Ste4:Ste18/Ste5 interactions and Ste5 dimerization/oligomerization

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Ste5 Dimerization/Oligomerization

  • A Ste5 mutant that is defective for Fus3/Kss1 binding (ΔC239-R335) when coexpressed with a Ste5 mutant that is defective for Ste7 binding (ΔV586-R746) is able to restore response to pheromone in a ste5Δ background. This suggests that Ste5 functions as a dimer or oligomer. Yablonski et al. 1996 PMID 8943027
    • Bhattacharyya et al. (2006 PMID 16424299) have since demonstrated that mutation of a Fus3 binding site on Ste5 (by changing 6 amino acids between 288 and 315 to alanine) does not by itself abolish signaling (as the ste5ΔC239-R335 does), suggesting either that the deletion causes a defect in a Ste5 function in addition to Fus3 binding (e.g., dimerization or membrane recruitment) or that the full-length multi-substitution mutant of Ste5 still retains some binding to Fus3 even though the peptide corresponding to the 288-315 region can no longer bind to Fus3.
  • When overexpressed, Ste5-Myc and GST-Ste5 coimmunoprecipitate. Overexpression of Ste5 is sufficient to activate the pathway as judged by cell morphology. Yablonski et al. 1996 PMID 8943027
  • Ste5/Ste5 interactions can be detected by yeast two-hybrid only when overexpressed in a ste5Δ ste7Δ background. Yablonski et al. 1996 PMID 8943027
  • Either of two Ste5 mutants defective for Ste11 binding (F514L and I504T) when coexpressed with a Ste5 mutant that is defective for Ste7 binding (V768A S861P) is able to restore response to pheromone in a ste5Δ background. This suggests that Ste5 functions as a dimer or oligomer. Inyoue et al. 1997 PMID 9335587
  • RING-H2 domain (residues 177-229) is implicated both in Ste5 dimerization/oligomerization and in Ste5 binding to Ste4. Inouye et al. 1997 PMID 9311911
    • Ste5(C177S) and Ste5(C177A C180A) are both unable to rescue mating in a ste5Δ strain.
    • Ste5(C177S) and Ste5(C177A C180A) both bind Ste11, Ste7 and Fus3 with WT efficiency as judged by coimmunoprecipitation. Ste4 does not immunoprecipitate with either of these mutants (Ste4 does IP with WT Ste5) .
    • Ste5(C177A C180A) can be made competent for mating response by fusing GST (which is known to homodimerize) to the C-terminus.
    • Ste5-GST does not rescue mating in a ste5Δ ste4Δ strain, suggesting that dimerization is not sufficient for signaling.
    • Ste4 does not immunoprecipitate with Ste5(C177A C180A)-GST, suggesting that for normal activation, Ste5 is first recruited to Ste4, which subsequently induces dimerization/oligomerization.
  • There is evidence that Ste5 oligomerization is separable from Ste5 binding to Ste4. Feng et al. 1998 PMID 9501067
    • Ste5(C180A) is impaired in binding to Ste4, but can oligomerize; bind Ste11, Ste7, and Fus3; facilitate basal activation of Ste11 and relay the Ste11 signal to the MAP kinases.
    • Ste5(C180A) cannot activate Ste11 in response to pheromone
    • The inability of Ste5(C180A) to bind Ste4, and the ability of Ste5(C180A) to oligomerize may be somewhat debatable due to variable basal signal in two-hybrid studies.
    • Oligomerization was also measured by co-IP of Myc tagged Ste5 (WT or mutant) with GST tagged Ste5 (WT or mutant). These experiments could be confounded because GST was always used for the pulldowns, and GST causes near complete homo-oligomerization of WT Ste5.
  • Most Ste5 is monomeric in the absence of pheromone. Wang & Elion 2003 PMID 12808050
    • Results suggest most Ste5 is present as inactive monomers with inaccessible RING-H2 domains and Ste11 binding sites, consistent with the idea that activation of Ste5 coincides with homo-oligomerization and recruitment to Ste4.
    • The RING-H2 domain is the most important region of Ste5 for oligomerization.
    • A leucine-rich region within residues 335-586 is also important for Ste5 homo-oligomerization, though not as important as the RING-H2 domain.
  • Fusion of a GST domain (which can homo-dimerize) to Ste5 induces near complete oligomerization of the Ste5 population. Wang and Elion. 2003 PMID 12808050
    • This mutant has higher pathway activation in the absence (40-fold) and presence (2-fold) of pheromone (measured by Fus1-LacZ reporter)
    • This mutant also localizes more strongly to the membrane in response to pheromone.
    • ste4Δ STE5-GST strain has the same Fus1-LacZ levels in the absence of pheromone as WT cells. This suggests that increased signaling though Ste5-GST required Ste4.
  • Overexpressed Ste5-GFP exists primarily as a dimer both in the absence and presence of pheromone. Slaughter et al. 2007 PMID 18077328
    • Photon counting histograms (PCH) were used to estimate dimerization state.
    • About 10% of Ste5 exists in dimers prior to pheromone treatment, and about 15% of Ste5 exist in dimers after treatment. It is not clear whether this difference is statistically significant.

