Unfair competition governs the interaction of pCPI-17 with myosin phosphatase (PP1-MYPT1)

  1. Joshua J Filter
  2. Byron C Williams
  3. Masumi Eto
  4. David Shalloway
  5. Michael L Goldberg  Is a corresponding author
  1. Cornell University, United States
  2. Sidney Kimmel Medical College at Thomas Jefferson University, United States
9 figures, 2 tables and 1 additional file

Figures

Models for MLCP reactivation.

(A) A simplistic model in which pCPI-17 is dephosphorylated only by other phosphatases (PPU) immediately after it dissociates from MLCP. The rate-limiting step for MLCP reactivation is the …

https://doi.org/10.7554/eLife.24665.002
Figure 2 with 1 supplement
Preparation of MLCP.

HEK-293 CRL-1573 cells were transformed with empty vector, with a construct overexpressing a FLAG-tagged version of the B56α regulatory subunit of PP2A for comparison, or with a construct …

https://doi.org/10.7554/eLife.24665.003
Figure 2—figure supplement 1
Phosphatase activities of the MLCP preparation shown in Figure 2.

Enzyme assays as described in Materials and methods were performed at 30°C with the following components in 6 μL reactions unless otherwise noted: (A) 0.5 μM myosin regulatory light chain …

https://doi.org/10.7554/eLife.24665.004
Figure 3 with 1 supplement
Dephosphorylation of pCPI-17 by MLCP as a function of pCPI-17 concentration.

All assays contained 0.1 nM MLCP and were performed for 3 min at 30°C in a total volume of 8 μL. The experiment was replicated in triplicate (n = 3); error bars represent standard deviations. Data …

https://doi.org/10.7554/eLife.24665.005
Figure 3—figure supplement 1
Dephosphorylation of pCPI-17 by MLCP as a function of pCPI-17 concentration.

This figure displays the same data as Figure 3, but with a different scale on the x-axis to better highlight the earliest timepoints.

https://doi.org/10.7554/eLife.24665.006
Determining the pCPI-17/MLCP Km by competitive inhibition.

(A) Inhibition of MLCP by okadaic acid (OA). Enzyme assays in the presence of increasing levels of OA were performed with the indicated substrates at the indicated concentrations; in all assays, the …

https://doi.org/10.7554/eLife.24665.007
In vitro demonstration of the unfair competition mechanism for MLCP and pCPI-17.

Enzyme assays containing 0.25 nM MLCP and 1.4 μM C-ERMAD were performed at 30°C for the indicated times, either in the absence of CPI-17 (inverted triangles) or in the presence of 50 nM pCPI-17 …

https://doi.org/10.7554/eLife.24665.008
Kinetic analysis of the dephosphorylation of pCPI-17 by PP2A-B56α, a candidate PPU enzyme.

(A). Linearity of assay employing PP2A-B56α enzyme (at 0.25 nM); the concentration of pCPI-17 was 1 μM. (B) Velocity of the dephosphorylation of pCPI-17 by 0.1 nM PP2A-B56α as a function of the …

https://doi.org/10.7554/eLife.24665.011
Figure 7 with 2 supplements
Okadaic acid inhibition of the dephosphorylation of pCPI-17 by dilute mouse uterus extracts.

Mouse uterus extracts at a dilution of 1:556 were prepared as described in Materials and methods and incubated at 30°C for 45 s with 687 nM 32P-labeled pCPI-17 plus the indicated concentrations of …

https://doi.org/10.7554/eLife.24665.012
Figure 7—figure supplement 1
Okadaic acid inhibition of the dephosphorylation of pCPI-17 by dilute mouse uterus extracts.

This experiment is identical to that shown in Figure 7 except that the mouse uterus extracts were diluted 1:278 and incubated at 30°C for 30 s with 32P-labeled pCPI-17 plus the indicated …

https://doi.org/10.7554/eLife.24665.013
Figure 7—figure supplement 2
Kinetic analysis of the dephosphorylation of pCPI-17 by PPU.

Highly diluted uterus extract was incubated with the indicated concentrations of 32P-labeled pCPI-17 for either 30 s (30°C) or 3 min (0°C) in 4 μl reactions. Values and standard deviations are for …

https://doi.org/10.7554/eLife.24665.014
Figure 8 with 2 supplements
Sequestration of pCPI-17 by MLCP in concentrated mouse uterus extracts.

Undiluted mouse uterus extracts (~14 mg/ml) were assayed for phosphatase activity using 32P-labeled pCPI-17 (0°C) or pC-ERMAD (30°C) substrates at the concentrations shown with the indicated …

https://doi.org/10.7554/eLife.24665.015
Figure 8—figure supplement 1
Dephosphorylation of pC-ERMAD by mouse uterus extracts.

Highly diluted (1:740) mouse extracts were incubated with 1.05 μM 32P-labeled pC-ERMAD and the indicated concentrations of okadaic acid at 30°C for 4 min. Values (n = 3) are presented as a …

https://doi.org/10.7554/eLife.24665.016
Figure 8—figure supplement 2
Determination of the ‘strength’ of competing phosphatase substrates and inhibitors in mouse uterus extracts.

