MAPK/phosphatase interactions

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Contents 1 Ptp2/Ptp3 dephosphrylation of MAPK 1.1 Reaction Definition 2 Msg5 dephosphorylation of MAPK 2.1 Reaction Definition

Ptp2/Ptp3 dephosphrylation of MAPK

• A mammalian MAPK (ERK2) is protected from dephosphorylation while bound to at least one of it's substrates. Wang et al. 2002 PMID 12056917

• Deletion of Msg5 causes increased levels of active Fus3 in the absence of pheromone. Deletion of Ptp2 and Ptp3 has does not appreciably affect the levels of active Fus3. Andersson et al. 2004 PMID 15192700

• Deletion of Ptp3 causes increased levels of active Kss1 in the absence of pheromone. Deletion of Ptp2 and Msg5 has does not appreciably affect the levels of active Kss1. Andersson et al. 2004 PMID 15192700

• Ptp3 interacts with Fus3 via the CH2 domain on Ptp3's N-terminal non-catalytic domain. Zhan and Guan. 1999 PMID 10557209
  • Ptp3 interacts more strongly with Fus3 than Ptp2.
  • Ptp3 binds GST-Fus3 in vitro with a Kd of 200nM.

• Tyrosine dephosphorylation of Fus3 is primarily regulated by Ptp3. Zhan et al. 1997 PMID 9224718
  • In the presence of pheromone, Ptp2 deletion only has a detectable effect when combined with Ptp3 deletion (measure by the amount of tyrosine-phosphorylated Fus3, and Fus3 activity).

Reaction Definition

We will lump Ptp2 and Ptp3 together into one species, Ptp, since we are not treating the cytoplasm and nucleus as separate compartments. Assumptions:

• The phosphatases bind Fus3 and Kss1 competitively with the MAPK's other binding partners, so the phosphatases can only bind free Fus3 and Kss1.
• Ptp can only bind Fus3 or Kss1 molecules that are phosphorylated on the tyrosine residue on the activation loop, and they bind independently of phopsohrylation on the threonine residue.
• Ptp binds and dephosphorylates Fus3 and Kss1 with equal efficiency

Ptp(MAPK_site) + Fus3(docking_site, Y182~PO4) <-> Ptp(MAPK_site!1).Fus3(docking_site!1, Y182~PO4)

Ptp(MAPK_site) + Kss1(docking_site, Y185~PO4) <-> Ptp(MAPK_site!1).Kss1(docking_site!1, Y185~PO4)

• Forward rate constant kon_Ptp_MAPK

• Reverse rate constant koff_Ptp_MAPK

Ptp(MAPK_site!1).Fus3(docking_site!1, Y182~PO4) -> Ptp(MAPK_site) + Fus3(docking_site, Y182~none)

Ptp(MAPK_site!1).Kss1(docking_site!1, Y185~PO4) -> Ptp(MAPK_site) + Kss1(docking_site, Y185~none)

• Dephosphorylation rate constant kcat_Ptp_MAPK_PO4

Msg5 dephosphorylation of MAPK

• Msg5 is a dual-specificity phosphatase. Doi et al. 1994 PMID 8306972
  • Msg5 contains sequence homology the conserved active site of protein tyrosine phosphatases, as well as a C-terminal sequence that is homologous to a dual-specificity phosphatase.
  • In vitro, Msg5 is able to dephosphorylate phospho-serine, phospho-threonine, and phospho-tyrosine on Fus3.

• Msg5 downregulates Fus3 activity, but not Ste7. Doi et al. 1994 PMID 8306972
  • Disruption of Msg5 greatly increases the activity of Fus3 that is isolated from vegetatively and pheromone-treated cells.
  • Disruption of Msg5 does not affect the activity of Ste7 that is isolated from vegetatively and pheromone-treated cells.

• A mammalian MAPK (ERK2) is protected from dephosphorylation while bound to at least one of its substrates. Wang et al. 2002 PMID 12056917

• The dual specificity phosphatase MKP3 has a Kd = 0.17 μM for unphosphorylated ERK2. Zhou et al. 2001 PMID 11104775

• Based on initial rates of release of radioactive phosphate, the dual specificity phosphatase MKP3 has the following kinetic parameters for ERK2: (Zhao & Zhang. 2001 PMID 11432864)

	Km (phosphatase MKP3)	kcat (phosphatase MKP3)
ERK2/pTpY	0.022 ± 0.005 μM	0.084 ± 0.009 s-1
ERK2/pY	0.034 ± 0.004 μM	0.108 ± 0.004 s-1
ERK2/pT	0.018 ± 0.005 μM	0.060 ± 0.006 s-1