Reaction Definition

Ste5 oligomerization appears to be linked to Ste4 binding.

Assumptions:

  • Ste5 dimerizes, but does not form higher order oligomers.
  • Ste5 dimerization and binding to Ste4 are cooperative processes. Ste5 dimerizes weakly in the absence of Ste4, more strongly when one of the Ste5 monomers is bound to Ste4:Ste18, and even more strongly when both Ste5 monomers are bound to Ste4:Ste18 dimers.
  • Ste5 dimerization is unaffected by Ste11, Ste7 and Fus3/Kss1 binding (nor by their phosphorylation states).
  • Differences in binding affinity occur only due to differences in dissociation rates.

See this section in the BioNetGen wiki for a description for how BioNetGen handles handles the symmetric dimerization reactions (where the reactants are identical).

Note that although we are assuming that both Ste5 molecules in a dimer can bind different Ste4:Ste18 molecules, it is easy to alter the model such that a Ste5 dimer can only bind a single Ste4:Ste18. By setting Ste4Ste18Ste5_Ste4Ste18Ste5_coop_factor close to zero (say 1e-3), then the association of Ste5:Ste4:Ste18:Ste5 with an additional Ste4:Ste18, and association of Ste5:Ste4:Ste18 with another Ste5:Ste4:Ste18 would both be essentially eliminated (see Ste5 dimerization and Ste4 binding rate constant constraints). Since we are assuming that the differences in affinity are due to differences in dissociation rate, this would manifest itself as very high dissociation rate of Ste4:Ste18:Ste5:Ste5:Ste4:Ste18 complexes. Alternatively, one could set both kon_Ste4Ste18_Ste4Ste18Ste5Ste5 and kon_Ste4Ste18Ste5_Ste4Ste18Ste5 to zero. 


Ste5(Ste5_site, Ste4_site) + Ste5(Ste5_site, Ste4_site) <-> 
Ste5(Ste5_site!1, Ste4_site).Ste5(Ste5_site!1, Ste4_site)





Ste5(Ste5_site, Ste4_site!+) + Ste5(Ste5_site, Ste4_site) <-> 
Ste5(Ste5_site!1, Ste4_site!+).Ste5(Ste5_site!1, Ste4_site)





Ste5(Ste5_site, Ste4_site!+) + Ste5(Ste5_site, Ste4_site!+) <-> 
Ste5(Ste5_site!1, Ste4_site!+).Ste5(Ste5_site!1, Ste4_site!+)


See Ste5 dimerization and Ste4 binding rate constant constraints.

