Imported:YPM/MAPK phosphorylation cascade

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Ste11 Phosphorylation by Ste20

  • Ste20 CRIB domain at residues 334-369 interacts the kinase domain of Ste20, inhibiting the kinase domain's activity. It's not known if the CRIB/kinase domain interactions occur inter- or intra-molecularly. Lamson et al. 2002 PMID 11940652
  • Ste20 CRIB domain interacts with GTP-bound Cdc42. The interaction with Cdc42 relieves the autoinhibition of the kinase domain. Lamson et al. 2002 PMID 11940652
  • Mutations within the CRIB domain that selectively eliminate the interaction between Ste20 and Cdc42 greatly decrease mating pathway activity. Lamson et al. 2002 PMID 11940652; Ash et al. 2003 PMID 12586692
    • This implies that Cdc42 plays an important role in Ste20 activation.
  • Deletion of the CRIB domain of Ste20 eliminates Cdc42-binding and has little effect on mating signal transduction (although greatly reduces response to nitrogen starvation and cell-cell adhesion during mating), making the role of Cdc42 somewhat unclear. Peter et al. 1996 PMID 9003780; Leberer et al. 1997 PMID 9009270; Lamson et al. 2002 PMID11940652
  • Ste20 is phosphorylated by Cln2-Cdc28 during the S phase of the cell cycle. This phosphorylation does not appear to affect in vitro Ste20 kinase activity using immunoprecipitated Ste20. Wu et al. 1998 PMID 9774429
  • The activity of immunoprecipitated Ste20 is largely unaffected by prior treatment of the cells with pheromone. Wu et al. 1995 PMID 7608157; Lamson et al. 2002 PMID 11940652
  • Phosphorylation of S302, and/or S306 and T307 by Ste20 is sufficient and necessary to activate Ste11. van Drogen et al. 2000 PMID 10837245
    • Mutation of all three sites to alanine results in a non-functioning pathway (as judged by halo assay, FUS1-lacZ transcriptional reporter, and mating assay).
    • Each single mutant (S302, S306 or T307 to alanine) was able to function in the pathway (as judged by halo assay, FUS1-lacZ transcriptional reporter, and mating assay).
    • Mutation of all three sites to aspartic acid (mimicking phosphorylation) results in a constitutive phenotype whose activation of transcription is independent of Ste20, but dependent on Ste7 (this mutant is also able to constitutively activate the high osmolarity pathway).
  • Ste11's amino-terminal domain inhibits the activity of Ste11's carboxy-terminal catalytic domain when S302, S306 and T307 are not phosphorylated. When these residues are phosphorylated, this inhibition is relieved. van Drogen et al. 2000 PMID 10837245
    • Ste11 amino-terminal domain (residues 1-424) mutant Ste11(S302A S306A T307A) interacts with the Ste11 carboxy-terminal catalytic domain (residues 424-738) by yeast two-hybrid, whereas the Ste11 amino-terminal domain (residues 1-424) mutant Ste11(S302D S306D T307D) does not.

Reaction Definition

The activity Ste20 does not appear to be regulated in either a cell-cycle or pheromone dependent manner, so we will not explicitly include the activation of Ste20. Since the cell-cycle dependent phosphorylation of Ste20 has no known effect on pheromone response, these reactions can be ignored as well.

Assumptions:

  • Phosphorylation of at least two of S302, S306 and T307 is necessary and sufficient for full activation of Ste11.
  • The phosphorylaion of Ste11 is ordered, thus we can just consider whether Ste11 has been phosphorylated zero times, once (pS), twice (pSpS), or three times (pSpSpT).
  • Each phosphorylation event occurs at the same rate.
  • Ste11 is only phosphorylated by Ste20 when the Ste5 molecule that Ste11 is bound to is associated with the same Ste4:Ste18 dimer as Ste20.
  • Feedback phosphorylation by Fus3 does not affect the phosphorylation rate.
  • Ste5 dimerization, and Ste5 binding to Ste7 and Fus3 do not affect the phosphorylation rate.

