Ste20-Ste4 bridge).
    • Because Ste11 and Ste5 interact tightly (see Choi et al. 1994 PMID 8062390), it is unlikely that the interaction mediated by this protein bridge is stronger than the direct interaction, so the actual Kd is likely on the order of 89 nM.
    • This apparent affinity was not affected by pheromone treatment.
  • Using FCS to estimate concentrations of Ste11 and Ste5, and FCCS to estimate the concentration of Ste11:Ste5, the apparent affinity of interaction of Ste11 and Ste5 is 84 nM in the absence of pheromone, and 161 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 (perhaps via a Ste50-Cdc42-Ste20-Ste4 bridge).
    • Because Ste11 and Ste5 interact tightly (see Choi et al. 1994 PMID 8062390), it is unlikely that the interaction mediated by this protein bridge is stronger than the direct interaction.
    • Assuming that the interactions isn't pheromone regulated (the data from Meader et al. 2007 PMID 17952059 suggests that it isn't), and thus the difference in the measured values is due to experimental error, the average Kd is 123 nM.

Reaction Definition

We assume that the Kd_Ste5_Ste11 = 106 nM, as measured by FCCS. Assumptions:

  • Binding is independent of whether Ste5 is bound to Ste4:Ste18, Ste7 and Fus3/Kss1, as well as whether Ste5 is dimerized (despite the data that suggests this might not be true).
  • Ste11's affinity for Ste5 is unchanged by whether Ste11 has been phosphorylated by Ste20 on S302, S306 and T307, or phosphorylated by Fus3 on an unspecified site.
  • Binding between Ste11 and Ste5 is unchanged by binding of Ste50 to Ste11.
  • Ste11 cannot bind Ste5 while Ste11 is bound by Fus3.
Ste5(Ste11_site) + Ste11(Ste5_site, MAPK_site) <-> Ste5(Ste11_site!1).Ste11(Ste5_site!1, MAPK_site)


Ste5 interactions with Ste7

  • Ste7-myc copurifies with GST-Ste5 in the absence of Fus3 in the presence and absence of pheromone. Only the faster migrating species of Ste7 (presumably hypophosphorylated) copurifies with Ste5. Choi et al. 1994 PMID 8062390
  • Ste5-CTM (which is constitutively localized to the cell membrane in a Ste4 independent manner, and expressed off the Gal promoter) causes localization of Ste7-GFP (expressed from the Gal promoter) to the cell membrane in cells also containing WT Ste5 and Ste7. van Drogen et al. 2001 PMID 11781566
  • Little if any Ste7 cosediments with Ste5 in density gradient sedimentation. Choi et al. 1994 PMID 8062390
  • Mutation of Ste5 (V763A S861P) such that it no longer binds Ste7 only slightly decreases Kss1 phosphorylation in response to pheromone, whereas phosphorylation of Fus3 is completely blocked by this mutation. Flatauer et al. 2005 PMID 15713635
    • This suggests that Fus3 cannot be phosphorylated off the scaffold, but Kss1 can.
  • Mutation of either of Ste7's MAPK consensus binding sites has a moderate effect on pathway output (measured by Fus1-GFP, Fus3 phosphorylation and mating efficiency), whereas simultaneous mutation of both MAPK consensus binding sites reduced pathway output to background levels (similar to Ste7 deletion mutants). Bhattacharyya et al. 2006 PMID 16424299
    • The crystal structure of Fus3 bound to Ste5(287-316) shows that this peptide binds in part to the same docking groove that Ste7 peptides bind. This suggests that Fus3 cannot simultaneously bind Ste7 and Ste5.
  • Ste5 and Ste7 may compete for binding to Fus3, as suggested by competition binding assays in vitro. Kusari et al. 2004 PMID 14734536
  • 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 Ste7 and Ste5, and FCCS to estimate the concentration of Ste7:Ste5, the apparent affinity of interaction of Ste7 and Ste7 is 118 nM. Maeder et al. 2007 PMID 17952059
    • This Kd represents the overall affinity of interaction via direct interaction and indirect interaction.
    • Because there aren't any clear indirect interactions that would cause association of Ste7 with Ste5, the actual Kd is likely on the order of 118 nM.
    • This apparent affinity was not affected by pheromone treatment.
  • Using FCS to estimate concentrations of Ste7 and Ste5, and FCCS to estimate the concentration of Ste7:Ste5, the apparent affinity of interaction of Ste7 and Ste5 is 63 nM in the absence of pheromone, and 104 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.
    • Assuming that the interaction isn't pheromone regulated (the data from Meader et al. 2007 PMID 17952059 suggests that it isn't), and thus the difference in the measured values is due to experimental error, the average effective Kd is 84 nM.
    • Because there aren't any clear indirect interactions that would cause association of Ste7 with Ste5, the actual Kd is likely on the order of 84 nM.

Reaction Definition

See Fus3 phosphorylation by Ste7 for comments on binding between Ste7 and Ste5. We assume that Kd_Ste5_Ste7 = 101 nM (the average of the measured values of 118 nM and 84 nM).