Ste4:Ste18 interactions with Ste5

  • Overexpression of Ste4 causes constitutive activation of the pathway.
  • The Ste4:Ste18 is thought to serve mainly to recruit Ste5 and its associated kinases to the membrane and into the vicinity of Ste20. Pryciak and Hunter. 1998 PMID 9732267
    • Recruitment of Ste5 to the membrane via lipid modification is sufficient for pathway activation in ste4Δ cells, but not in ste20Δ, ste11Δ or ste7Δ cells. Temperature sensitive alleles of Cdc42 and Cdc24 were used to show that these proteins are also required for pathway activation in membrane targeted Ste5 mutants.
  • The pool of Ste5 that is associated with the membrane/shmoo tip decreases to background levels ~2.5 hours after pheromone treatment (50nM pheromone, bar1- strain). Mahanty et al. 1999 PMID 10481914
  • Ste4 interacts with the N-terminal 214 amino acids of Ste5 by yeast two-hybrid only when Ste18 is also expressed. Whiteway et al. 1995 PMID 7667635
  • Ste4 and Ste5 coimmunoprecipitate in cells treated with pheromone. Whiteway et al. 1995 PMID 7667635
  • The recovery half time for Ste5-GFP (overexpressed from the Gal promoter) that has been photobleached at the shmoo tip is 8.22s (+/-1.3s). van Drogen et al. 2001 PMID 11781566
    • Assuming that the recovery time is dissociation limited (i.e. dissociation is much slower than binding which is likely faster than normal because Ste5-GFP is overexpressed), then the off rate of Ste5 from Ste4 is 0.084 s-1.
  • Cdc42-1 and Cdc24-4 temperature sensitive mutants each cause defects in Ste5 localization to the membrane (and thus pathway activation), in the absence and presence of pheromone, and at the restrictive and permissive temperature. Wang et al. 2005 PMID 15657049
Figure 8b from Winters et al. 2005 PMID 16209942
Figure 8b from Winters et al. 2005 PMID 16209942
  • Proper membrane recruitment and activation of Ste5 requires a weak membrane interaction through a membrane associating domain (PM/NLS domain) in addition to a weak membrane-associated protein interaction (interaction with Ste4), neither of which is sufficient on its own to induce signaling (see figure to the right). Winters et al. 2005 PMID 16209942
    • Tethering of Ste11 to the membrane via a membrane targeting sequence causes constitutive pathway activation (FUS1-lacZ reporter). Deletion of the Ste4 binding site does not affect this constitutive activation, but deletion of the PM/NLS domain (which causes association with the plasma membrane) eliminated signaling.
    • Replacement of the PM/NLS domain with a series of pleckstrin homology (PH) domains was sufficient to restore Ste5 function.
  • Overexpression of a GFP-tagged N-terminal Ste5 fragment (residues 1-214; contains the PM/NLS domain and the Ste4 binding site) is sufficient to inhibit response to pheromone (dominant-negative phenotype). Winters et al. 2005 PMID 16209942
    • Unlike WT Ste5, this fragment constitutively associates with the membrane and it is thought to compete with WT Ste5 for binding to free Ste4 upon stimulation with pheromone.
    • Deletion of part of the PM/NLS domain (residues 48-67) in the N-terminal fragment is sufficient to eliminate membrane association and the dominant-negative phenotype. This suggests that association with the membrane is important for Ste5's ability to bind Ste4.
    • The membrane associating domain (PM/NLS domain) may be masked in Ste5 prior to Ste4 binding.
  • The PM/NLS domain and the Ste4 binding site on Ste5 are insufficient for Ste5 membrane localization and function. In addition to these two sites, a third site is needed. This site is a pleckstrin-homology (PH) domain (residues 388-518). Garrenton et al. 2006 PMID 16847350
    • This PH domain binds phosphoinositides in vitro.
    • The R407S K411S mutation of Ste5 or a fragment of Ste5 abolishes phosphoinositide binding, membrane recruitment, and Ste5 function, but does not reduce binding to Ste4 or Ste11 or itself.
    • Artificial membrane targeting of this mutant Ste5 restores signaling.
  • Efficient signaling requires that Ste5 bring Ste11 into close proximity of Ste20 by binding Ste4 at the membrane, and that Ste5 dimerize and/or bind to the membrane (through Ste4 or other means). Lamson et al. 2006 PMID 16546088
    • Directing Ste5 (via CTM motif) to plasma membrane is sufficient for pathway activation in otherwise WT cells. In cells with hyperactive Ste11, directing Ste5 to internal, membranes (like ER) is also sufficient for pathway activation
    • In cells with hyperactive Ste11, directing Ste18 (and thus Ste4) to internal membranes is sufficient for pathway activation. Cytoplasmic Ste18 (missing any membrane targeting sequence) does not cause pathway activation.
    • This membrane localization effect is independent of Ste5 oligomerization, because N-terminal deletions (lacking the RING-H2 domain) still propagates the signal when tagged with CTM motif.
    • Forcing Ste5 dimerization via GST domains results in the same pathway activation as membrane targeting in cells with constitutive Ste11. This effect is occurs in the absence of Ste4 and with deletion of the membrane binding domain (Ste5Δ48-67).
  • Fus3 phosphorylation of Ste5 may act to attenuate signaling. Bhattacharyya et al. 2006 PMID 16424299
    • When Ste5(280-321) is incubated with Fus3 the Ste5 peptide is phosphorylated.
    • 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.
  • Elimination of Ste5-Fus3 binding increases the localization of Ste5 to the shmoo tip in response to pheromone. Maeder et al. 2007 PMID 17952059
    • Because Fus3 is able to phosphorylate Ste5, and either of elimination of the phosphorylation site on Ste5, or mutation of the MAPK binding site on Ste5 is sufficient to increase signaling, this suggests that Fus3 phosphorylation of Ste5 may attenuate signaling by reducing Ste5's membrane association.
    • The phosphorylation could reduce membrane localization by interfering with dimerization, Ste4 binding, or interactions between the PM or PH domains and the plasma membrane itself.