<modelRxnFull><modelRxnRule>

Ste20(Ste4_site!2).Ste4(Ste5_site!3, Ste20_site!2).Ste5(Ste4_site!3, Ste11_site!4).Ste11(Ste5_site!4, S302_S306_T307~none) -> 
     Ste20(Ste4_site!2).Ste4(Ste5_site!3, Ste20_site!2).Ste5(Ste4_site!3, Ste11_site!4).Ste11(Ste5_site!4, S302_S306_T307~pS)

</modelRxnRule>

<modelRxnFull><modelRxnRule>

Ste20(Ste4_site!2).Ste4(Ste5_site!3, Ste20_site!2).Ste5(Ste4_site!3, Ste11_site!4).Ste11(Ste5_site!4, S302_S306_T307~pS) -> 
     Ste20(Ste4_site!2).Ste4(Ste5_site!3, Ste20_site!2).Ste5(Ste4_site!3, Ste11_site!4).Ste11(Ste5_site!4, S302_S306_T307~pSpS)

</modelRxnRule>

<modelRxnFull><modelRxnRule>

Ste20(Ste4_site!2).Ste4(Ste5_site!3, Ste20_site!2).Ste5(Ste4_site!3, Ste11_site!4).Ste11(Ste5_site!4, S302_S306_T307~pSpS) -> 
     Ste20(Ste4_site!2).Ste4(Ste5_site!3, Ste20_site!2).Ste5(Ste4_site!3, Ste11_site!4).Ste11(Ste5_site!4, S302_S306_T307~pSpSpT)

</modelRxnRule>

Ste7 phosphorylation by Ste11

  • Purified constitutively active Ste11 (Ste11-GST) is able to phosphorylate purified Ste7. Neiman and Herskowitz 1994 PMID 8159759
  • Phosphorylation at T363 is important for Ste7 activity. Neiman and Herskowitz 1994 PMID 8159759
  • Phosphorylation of S359 and T363 are required for Ste7 activation. Zheng and Guan. 1994 PMID 8131746
  • Ste5 binding to Ste11 may relieve the inhibitory effect of the N-terminal region of Ste11 on the kinase domain of Ste11. Cairns et al. 1992 PMID 1628833 and Stevenson et al. 1992 PMID 1628832.
  • Cell with hyperactive Ste11 or Ste20 mutants have increased basal pathway activity, but still respond to pheromone, suggesting that Ste11 activation isn’t sufficient for maximal pathway activation. Pathway activity in these strains is Ste5 dependent. Lamson et al. 2006 PMID 16546088
  • 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).
  • 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


Reaction Definition

There is strong evidence that Ste5 dimerization is required for efficient signal transduction (see Ste4:Ste18/Ste5 interactions and Ste5 dimerization/oligomerization). Ste5 mutants that are defective in Ste11- or Ste7-binding, when coexpressed, restore response to pheromone in a ste5Δ background, which suggests that Ste11 can phosphorylate Ste7 in trans across a Ste5 dimer. It seems unlikely that Ste11 and Ste7 would be positioned (or have enough range of motion) such that Ste11 could phosphorylate Ste7 both in trans across a Ste5 dimer, as well as in cis on the same Ste5 monomer, suggesting that perhaps Ste11 can only phosphorylate Ste7 in trans across a Ste5 dimer.

Assumptions:

  • Active Ste11 can only phosphorylate Ste7 in trans across a Ste5 dimer.
  • As assumed above, phosphorylation of two of Ste11's three phosphorylation sites is sufficient for full activation.
  • The phosphorylation rate is independent of whether Ste5 is bound to Ste4, or bound to Fus3 or Kss1.
  • Ste11 can only phosphorylate Ste7 that is not bound to Fus3 or Kss1.
  • Singly phosphorylated Ste11 phoshphorylates Ste7 more slowly (by a constant factor Ste11_pS_only_PO4_factor) than fully active (doubly or triply phosphorylated) Ste11 does.
  • Ste7 is phosphorylated in an ordered manner, S359 first, and then T363.