Assumptions:

  • Ste7's affinity for Ste5 is unchanged by whether Ste7 has been phosphorylated on S359 and T363 by Ste11 and unchanged by whether Ste7 has been phosphorylated on multiple sites by Fus3.
Ste5(Ste7_site) + Ste7(Ste5_site, MAPK_site) <-> Ste5(Ste7_site!1).Ste7(Ste5_site!1, MAPK_site)


Ste5 interactions with MAPK

Ste5 interactions with Fus3

  • Ste5 interacts with Fus3 as judged by yeast two-hybrid. Choi et al. 1994 PMID 8062390
    • Deletion of the Ste5 domain that interacts with Fus3 by two-hybrid (143-309) essentially eliminates the kinase activity of Fus3-HA purified from cells pre- and post-pheromone treatment.
  • Fus3-Myc interacts strongly with Ste5 in the absence of Ste7 (determined by coimmunoprecipitation). Proteins were expressed and purified from insect cells. Breitkreutz et al. 2001 PMID 11525741
  • Ste5-CTM (which is constitutively localized to the cell membrane in a Ste4 independent manner, and expressed off the Gal promoter) causes localization of Fus3-GFP (expressed from the Gal promoter) to the cell membrane in WT cells (containing WT Ste5 and Fus3). Catalytically inactive (K42R) and unphospohrylatable (T180A Y182F) Fus3-GFP were similarly localized. Fus3-GFP plasma membrane localization is abolished by deletion of the Fus3 binding site from Ste5-CTM (Δ241-336). van Drogen et al. 2001 PMID 11781566
  • Again using Ste5-CTM and Fus3-GFP (expressed off Gal promoters in WT cells), a region of the plasma membrane was continually photobleached, and nuclear fluorescence was monitored to observe the rate of exchange between Fus3-GFP at the membrane and in the nucleus. Catalytically inactive Fus3-GFP (K42R) and WT showed similar loss of fluorescence in the nucleus, whereas unphosphorylatable Fus3-GFP (T180A Y182F) showed a slower rate of exchange. In ste11Δ cells, the WT, K42R and T180A Y182F alleles all had similar loss of fluorescence in the nucleus. van Drogen et al. 2001 PMID 11781566
    • Since nuclear import is similar between WT, K42R and T180A Y182F alleles, this difference is thought to be due to different dissociation rates from Ste5-CTM. Thus the hypothesis is that phosphorylation at T180 and Y182 (on the T-loop) on Fus3-GFP results in faster dissociation from Ste5-CTM.
    • I'm not sure why they didn't just use WT Ste5 allele, expose the cells to pheromone, and do FRAP at the shmoo tip for these alleles...
  • The recovery half-time for Fus3-GTP that has been photobleached at the shmoo tip is 0.32s (+/-0.8s). The half-time of recovery of Ste5-GFP at the shmoo tip is significantly longer (8.22s +/-1.3s), suggesting that Fus3 may rapidly dissociate from Ste5. van Drogen et al. 2001 PMID 11781566
  • Fus3 binds to GST-Ste5(241-336) with a Kd of 1 μM (via cosedimentation assay). Kusari et al. 2004 PMID 14734536
    • Introduction of Ste7(2-22) (which binds Fus3) inhibited the interactions between Fus3 and GST-Ste5(241-336), suggesting that Ste7 and Ste5 may compete for binding to Fus3.
  • Fus3(T180V Y182F) binds a peptide from Ste5 (residues 287-316: the minimal Ste5 peptide that binds Fus3 efficiently) with a Kd = 4.2 ± 1.4 μM. Bhattacharyya et al. 2006 PMID 16424299
    • The crystal structure of Fus3 bound to Ste5(287-316) shows that this peptide binds in part to the same docking groove that Ste7, Msg5 and Far1 peptides bind, suggesting that Ste5 binds competitively with these other Fus3 binding partners.
    • Mutation of Ste5 such that it no longer binds Fus3 (Ste5(Q292A I294A Y295A L307A p310A N315A) causes a 2-fold increase in pathway output (as judged by Fus1-GFP expression), suggesting that Ste5 binding to Fus3 may actually attenuate signaling.
    • FCCS confirms that this full-length Ste5 mutant has a greatly reduced affinity for Fus3. Maeder et al. 2007 PMID 17952059
  • When Ste5(280-321) is incubated with Fus3, in addition to Fus3 autophosphorylation (see MAPK_phosphorylation_cascade), the Ste5 peptide is phosphorylated. Bhattacharyya et al. 2006 PMID 16424299
    • T287V mutation is sufficient to eliminate the phosphorylation of the Ste5 peptide. This mutant peptide still binds Fus3 with the same affinity, suggesting that T287 is the site of phosphorylation.
    • Mutation of Ste5 such that it no longer binds Fus3 (Ste5(Q292A I294A Y295A L307A p310A N315A)) causes a 2-fold increase in pathway output (as judged by Fus1-GFP expression).
    • Mutation of Ste5 such that it is not phosphorylated by Fus3 at T287 causes a moderate increase in pathway output (as judged by Fus1-GFP expression).
    • Strains with Ste5 mutants that neither bind Fus3 nor can be phosphorylated at T287 by Fus3 behave identically to strains with Ste5 mutants that can't bind Fus3.
    • This suggests that in wild-type cell, Fus3 binds Ste5, autophosphorylates Fus3 Y182, and subsequently phosphorylates Ste5 T287.
    • Ptc1 phosphatase is recruited to Ste5 upon pheromone stimulation and dephosphorylates Ste5 T268, S276, T287 and S329. This dephosphorylation competes with Fus3 phosphorylation of the same residues. Dephosphorylation of Ste5 is required for Fus3 to be activated by Ste7 and dissociate from Ste5. Malleshaiah et al 2010 doi:10.1038/nature08946
  • Using FCS to estimate concentrations of Fus3 and Ste5, and FCCS to estimate the concentration of Fus3:Ste5, the apparent affinity of interaction of Fus3 and Ste5 is 910 nM. Maeder et al. 2007 PMID 17952059
    • This Kd represents the overall affinity of interaction via direct interaction and indirect interaction.
    • Fus3 and Ste7 might be able to interact while Ste7 is bound to Ste5, making this one indirect interaction that this affinity may represent.
    • Both the Fus3-Ste7 and Ste7-Ste5 interactions are higher affinity than 910 nM.
    • This apparent affinity was not affected by pheromone treatment.
  • Using FCCS, the interaction between Fus3 and Ste5 was not detected in the absence or presence of pheromone, in the cytosol or nucleus. Slaughter et al. 2007 PMID 18077328
    • The authors estimate that the affinity of this interaction must be > 400 nM.
  • Fus3 may interact preferentially with Ste5 dimers. Slaughter et al. 2007 PMID 18077325
    • Fus3 interacts with GST-Ste5, which forms homodimers, with an affinity of 144 nM in the cytosol in the absence of pheromone, 57 nM in the cytosol 2 hours after pheromone treatment, 257 nM in the nucleus in the absence of pheromone, and 158 nM in the nucleus 2 hours after pheromone treatment.
    • This suggests that Fus3 may interact more strongly with Ste5 dimers than Ste5 monomers.
    • It is unclear if the differences between the affinities in the absence and presence of pheromone are statistically significant. If they are, this could indicate that Fus3 might also interact preferentially with Ste5 in the presence of pheromone.
    • A weak FRET signal can also be detected between Ste5 and Fus3 at the cell periphery in response to pheromone treatment, but not elsewhere in the cell. Assuming that Ste5 dimerizes upon membrane relocalization, this further supports the notion that Fus3 binds preferentially to Ste5 dimers.

