Imported:YPM/MAPK/target interaction properties
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Fus3/Kss1 Differences
Interaction with Ste5
- Fus3 binds to GST-Ste5(241-336) with a Kd of 1 μM, whereas Kss1 binds to GST-Ste5(241-336) with a Kd of 6.5 μM (via cosedimentation assay). Kusari et al. 2004 PMID 14734536
- Fus3(T180V Y182F) binds a smaller 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). We assume that the affinity measured by Bhattacharyya et al. is weaker than the affinity measured by Kusari et al. because Bhattacharyya et al. used a smaller (possibly not as well foldeded) fragment of Ste5.
Thus, Fus3's affinity for Ste5 is 6.5-fold higher than Kss1's affinity for Ste5.
Interaction with Ste7
- Fus3 binds to GST-Ste7(1-98) with a Kd of 0.1 μM, and Kss1 binds to GST-Ste7(1-98) with a Kd of 0.1 μM (via cosedimentation assay). Kusari et al. 2004 PMID 14734536
- 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. 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. Measurements made by competition fluorescence polarizing assays. Remenyi et al. 2005 PMID 16364914
Thus Fus3 and Kss1 have the same affinity for Ste7.
Interaction with Msg5
- Fus3 binds a peptide with the MAPK consensus binding motif from Msg5 (residues 23-37; PRSLQNRNTKNLSLD) with a Kd = 2 ± 0.5 μM. Kss1 binds a peptide with the MAPK consensus binding motif from Msg5 (residues 23-37; PRSLQNRNTKNLSLD) with a Kd = 0.8 ± 0.15 μM (measurements made by competition fluorescence polarizing assay). Remenyi et al. 2005 PMID 16364914
Thus Fus3's affinity for Msg5 is about 2.5-fold weaker than Kss1's affinity for Msg5.
Interaction with Ste12
- Activated Kss1-Myc phosphorylates Ste12-Flag at half 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
- Fus3 binding to GST-Ste12(298-473) is not detectable (whereas Kss1 binding is), and Fus3 shows a weaker association with Ste12 than Kss1 in yeast cell extracts. Bardwell et al. 1998 PMID 9744865
- Unphosphorylated Kss1 interacts with Ste12 with a Kd of ~ 400 nM (determined by cosedimentation). Bardwell et al. 1998 PMID 9744865
The data suggest that Kss1 may have a higher affinity than Fus3 for Ste12, but conversely, Fus3 may phosphorylate Ste12 more efficiently than Kss1 does. We will assume that the affinity Ste12's MAPK binding site for Kss1 and Fus3 is the same.
Interaction with Dig1
- Fus3 interacts with GST-Dig1(213-452) with about the same efficiency as Kss1. Bardwell et al. 1998 PMID 9744865
- Kss1 binds both GST-Dig1(93-111) and GST-Dig2(98-114) about 17-fold more weakly than Fus3 binds these targets. Kusari et al. 2004 PMID 14734536
Thus, Fus3's affinity for Dig1 is 17-fold higher than Kss1's affinity for Dig1.
Interaction with Far1
- Fus3 binds a peptide with the MAPK consensus binding motif from Far1 (residues 72-83; KRGNIPKPLNLS) with a Kd = 7 ± 1 μM. 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
- Activated Kss1-Myc phosphorylates His-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
Thus, Fus3's affinity for Ste5 is 6.6-fold higher than Kss1's affinity for Ste5.
Modeling Assumptions
- In order to simplify the model, we will assume that the only differences between Kss1 and Fus3 are in their binding affinities for other proteins. So when they are bound to another protein, Fus3 and Kss1 behave identically (whether they are being phosphorylated by Ste7, phosphorylating a target protein, being dephosphorylated by a phosphatase, or stabilizing a complex).
- By solving a series of crystal structures, Remenyi et al. (2005 PMID 16364919) and Bhattacharyya et al. (2006 PMID 16424299) found that Ste5 and Far1's MAPK binding motif peptides induce a subtle rearrangement of residues in Fus3's docking groove upon binding, and this rearrangement is not seen upon binding of Ste7 and Msg5's MAPK binding motif peptides. Remenyi et al. 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. Based on this work, we will assume that Fus3/Kss1 binding partners fall into two discrete classes, those that bind Fus3 and Kss1 with equal affinity, and those that require a subtle rearrangement of the MAPK docking grove, and thus bind Fus3 with higher affinity than they bind Kss1.
- Fus3 is known to interact with Ste5, Dig1 and Far1 with higher affinity than Kss1. On average for these three interactions, Fus3's affinity is 10-fold higher than Kss1's affinity. In all other cases listed above, Kss1 and Fus3's affinities are within a factor of 2.5x of eachother. In these cases, and any other case in which we do not know that there is a difference in affinity, we will assume that Fus3 and Kss1 bind with equal affinity.
- When modeling a difference in affinity of Fus3 and Kss1 for a binding partner, the difference in affinity manifests itself solely in the dissociation rate constants. When there is a known difference in binding affinity, but that difference has not been measured, assume that kon_Kss1_target = kon_Fus3_target, koff_Kss1_target = Fus3_Kss1_Kd_preference_factor * koff_Fus3_target, and Fus3_Kss1_Kd_preference_factor = 10 (the average of the measured differences).
Effect of MAPK phosphorylation state on binding and kinase activity
- Erk2 activity in the table below. Zhou and Zhang. 2002 PMID 11839761
- For two different substrates, Erk2 has ~10x lower activity when singly phosphorylated than when doubly phosphorylated, and ~10000x lower activity when unphosphorylated than it does when fully phosphorylated.