<modelRxnFull><modelRxnRule>

Ste11(Ste5_site!2, S302_S306_T307~pS).Ste5(Ste11_site!2, Ste5_site!3).Ste5(Ste5_site!3, Ste7_site!4).Ste7(Ste5_site!4, S359_T363~none) ->
    Ste11(Ste5_site!2, S302_S306_T307~pS).Ste5(Ste11_site!2, Ste5_site!3).Ste5(Ste5_site!3, Ste7_site!4).Ste7(Ste5_site!4, S359_T363~pS)

</modelRxnRule>

<modelRxnFull><modelRxnRule>

Ste11(Ste5_site!2, S302_S306_T307~pS).Ste5(Ste11_site!2, Ste5_site!3).Ste5(Ste5_site!3, Ste7_site!4).Ste7(Ste5_site!4, S359_T363~pS) ->
    Ste11(Ste5_site!2, S302_S306_T307~pS).Ste5(Ste11_site!2, Ste5_site!3).Ste5(Ste5_site!3, Ste7_site!4).Ste7(Ste5_site!4, S359_T363~pSpT)

</modelRxnRule>

<modelRxnFull><modelRxnRule>

Ste11(Ste5_site!2, S302_S306_T307~pSpS).Ste5(Ste11_site!2, Ste5_site!3).Ste5(Ste5_site!3, Ste7_site!4).Ste7(Ste5_site!4, S359_T363~none) ->
    Ste11(Ste5_site!2, S302_S306_T307~pSpS).Ste5(Ste11_site!2, Ste5_site!3).Ste5(Ste5_site!3, Ste7_site!4).Ste7(Ste5_site!4, S359_T363~pS)

</modelRxnRule>

<modelRxnFull><modelRxnRule>

Ste11(Ste5_site!2, S302_S306_T307~pSpS).Ste5(Ste11_site!2, Ste5_site!3).Ste5(Ste5_site!3, Ste7_site!4).Ste7(Ste5_site!4, S359_T363~pS) ->
    Ste11(Ste5_site!2, S302_S306_T307~pSpS).Ste5(Ste11_site!2, Ste5_site!3).Ste5(Ste5_site!3, Ste7_site!4).Ste7(Ste5_site!4, S359_T363~pSpT)

</modelRxnRule>

<modelRxnFull><modelRxnRule>

Ste11(Ste5_site!2, S302_S306_T307~pSpSpT).Ste5(Ste11_site!2, Ste5_site!3).Ste5(Ste5_site!3, Ste7_site!4).Ste7(Ste5_site!4, S359_T363~none) ->
    Ste11(Ste5_site!2, S302_S306_T307~pSpSpT).Ste5(Ste11_site!2, Ste5_site!3).Ste5(Ste5_site!3, Ste7_site!4).Ste7(Ste5_site!4, S359_T363~pS)

</modelRxnRule>

<modelRxnFull><modelRxnRule>

Ste11(Ste5_site!2, S302_S306_T307~pSpSpT).Ste5(Ste11_site!2, Ste5_site!3).Ste5(Ste5_site!3, Ste7_site!4).Ste7(Ste5_site!4, S359_T363~pS) ->
    Ste11(Ste5_site!2, S302_S306_T307~pSpSpT).Ste5(Ste11_site!2, Ste5_site!3).Ste5(Ste5_site!3, Ste7_site!4).Ste7(Ste5_site!4, S359_T363~pSpT)