Ste5 interactions with Kss1

  • Ste5 interacts with Kss1 as judged by yeast two-hybrid. This was done in cells that contained the proteins in the pathway. Choi et al. 1994 PMID 8062390
  • Kss1-Myc does not detectably interact with Ste5 in the absence of Ste7, whereas it does in the presence of Ste7 (determined by coimmunoprecipitation). Proteins were expressed and purified from insect cells. Breitkreutz et al. 2001 PMID 11525741
  • Ste5-CTM (which is constitutively localized to the membrane in a Ste4 independent manner, and expressed off the Gal promoter) fails to recruit Kss1-GFP (expressed from the Gal promoter in WT cells) to the membrane in WT cells (containing WT Ste5 and Kss1). Fus3-GFP is successfully recruited under the same circumstances. van Drogen et al. 2001 PMID 11781566
  • Kss1 binds to GST-Ste5(241-336) with a Kd of 6.5 μM (via cosedimentation assay). Kusari et al. 2004 PMID 14734536

Reaction Definition

Fus3 binds Ste5(287-316) with an affinity of 4.2 ± 1.4 μM, and it binds Ste5(241-336) with an affinity of 1 μM. We will assume Kd_Ste5_Fus3 = 1.0 μM, since this was measured for a larger Ste5 fragment, which presumably is a better predictor of the affinity of full length Ste5 for Fus3. Fus3-Ste5 binding was also estimated by FCCS, and although this measurement also takes into account indirect interaction, the measured affinity (910 nM) agrees well with the 1 μM we chose here. Kss1 binds Ste5(241-336) with a Kd_Ste5_Kss1 = 6.5 μM. Fus3 appears to dissociate from Ste5 with a half time of ~ 0.3 s-1, which corresponds to a dissociation rate of 2.3 s-1. In the absence of information to the contrary, we will assume that Kss1 has the same association rate as Fus3, but a faster dissociation rate.

Assumptions:

  • The MAPKs affinity for Ste5 is unaffected by Ste5 binding to Ste7, Ste11, and Ste4:Ste18, as well as by Ste5 dimerization.
  • MAPKs affinity for Ste5 is unaffected by MAPK phosphorylation.
Ste5(MAPK_site) + Fus3(docking_site) <-> Ste5(MAPK_site!1).Fus3(docking_site!1)


Ste5(MAPK_site) + Kss1(docking_site) <-> Ste5(MAPK_site!1).Kss1(docking_site!1)

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