- For two different substrates, Erk2 has ~2x higher Km in singly phosphorylated form than it does in doubly phosphorylated form, and ~2-10x higher Km when unphosphorylated than it does when fully phosphorylated.
Km (substrate - MBP) | kcat (substrate - MBP) | Km (substrate - Elk-1) | kcat (substrate - Elk-1) | |
Erk2/pTpY | 10.0 μM | 6.5 s-1 | 1.95 μM | 10.2 s-1 |
Erk2/pT | 15.2 μM | 0.45 s-1 | 3.6 μM | 0.94 s-1 |
Erk2/pY | 15.5 μM | 0.165 s-1 | 4.7 μM | 0.30 s-1 |
Erk2 | 23 μM | 7.3 × 10-4 s-1 | 7.5 μM | 10 × 10-4 s-1 |
- Erk2 activity in the table below. Wang et al. 2002 PMID 12056917
- For Elk-1, ERK2 has ~4x lower activity when singly phosphorylated than when doubly phosphorylated, and ~100x lower activity when unphosphorylated than it does when fully phosphorylated.
- For Elk-1, Erk2 has ~3x higher Km in singly phosphorylated form than it does in doubly phosphorylated form, and ~75x higher Km when unphosphorylated than it does when fully phosphorylated.
Km (substrate - Elk-1) | kcat (substrate - Elk-1) | |
Erk2/pTpY | 29.3 μM | 0.477 s-1 |
Erk2/pT | 80.6 μM | 0.129 s-1 |
Erk2 | 2180 μM | 4.84 × 10-3 s-1 |
- Using myelin basic protein as a model substrate, Fus3's activity in the unphosphorylated form, monophosphorylated form (Fus3/pY), and doubly phosphorylated form (Fus3/pTpY) have relative activities estimated to be in the ratio of 1:25:120. Bhattacharyya et al. 2006 PMID 16424299
- mCherry-tagged Fus3 localizes strongly to the shmoo tip in response to pheromone treatment, and this localization was greatly reduced when a non-phosphorylatable Fus3 (T180A Y182A) is expressed. Maeder et al. 2007 PMID 17952059
- Shmoo tip localization is probably in part mediated by Fus3's interactions with target proteins, and this mutation suggests that phosphorylated Fus3 may have a higher affinity for those targets.
- Supporting this, more Fus3 localizes to the shmoo tip when Msg5 is underexpressed, and less Fus3 localizes to the shmoo tip when Msg5 is overexpressed.
Modeling Assumptions
MAPK/target binding
- The affinity of Fus3pY and Kss1pY (only phosphorylated on the tyrosine residue) for their targets will always be a constant factor MAPK_pY_only_Kd_factor less than the affinity of Fus3pTpY and Kss1pTpY for the same targets (e.g., Kd_Fus3pY_target = MAPK_pY_only_Kd_factor * Kd_Fus3pYpT_target).
- The affinity of Fus3pT and Kss1pT (only phosphorylated on the threonine residue) for their targets will always be a constant factor MAPK_pT_only_Kd_factor less than the affinity of Fus3pTpY and Kss1pTpY for the same targets (e.g., Kd_Fus3pT_target = MAPK_pT_only_Kd_factor * Kd_Fus3pYpT_target).
- The difference in affinity for singly and doubly phosphorylated MAPK for their targets manifests itselt entirely in differences in the dissociation rates (e.g., kon_Fus3pY_target = kon_Fus3pT_target = kon_Fus3pYpT_target, koff_Fus3pY_target = MAPK_pY_only_Kd_factor * koff_Fus3pYpT_target, koff_Fus3pT_target = MAPK_pT_only_Kd_factor * koff_Fus3pYpT_target).
- We will assume that the differencesin Km equate to the same differences in Kd. For ERK2, MAPK_pY_only_Kd_factor and MAPK_pT_only_Kd_factor are thus ~1.5-2.5. We will use MAPK_pY_only_Kd_factor = MAPK_pT_only_Kd_factor = 2.
Target phosphorylation
- We will assume that the phosphorylation rate of targets by Fus3pY and Kss1pY (only phosphorylated on the tyrosine residue) will always be a constant factor MAPK_pY_only_PO4_factor less than the phosphorylation rate of the same targets by Fus3pTpY and Kss1pTpY. (e.g., kcat_Fus3pY_target_PO4 = kcat_Fus3pYpT_target_PO4 / MAPK_pY_only_PO4_factor)
- The phosphorylation rate of targets by Fus3pT and Kss1pT (only phosphorylated on the threonine residue) will always be a constant factor MAPK_pT_only_PO4_factor less than the phosphorylation rate of the same targets by Fus3pTpY and Kss1pTpY. (e.g., kcat_Fus3pT_target_PO4 = kcat_Fus3pYpT_target_PO4 / MAPK_pT_only_PO4_factor)
- For Fus3, MAPK_pY_only_PO4_factor was estimated to be ~5 (for MBP). For ERK2, MAPK_pY_only_PO4_factor was measured to be ~35-40. We will use the MAPK_pY_only_PO4_factor measured for Fus3. Therefore, we will use MAPK_pY_only_PO4_factor = 5.
- For ERK2, MAPK_pT_only_PO4_factor was measured to be ~10-15, lower than the factor measured for ERKpY. Therefore, based on the Fus3 MAPK_pT_only_PO4_factor (= 5), we will use MAPK_pT_only_PO4_factor = 3.