</modelRxnRule>

Fus3 phosphorylation by Ste7

  • Purified phosphorylated Ste7 is able to phosphorylate purified Fus3. Neiman and Herskowitz 1994 PMID 8159759
  • Ste5(Δ143-309) does not bind Fus3 (by two-hybrid), and also suppresses activation of Fus3 in the presence of pheromone, suggesting that Fus3 binding to Ste5 is required for its activation. Choi et al. 1994 PMID 8062390
  • Ste5-CTM (Ste5 fused to a transmembrane domain) localizes Ste7-GFP and Fus3-GFP to the plasma membrane, whereas Ste5(Δ241-336)-CTM (which is lacking its MAPK binding site), localizes Ste7-GFP but not Fus3-GFP to the plasma membrane. van Drogen et al. 2001 PMID 11781566
  • MAPKs tend to be phosphorylated on the tyrosine before they get phosphorylated on the threonine on their activation loop. Ferrell and Bhatt. 1997 PMID 9228083
  • 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
    • This suggests an extra level of specificity; Ste7 needs to be able to bind Ste5 and Fus3 for efficient signal transmission.
  • Phosphorylated Fus3 levels peak at about 15 minutes after treatment with 150 nM pheromone and drop off somewhere in the 3 to 4 hour range. Sabbagh et al. 2001 PMID 11583629
    • This seems highly variable, because in the same paper they show drop off in phosphorylated Fus3 levels between 1 and 2 hoursafter treatment with 250 nM pheromone
    • These experiments were done in a BAR1 strain, so Bar1 was degrading pheromone throughout the experiment
  • Purified Ste7-Myc phosphorylates purified Fus3-Myc primarily on T180 and Y182. Errede et al. 1993 PMID 8384702
  • Phosphorylation of Fus3 at T180 and Y182 is necessary for its activation. Errede et al. 1993 PMID 8384702
  • Phosphorylation of Fus3 in response to pheromone requires Ste5's adaptor function as well as its scaffolding function. Flatauer et al. 2005 PMID 15713635
    • Deletion of Ste5 is sufficient to block phosphorylation of Fus3, as is mutation of Ste5 (F514L) such that it no longer binds Ste11, demonstrating that the adaptor function of Ste5 is required for activation of Fus3 in response to pheromone.
    • Mutation of Ste5 (V763A S861P) such that it no longer binds Ste7 blocks phosphorylation of Fus3 in response to pheromone.
    • Deletion of Ste5 or mutation of Ste5 (V763A S861P) such that it no longer binds Ste7 blocks phosphorylation of Fus3 in a strain with a hyperactive Ste11 allele (Ste11-4).
  • Phosphorylation of Fus3 requires activation of Ste5 and Ste11. Flatauer et al. 2005 PMID 15713635
    • Expression of the Ste11-4 (hyperactive) mutant results in a small increase in Fus3 phosphorylation, as does low-level expression of GST-Ste5 (which when overexpressed can activate the pathway). When Ste11-4 and GST-Ste5 are co-expressed (GST-Ste5 at the same low levels as before), Fus3 phosphorylation is greatly enhanced.
  • Mutation of either of the MAPK consensus binding sites on Ste7 had a moderate effect on pathway output (measured by Fus1-GFP), whereas simultaneous mutation of both MAPK consensus binding sites greatly reduced pathway output to near 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.
  • The crystal structure of Fus3 bound to Ste5(287-316) shows that this peptide binds in part to the same docking groove on Fus3 that Ste7's consensus MAPK binding site peptides bind. Bhattacharyya et al. 2006 PMID 16424299
  • One of the Ste7 peptides that binds Fus3 can compete with and reduce binding between Ste5(241-336) and Fus3. Kusari et al. 2004 PMID 1473453
    • This suggests that Fus3 cannot bind simultaneously to Ste5 and Ste7 via its docking interactions.
  • Evidence suggests the same docking groove on Fus3 binds to a large number of proteins, including its upstream kinase Ste7, its phosphatases Msg5 and Ptp3, its scaffold Ste5, and its substrates Far1 and Ste7 and Dig1 and Dig2. Kss1 binds some but not all of these proteins. Kusari et al. 2004 PMID 1473453 Remenyi et al. 2006 PMID 16364919 Zhan & Guan 1999 PMID 10557209
    • This suggests that Fus3 may bind transiently and serially to its many interacting partners via its docking interactions.
  • Rate of ERK phosphorylation by MEK in the table below. Markevich et al. 2004 PMID 14744999
    • These numbers were obtained by fitting a model to data from Ferrell & Bhatt. 1997 PMID 9228083, and Burack & Sturgill. 1997 PMID 9166761
kon koff kcat
ERK -> ERK/pY 0.005 nM-1s-1 1 s-1 1.08 s-1
ERK/pY -> ERK/pYpT 0.025 nM-1s-1 1 s-1 0.007 s-1
ERK -> ERK/pT 0.05 nM-1s-1 1 s-1 0.008 s-1
ERK/pT -> ERK/pYpT 0.005 nM-1s-1 1 s-1 0.45 s-1


  • 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). Bhattacharyya et al. 2006 PMID 16424299
    • The authors only showed that a peptide fragment of this Ste5 mutant, and not the entire Ste5 mutant, fails to bind Fus3
    • This suggests 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

Reaction Definition

New Proposed Mechanism

Ste7 must bind to Ste5 for efficient Fus3 phosphorylation and Fus3 must be able to bind Ste7 (which Fus3 can't do at the same time as binding to Ste5 according to the crystal structures) for efficient Fus3 phosphorylation. This suggests that Fus3 is phosphorylated while bound to Ste7 (which must in turn be bound to Ste5), not while Fus3 is bound directly to Ste5. Deletion of the MAPK binding domain on Ste5 eliminates response to pheromone. However, because it is a large deletion, it is difficult to say what other effects this deletion has. We will assume that the sterility of this Ste5 mutant is not due to its failure to bind Fus3, but due to secondary effects. Smaller mutations to Ste5 that eliminate binding between Ste5 and Fus3 result in increased signaling.