Mouse embryo extracts were assayed through the range of indicated concentrations (1 = undiluted concentrated extract) for their ability to dephosphorylate either (A) 1.25 μM 32P-pCPI-17 or (B) 0.89 …

https://doi.org/10.7554/eLife.24665.017
Figure 9 with 1 supplement
Comparing models for aorta relaxation upon application of vasodilator.

Black points with error bars reproduce data from Figure 4 in (Kitazawa et al., 2009) describing the temporal variation in the levels of (A) CPI-17 and (C) MRLC phosphorylation following the …

https://doi.org/10.7554/eLife.24665.018
Figure 9—figure supplement 1
Effect of ROCK thiophosphorylation on MLCP activity.

MLCP (0.5 nM) was thiophosphorylated with 2.5 nM ROCK for 90 min at 30°C, and then standard phosphatase assays were conducted with 1 μM pC-ERMAD or 100 nM pCPI-17 substrates for 20 min at 30°C. Data …

https://doi.org/10.7554/eLife.24665.019

Tables

Table 1

Phosphatase and substrate constants and concentrations.

https://doi.org/10.7554/eLife.24665.009
PhosphataseSubstrate/
Inhibitor
ConstantValueSource figure or (reference)
pCPI-17([CPI-17]+[pCPI-17])phys7 µM(Kitazawa and Kitazawa, 2012; Woodsome et al., 2001)
PhosphataseSubstrate/
Inhibitor
ConstantValueSource figure or (reference)
MLCP[MLCP]phys1 µM(Alessi et al., 1992; Shirazi et al., 1994)
pCPI-17Km0.48 ± 0.03 nMFigure 4
kcat0.06 ± 0.01 s−1Figure 2—figure supplement 1, Figures 3, 4 and 5
OAKi20 ± 2 nMFigure 4
calyculin AKi0.13–0.22 nMFigure 4
PhosphataseSubstrate/
Inhibitor
ConstantValueSource figure or (reference)
PP2A-B56αpCPI-17Km4.2 ± 0.5 µMFigure 6
kcat29 ± 2 s−1Figure 6
PhosphataseSubstrate/
Inhibitor
ConstantValueSource figure or (reference)
PPUa[PPU]aphys~1.5 µMFigure 8
pCPI-17Km15 ± 2 µMFigure 7—figure supplement 2
kcat*41 ± 6 s−1Figure 7, Figure 7—figure supplement 1 and Figure 8
OAKi<0.5 nMFigure 7 and Figure 7—figure supplement 1
PhosphataseSubstrate/
Inhibitor
ConstantValueSource figure or (reference)
PPUbpCPI-17Km≥15 µMFigure 7—figure supplement 2
kcat [PPU]bphys/Km~0.7 s−1Figure 7 and Figure 7—figure supplement 1
OAKi~450 nMFigure 7 and Figure 7—figure supplement 1
  1. All parameters are at 30°C.

  2. phys: Physiological total (bound plus unbound) concentration; these values are computed using an estimated physiological total protein concentration of 170 mg/ml (Wiśniewski et al., 2014).

  3. PPUa is the PP2A-like component of PPU; it contributes 85% of the pCPI-17-directed PPU phosphatase activity. PPUb is the minor OA-resistant component.

  4. *Computed from kcat [PPU]a/M/Km, [PPU]a/M, and K(see Table 2).

Table 2

Additional phosphatase constants.

https://doi.org/10.7554/eLife.24665.010
PhosphataseSubstrateConstantValueSource figure or (reference)
MLCPΣphys*~15Figure 8—figure supplement 2
pMyBPKm~1.7 μMFigure 2—figure supplement 1
kcat~0.18 s−1Figure 2—figure supplement 1
pC-ERMADKm>2.5 μMFigure 2—figure supplement 1
kcat /Km2.6 ± 0.03 × 10−4 nM−1 s−1Figure 2—figure supplement 1
pMRLCKm~16 μM(Kawano et al., 1999)
PhosphataseSubstrateConstantValueSource figure or (reference)
PPUΣphys*~24Figure 8—figure supplement 2
pCPI-17Km15 ± 2 μMFigure 7—figure supplement 2
Km(0°C)14 ± 3 μMFigure 7—figure supplement 2
PhosphataseSubstrateConstantValueSource figure or (reference)
PPUapCPI-17kcat (30°C)/kcat (0°C)8.3 ± 0.8Figure 7—figure supplement 1
kcat [PPU]a/M/Km0.024 ± 0.001 s−1 ml mg−1Figure 7 and Figure 7—figure supplement 1
[PPU]a/M8.9 ± 0.9 pmol/mgFigure 8
PhosphataseSubstrateConstantValueSource figure or (reference)
PPUbpCPI-17kcat [PPU]b/M/Km0.0045 ± 0.0002 s−1 ml mg−1Figure 7 and Figure 7—figure supplement 1
  1. All parameters are at 30°C except as specified.

  2. phys: Physiological total (bound plus unbound) concentration; these values are computed using an estimated physiological total protein concentration of 170 mg/ml (Wiśniewski et al., 2014).

  3. M: total extract protein concentration

  4. Σ: Competition factor from other substrates (see Supplementary file 1)

Additional files

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