• MKP3 appears to dephospohrylate ERK2 via an ordered distributive mechnism, dephosphorylating tyrosine residue before the threonine residue. Zhao & Zhang. 2001 PMID 11432864

• Deletion of Msg5 causes increased levels of active Fus3 in the absence of pheromone. Deletion of Ptp2 and Ptp3 has does not appreciably affect the levels of active Fus3. Andersson et al. 2004 PMID 15192700

• Deletion of Ptp3 causes increased levels of active Kss1 in the absence of pheromone. Deletion of Ptp2 and Msg5 has does not appreciably affect the levels of active Kss1. Andersson et al. 2004 PMID 15192700

• Fus3 binds a peptide with the MAPK consensus binding motif from Msg5 (residues 23-37; PRSLQNRNTKNLSLD) with a Kd = 2 ± 0.5 μM. Remenyi et al. 2005 PMID 16364919

• 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 16364919

• Fus3's dephosphorylation by Msg5 is at least partially dependent on docking site/docking groove interactions. Remenyi et al. 2005 PMID 16364914
  • Mutation of the Fus3 docking grove elimintates its in vitro dephosphorylation by Msg5, though this mutant is still efficiently dephosphorylated by λ-phosphatase, which is known to not require substrate docking.

Reaction Definition

MKP3 binds ERK2 with near equal affinity when ERK2 is in any of its phopsohrylated states, and binds it ~10-fold more weakly when ERK2 is not phosphoryated. MKP3 also appears to dephosphorylate ERK2 with equal efficiency when ERK2 is in any of its phopsohrylated states. We will thus assume that Msg5 has equal affinity and activity on all phosphorylated forms of Fus3 and Kss1, and does not bind the unphosphorylated forms of the MAPKs. Thus, based on the binding of Fus3 and Kss1 to the MAPK binding motif from Msg5, Kd_Msg5_MAPK = 1.4 μM. Also, we will assume that Msg5 acts in an ordered distributive mechanism (as MPK3 does), by first dephosphorylating tyrosine, and then threonine.

Assumptions:

• Msg5 binds Fus3 and Kss1 competitively with their other binding partners, so Msg5 can only bind free Fus3 and Kss1.
• Msg5 binds and dephosphorylates Fus3 and Kss1 with equal efficiency

Msg5(MAPK_site) + Fus3(docking_site, T180~PO4, Y182~none) <-> 
   Msg5(MAPK_site!1).Fus3(docking_site!1, T180~PO4, Y182~none)

Msg5(MAPK_site) + Fus3(docking_site, T180~none, Y182~PO4) <-> 
    Msg5(MAPK_site!1).Fus3(docking_site!1, T180~none, Y182~PO4)

Msg5(MAPK_site) + Fus3(docking_site, T180~PO4, Y182~PO4) <-> 
    Msg5(MAPK_site!1).Fus3(docking_site!1, T180~PO4, Y182~PO4)

Msg5(MAPK_site) + Kss1(docking_site, T183~PO4, Y185~none) <-> 
    Msg5(MAPK_site!1).Kss1(docking_site!1, T183~PO4, Y185~none)

Msg5(MAPK_site) + Kss1(docking_site, T183~none, Y185~PO4) <-> 
    Msg5(MAPK_site!1).Kss1(docking_site!1, T183~none, Y185~PO4)

Msg5(MAPK_site) + Kss1(docking_site, T183~PO4, Y185~PO4) <-> 
    Msg5(MAPK_site!1).Kss1(docking_site!1, T183~PO4, Y185~PO4)

• Forward rate constant kon_Msg5_MAPK

• Reverse rate constant koff_Msg5_MAPK

Msg5(MAPK_site!1).Fus3(docking_site!1, Y182~PO4) -> 
   Msg5(MAPK_site) + Fus3(docking_site, Y182~none)

Msg5(MAPK_site!1).Fus3(docking_site!1, T180~PO4, Y182~none) -> 
    Msg5(MAPK_site) + Fus3(docking_site, T180~none, Y182~none)

Msg5(MAPK_site!1).Kss1(docking_site!1, Y185~PO4) -> 
    Msg5(MAPK_site) + Kss1(docking_site, Y185~none)

Msg5(MAPK_site!1).Kss1(docking_site!1, T183~PO4, Y185~none) -> 
    Msg5(MAPK_site) + Kss1(docking_site, T183~none, Y185~none)

• Dephosphorylation rate constant kcat_Msg5_MAPK_PO4