Because this mechanism goes against conventional wisdom, and has not been proven explicitly, we will not implement it. However, if our arguments above seem convincing to others, please leave comments below.

If we were to implement this mechanism, we would use the same phosphorylation rates measure for MEK/ERK, from the table above.

If we were to implement this mechanism, Ste7/MAPK interactions and Ste5/MAPK cascade interactions would have to be modified to allow co-binding of Ste7 to Ste5 and Fus3/Kss1.

Assumptions (that we would make were we to implement this mechanism):

  • Fus3 is not phosphorylated while bound directly to Ste5, but rather while it is bound to Ste5 via a Ste7 'bridge'.
  • Fus3 is only phosphorylated by Ste7 when active Ste7 is bound to Ste5.
  • The phosphorylation rate is not affected by Ste11 or Ste4 binding to Ste5, nor by Ste5 dimerization.
  • Singly phosphorylated Ste7 phosphorylates Fus3 more slowly (by a constant factor Ste7_pS_only_PO4_factor) than fully active Ste7 does.

Conventional Mechanism

Conventional evidence and wisdom is that both Ste7 and Fus3 must be bound to Ste5 in order for Ste7 to phosphorylate Fus3.

Assumptions:

  • The phosphorylation rate is not affected by Ste11 or Ste4 binding to Ste5, nor by Ste5 dimerization.

<modelRxnFull><modelRxnRule>

Ste7(Ste5_site!2, MAPK_site, S359_T363~pS).Ste5(Ste7_site!2, MAPK_site!3).Fus3(docking_site!3, T180~none, Y182~none) ->
     Ste7(Ste5_site!2, MAPK_site, S359_T363~pS).Ste5(Ste7_site!2, MAPK_site!3).Fus3(docking_site!3, T180~none, Y182~PO4)

</modelRxnRule>

<modelRxnFull><modelRxnRule>

Ste7(Ste5_site!2, MAPK_site, S359_T363~pS).Ste5(Ste7_site!2, MAPK_site!3).Fus3(docking_site!3, T180~none, Y182~PO4) ->
     Ste7(Ste5_site!2, MAPK_site, S359_T363~pS).Ste5(Ste7_site!2, MAPK_site!3).Fus3(docking_site!3, T180~PO4, Y182~PO4)

</modelRxnRule>

<modelRxnFull><modelRxnRule>

Ste7(Ste5_site!2, MAPK_site, S359_T363~pS).Ste5(Ste7_site!2, MAPK_site!3).Fus3(docking_site!3, T180~none, Y182~none) ->
     Ste7(Ste5_site!2, MAPK_site, S359_T363~pS).Ste5(Ste7_site!2, MAPK_site!3).Fus3(docking_site!3, T180~PO4, Y182~none)

</modelRxnRule>

<modelRxnFull><modelRxnRule>

Ste7(Ste5_site!2, MAPK_site, S359_T363~pS).Ste5(Ste7_site!2, MAPK_site!3).Fus3(docking_site!3, T180~PO4, Y182~none) ->
     Ste7(Ste5_site!2, MAPK_site, S359_T363~pS).Ste5(Ste7_site!2, MAPK_site!3).Fus3(docking_site!3, T180~PO4, Y182~PO4)

</modelRxnRule>

<modelRxnFull><modelRxnRule>

Ste7(Ste5_site!2, MAPK_site, S359_T363~pSpT).Ste5(Ste7_site!2, MAPK_site!3).Fus3(docking_site!3, T180~none, Y182~none) ->
     Ste7(Ste5_site!2, MAPK_site, S359_T363~pSpT).Ste5(Ste7_site!2, MAPK_site!3).Fus3(docking_site!3, T180~none, Y182~PO4)