Cell Cycle Regulation of Ste5 Membrane Association

  • Cln2-Cdc28 represses mating response via Ste5's PM domain. Strickfaden et al. 2007 PMID 17289571
    • Overexpressed Ste5(Q59L) or Ste11-Cpr, which are sufficient for pathway activation but require the Ste5 PM domain, can be repressed by overexpression of Cln2.
    • Overexpressed Ste5-CTM or Ste11ΔN, which are sufficient for pathway activation but do not require the Ste5 PM domain, cannot be repressed by overexpression of Cln2.
  • Cln2-Cdc28 regulates mating response by phosphorylating Ste5 at 8 different sites around Ste5's PM domain. Strickfaden et al. 2007 PMID 17289571
    • Ste5 has 8 CDK phosphorylation sites (SP or TP) near its PM domain.
    • Mutation of these 8 phosphorylation sites to alanines (AP) eliminates cell cycle dependent Fus1-lacZ expression and Fus3 activation, and the ability of overexpressed Cln2 to repress mating response and membrane localization of Ste5.
    • The Ste5(Q59L) mutant, which is Gβγ-independent, can also be rendered immune to Cln2 repression via the same 8A mutations, indicating that Cln2 does not act at the level of the Ste5/Gβγ interaction.
    • All single alanine mutants displayed a small amount of resistance to Cln2-mediated signal repression, suggesting that phosphorylation at all 8 sites is required for full Cln2-mediated repression.
    • Double, triple, and quadruple alanine mutants displayed increasing resistance to Cln2-mediated signal repression.
    • Only the 8A mutant (5A, 6A, and 7A were not tested) displayed full resistance to Cln2-mediated signal repression.
    • Purified Cln2-Cdc28 phosphorylates purified Ste5(1-125) (which contains the PM domain and surrounding phosphorylation sites), but only weakly phosphorylates Ste5(1-125)8A.
  • Cln2-mediated inhibition of mating signaling is caused by added negative charges of phosphate groups, and acts specifically on the PM domain. Strickfaden et al. 2007 PMID 17289571
    • Mutation of the 8 phosphorylation sites to glutamates causes partial inhibition of mating signal.
    • Mutation of the 8 SP/TP pairs to EE pairs (16E mutant), to mimic the two negative charges per SP/TP pair caused by phosphorylation, represses response to pheromone as much as Cln2 overexpression.
    • The 14E mutant is slightly more sentitive to pheromone than the 16E mutant, or than WT Ste5 with Cln2 overexpression, suggesting that full phosphorylation is necessary for full signal repression.
    • The 16E mutant is expressed at WT levels, and immunoprecipitates with Ste4 as well as WT Ste5 does.
    • The 16E mutant repression is bypassed by other mutants that activate the pathway in the absence of the Ste5 PM domain.
  • A simple model of Ste5 phosphorylation and membrane association suggests that the ability of Ste5 to associate with the membrane could drop sharply over a narrow range of Cln2-Cdc28 activity. Serber and Ferrell. 2007 PMID 17289565
    • Using a previously published estimate of a 10x reduction in binding per phosphorylation event, a simple kinetic model predicts a switch-like drop in membrane-associated Ste5 in response to changes in active CDK concentration, with a Hill coefficient of 4.1.
  • Upon chemical inhibition of Cdc28, asynchronous population of cells have a similar response to pheromone as a synchronous population of cell in G1 phase (not treated with the inhibitor). Colman-Lerner et al. 2005 PMID 16170311
    • This suggests that dephosphorylation of Ste5 is rapid.

Reaction Definition

Ste5 oligomerization appears to be linked to Ste4:Ste18 binding. Ste5 can only bind Ste4:Ste18 dimers that are not bound to Gpa1. Since above we are assuming that Ste5 binding to Ste4:Ste18 can affect Ste5 dimerization, therefore, Ste5 dimerization must have a reciprocal effect on Ste5 binding to Ste4:Ste18.

Assumptions: 

Ste4(Gpa1_site, Ste5_site) + Ste5(Ste5_site, Ste4_site) <-> 
Ste4(Gpa1_site, Ste5_site!1).Ste5(Ste5_site, Ste4_site!1)





Ste4(Gpa1_site, Ste5_site) + Ste5(Ste5_site!2, Ste4_site).Ste5(Ste5_site!2, Ste4_site) <-> 
Ste4(Gpa1_site, Ste5_site!1).Ste5(Ste5_site!2, Ste4_site!1).Ste5(Ste5_site!2, Ste4_site)





Ste4(Gpa1_site, Ste5_site) + Ste5(Ste5_site!2, Ste4_site).Ste5(Ste5_site!2, Ste4_site!+) <-> 
Ste4(Gpa1_site, Ste5_site!1).Ste5(Ste5_site!2, Ste4_site!1).Ste5(Ste5_site!2, Ste4_site!+)


See Ste5 dimerization and Ste4 binding rate constant constraints.

moleculizer-Ste5-blocks-Gpa1-binding

moleculizer-Gpa1-blocks-Ste5-binding

moleculizer-Ste4-Ste5-binding-rates

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