</modelRxnRule>

<modelRxnFull><modelRxnRule>

Ste7(Ste5_site!2, MAPK_site, S359_T363~pSpT).Ste5(Ste7_site!2, MAPK_site!3).Fus3(docking_site!3, T180~none, Y182~PO4) ->
     Ste7(Ste5_site!2, MAPK_site, S359_T363~pSpT).Ste5(Ste7_site!2, MAPK_site!3).Fus3(docking_site!3, T180~PO4, Y182~PO4)

</modelRxnRule>

<modelRxnFull><modelRxnRule>

Ste7(Ste5_site!2, MAPK_site, S359_T363~pSpT).Ste5(Ste7_site!2, MAPK_site!3).Fus3(docking_site!3, T180~none, Y182~none) ->
     Ste7(Ste5_site!2, MAPK_site, S359_T363~pSpT).Ste5(Ste7_site!2, MAPK_site!3).Fus3(docking_site!3, T180~PO4, Y182~none)

</modelRxnRule>

<modelRxnFull><modelRxnRule>

Ste7(Ste5_site!2, MAPK_site, S359_T363~pSpT).Ste5(Ste7_site!2, MAPK_site!3).Fus3(docking_site!3, T180~PO4, Y182~none) ->
     Ste7(Ste5_site!2, MAPK_site, S359_T363~pSpT).Ste5(Ste7_site!2, MAPK_site!3).Fus3(docking_site!3, T180~PO4, Y182~PO4)

</modelRxnRule>

Kss1 phosphorylation by Ste7

  • Kss1 is phosphorylated at Thr183 and Tyr185 in response to pheromone. Tyr185 appears to be more strongly phosphorylated than Thr183. Ma et al. 1995 PMID 7579701
  • MAPKs tend to be phosphorylated on the tyrosine before they get phosphorylated on the threonine on their activation loop. Ferrell and Bhatt. 1997 PMID 9228083
  • Phosphorylated Kss1 levels peak at about 15 minutes after treatment with pheromone and drop off somewhere in the 1 to 2 hour range (150 nM pheromone), or 2 to 3 hour range (250 nM pheromone). Sabbagh et al. 2001 PMID 11583629
    • This seems highly variable, especially when considering the variation in drop off in phosphorylated Fus3 levels (see above).
  • Phosphorylation of Kss1 in response to pheromone requires only Ste5's adaptor function, suggesting that it can or does happen efficiently off the scaffold. Flatauer et al. 2005 PMID 15713635
    • Deletion of Ste5 is sufficient to block phosphorylation of Kss1, as is mutation of Ste5 (F514L) such that it no longer binds Ste11, demonstrating that the adaptor function of Ste5 is required for activation of Kss1 in response to pheromone.
    • Mutation of Ste5 (V763A S861P) such that it no longer binds Ste7 only slightly decreases Kss1 phosphorylation.
  • Mutation of either of Ste7's consensus MAPK binding motifs (residues 7-19, and residues 61-72) 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 16364914
    • This suggests that stable binding to Ste7 is required for Kss1's phosphorylation.

Reaction Definition

New Proposed Mechanism

In contrast to Fus3 phosphorylation, Ste7 does not need to bind to Ste5 for efficient Kss1 phosphorylation. Kss1 must be able to bind Ste7 (which Kss1 likely can't do at the same time as binding to Ste5 according to the Fus3 crystal structures) for efficient Kss1 phosphorylation. This suggests that Kss1 is phosphorylated while bound to Ste7, but unlike phosphorylation of Fus3, Ste7 does not need to be bound to Ste5. Deletion of the MAPK binding domain on Ste5 eliminates response to pheromone. However, because it is a large deletion, it is difficult to say what other effects this deletion has. We will assume that the sterility of this Ste5 mutant is not due to its failure to bind Fus3, but due to secondary effects.

Because this mechanism goes against conventional wisdom, and has not been proven explicitly, we will not implement it. However, if our arguments above seem convincing to others, please leave comments below.

If we were to implement this mechanism, we would use the same phosphorylation rates measure for MEK/ERK, from the table above.

If we were to implement this mechanism, Ste7/MAPK interactions and Ste5/MAPK cascade interactions would have to be modified to allow cobinding of Ste7 to Ste5 and Fus3/Kss1.

Assumptions (that we would make were we to implement this mechanism):

  • Kss1 is not phosphorylated while bound to Ste5, but rather while it is directly bound to Ste7 (regardless of whether Ste7 is bound to Ste5).
  • If Ste7 is bound to Ste5, the phosphorylation rate is not affected by Ste11 or Ste4 binding to Ste5, nor by Ste5 dimerization.

Conventional Mechanism

In contrast to Fus3 phosphorylation, Ste7 does not need to bind to Ste5 for efficient Kss1 phosphorylation. We will thus assume that Ste7 can phosphorylate Kss1 when neither protein is bound to Ste5, or when both proteins are bound to Ste5.

Assumptions:

  • The rate at which Kss1 is phosphorylated is unaffected by whether Kss1 and Ste7 are both bound to Ste5 or not.


<modelRxnFull><modelRxnRule>

Ste7(MAPK_site!2, Ste5_site, S359_T363~pS).Kss1(docking_site!2, T183~none, Y185~none) ->
     Ste7(MAPK_site!2, Ste5_site, S359_T363~pS).Kss1(docking_site!2, T183~none, Y185~PO4)

</modelRxnRule> <modelRxnRule>

Ste7(MAPK_site, Ste5_site!2, S359_T363~pS).Ste5(Ste7_site!2,MAPK_site!3).Kss1(docking_site!3, T183~none, Y185~none) ->
     Ste7(MAPK_site, Ste5_site!2, S359_T363~pS).Ste5(Ste7_site!2,MAPK_site!3).Kss1(docking_site!3, T183~none, Y185~PO4)

</modelRxnRule>

  • Phosphorylation rate constant <modelRxnParam>kcat_Ste7pSKss1_pY</modelRxnParam></modelRxnFull>

<modelRxnFull><modelRxnRule>

Ste7(MAPK_site!2, Ste5_site, S359_T363~pS).Kss1(docking_site!2, T183~none, Y185~PO4) ->
     Ste7(MAPK_site!2, Ste5_site, S359_T363~pS).Kss1(docking_site!2, T183~PO4, Y185~PO4)

</modelRxnRule> <modelRxnRule>

Ste7(MAPK_site, Ste5_site!2, S359_T363~pS).Ste5(Ste7_site!2,MAPK_site!3).Kss1(docking_site!3, T183~none, Y185~PO4) ->
     Ste7(MAPK_site, Ste5_site!2, S359_T363~pS).Ste5(Ste7_site!2,MAPK_site!3).Kss1(docking_site!3, T183~PO4, Y185~PO4)

</modelRxnRule>

<modelRxnFull><modelRxnRule>

Ste7(MAPK_site!2, Ste5_site, S359_T363~pS).Kss1(docking_site!2, T183~none, Y185~none) ->
     Ste7(MAPK_site!2, Ste5_site, S359_T363~pS).Kss1(docking_site!2, T183~PO4, Y185~none)

</modelRxnRule> <modelRxnRule>

Ste7(MAPK_site, Ste5_site!2, S359_T363~pS).Ste5(Ste7_site!2,MAPK_site!3).Kss1(docking_site!3, T183~none, Y185~none) ->
     Ste7(MAPK_site, Ste5_site!2, S359_T363~pS).Ste5(Ste7_site!2,MAPK_site!3).Kss1(docking_site!3, T183~PO4, Y185~none)

</modelRxnRule>

  • Phosphorylation rate constant <modelRxnParam>kcat_Ste7pSKss1_pT</modelRxnParam></modelRxnFull>

<modelRxnFull><modelRxnRule>

Ste7(MAPK_site!2, Ste5_site, S359_T363~pS).Kss1(docking_site!2, T183~PO4, Y185~none) ->
     Ste7(MAPK_site!2, Ste5_site, S359_T363~pS).Kss1(docking_site!2, T183~PO4, Y185~PO4)

</modelRxnRule> <modelRxnRule>

Ste7(MAPK_site, Ste5_site!2, S359_T363~pS).Ste5(Ste7_site!2,MAPK_site!3).Kss1(docking_site!3, T183~PO4, Y185~none) ->
     Ste7(MAPK_site, Ste5_site!2, S359_T363~pS).Ste5(Ste7_site!2,MAPK_site!3).Kss1(docking_site!3, T183~PO4, Y185~PO4)

</modelRxnRule>

<modelRxnFull><modelRxnRule>

Ste7(MAPK_site!2, Ste5_site, S359_T363~pSpT).Kss1(docking_site!2, T183~none, Y185~none) ->
     Ste7(MAPK_site!2, Ste5_site, S359_T363~pSpT).Kss1(docking_site!2, T183~none, Y185~PO4)

</modelRxnRule> <modelRxnRule>

Ste7(MAPK_site, Ste5_site!2, S359_T363~pSpT).Ste5(Ste7_site!2,MAPK_site!3).Kss1(docking_site!3, T183~none, Y185~none) ->
     Ste7(MAPK_site, Ste5_site!2, S359_T363~pSpT).Ste5(Ste7_site!2,MAPK_site!3).Kss1(docking_site!3, T183~none, Y185~PO4)

</modelRxnRule>

<modelRxnFull><modelRxnRule>

Ste7(MAPK_site!2, Ste5_site, S359_T363~pS).Kss1(docking_site!2, T183~none, Y185~PO4) ->
     Ste7(MAPK_site!2, Ste5_site, S359_T363~pS).Kss1(docking_site!2, T183~PO4, Y185~PO4)

</modelRxnRule> <modelRxnRule>

Ste7(MAPK_site, Ste5_site!2, S359_T363~pSpT).Ste5(Ste7_site!2,MAPK_site!3).Kss1(docking_site!3, T183~none, Y185~PO4) ->
     Ste7(MAPK_site, Ste5_site!2, S359_T363~pSpT).Ste5(Ste7_site!2,MAPK_site!3).Kss1(docking_site!3, T183~PO4, Y185~PO4)

</modelRxnRule>

<modelRxnFull><modelRxnRule>

Ste7(MAPK_site!2, Ste5_site, S359_T363~pSpT).Kss1(docking_site!2, T183~none, Y185~none) ->
     Ste7(MAPK_site!2, Ste5_site, S359_T363~pSpT).Kss1(docking_site!2, T183~PO4, Y185~none)

</modelRxnRule> <modelRxnRule>

Ste7(MAPK_site, Ste5_site!2, S359_T363~pSpT).Ste5(Ste7_site!2,MAPK_site!3).Kss1(docking_site!3, T183~none, Y185~none) ->
     Ste7(MAPK_site, Ste5_site!2, S359_T363~pSpT).Ste5(Ste7_site!2,MAPK_site!3).Kss1(docking_site!3, T183~PO4, Y185~none)

</modelRxnRule>

<modelRxnFull><modelRxnRule>

Ste7(MAPK_site!2, Ste5_site, S359_T363~pS).Kss1(docking_site!2, T183~PO4, Y185~none) ->
     Ste7(MAPK_site!2, Ste5_site, S359_T363~pS).Kss1(docking_site!2, T183~PO4, Y185~PO4)

</modelRxnRule> <modelRxnRule>

Ste7(MAPK_site, Ste5_site!2, S359_T363~pSpT).Ste5(Ste7_site!2,MAPK_site!3).Kss1(docking_site!3, T183~PO4, Y185~none) ->
     Ste7(MAPK_site, Ste5_site!2, S359_T363~pSpT).Ste5(Ste7_site!2,MAPK_site!3).Kss1(docking_site!3, T183~PO4, Y185~PO4)

</modelRxnRule>

Fus3 autophosphorylation

  • Fus3 binding to Ste5(288-316) increases it's rate of autophosphorylation at least 50-fold. Bhattacharyya et al. 2006 PMID 16424299
    • Mass spec. analysis shows that this autophosphorylation only phosphorylates Y182, not T180.
    • Crystal structure of this peptide bound to Fus3 shows that binding to Ste5(288-316) causes a slight shift in the orientation of the C- and N-terminal lobes of Fus3.
    • Other peptides that bind Fus3 were tested and do not cause autophosphorylation.
    • This authophosphorylation is concentration independent, suggesting that it is occuring intra-molecularly (unpublished data).
    • In the presence of Ste5(288-316), Fus3 is autophosphorylated on Y182 with a half time of about 40 min (see fig 3a).

Reaction Definition

We will not model Fus3 autophosphorylation. This autophosphorylation rate is rather slow, and it is not know whether Kss1 also undergoes autophosphorylation while bound to Ste5.