(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 …
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 …
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 …
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 …
This figure displays the same data as Figure 3, but with a different scale on the x-axis to better highlight the earliest timepoints.
(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 …
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 …
(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 …
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 …
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 …
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 …
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 …
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 …
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 …
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 …
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 …
Phosphatase and substrate constants and concentrations.
Phosphatase | Substrate/ Inhibitor | Constant | Value | Source figure or (reference) |
---|---|---|---|---|
pCPI-17 | ([CPI-17]+[pCPI-17])phys | 7 µM | (Kitazawa and Kitazawa, 2012; Woodsome et al., 2001) |
Phosphatase | Substrate/ Inhibitor | Constant | Value | Source figure or (reference) |
---|---|---|---|---|
MLCP | [MLCP]phys | 1 µM | (Alessi et al., 1992; Shirazi et al., 1994) | |
pCPI-17 | Km | 0.48 ± 0.03 nM | Figure 4 | |
kcat | 0.06 ± 0.01 s−1 | Figure 2—figure supplement 1, Figures 3, 4 and 5 | ||
OA | Ki | 20 ± 2 nM | Figure 4 | |
calyculin A | Ki | 0.13–0.22 nM | Figure 4 |
Phosphatase | Substrate/ Inhibitor | Constant | Value | Source figure or (reference) |
---|---|---|---|---|
PP2A-B56α | pCPI-17 | Km | 4.2 ± 0.5 µM | Figure 6 |
kcat | 29 ± 2 s−1 | Figure 6 |
Phosphatase | Substrate/ Inhibitor | Constant | Value | Source figure or (reference) |
---|---|---|---|---|
PPUa | [PPU]aphys | ~1.5 µM | Figure 8 | |
pCPI-17 | Km | 15 ± 2 µM | Figure 7—figure supplement 2 | |
kcat* | 41 ± 6 s−1 | Figure 7, Figure 7—figure supplement 1 and Figure 8 | ||
OA | Ki | <0.5 nM | Figure 7 and Figure 7—figure supplement 1 |
Phosphatase | Substrate/ Inhibitor | Constant | Value | Source figure or (reference) |
---|---|---|---|---|
PPUb | pCPI-17 | Km | ≥15 µM | Figure 7—figure supplement 2 |
kcat [PPU]bphys/Km | ~0.7 s−1 | Figure 7 and Figure 7—figure supplement 1 | ||
OA | Ki | ~450 nM | Figure 7 and Figure 7—figure supplement 1 |
All parameters are at 30°C.
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).
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.
*Computed from kcat [PPU]a/M/Km, [PPU]a/M, and Km (see Table 2).
Additional phosphatase constants.
Phosphatase | Substrate | Constant | Value | Source figure or (reference) |
---|---|---|---|---|
MLCP | Σphys* | ~15 | Figure 8—figure supplement 2 | |
pMyBP | Km | ~1.7 μM | Figure 2—figure supplement 1 | |
kcat | ~0.18 s−1 | Figure 2—figure supplement 1 | ||
pC-ERMAD | Km | >2.5 μM | Figure 2—figure supplement 1 | |
kcat /Km | 2.6 ± 0.03 × 10−4 nM−1 s−1 | Figure 2—figure supplement 1 | ||
pMRLC | Km | ~16 μM | (Kawano et al., 1999) |
Phosphatase | Substrate | Constant | Value | Source figure or (reference) |
---|---|---|---|---|
PPU | Σphys* | ~24 | Figure 8—figure supplement 2 | |
pCPI-17 | Km | 15 ± 2 μM | Figure 7—figure supplement 2 | |
Km(0°C) | 14 ± 3 μM | Figure 7—figure supplement 2 |
Phosphatase | Substrate | Constant | Value | Source figure or (reference) |
---|---|---|---|---|
PPUa | pCPI-17 | kcat (30°C)/kcat (0°C) | 8.3 ± 0.8 | Figure 7—figure supplement 1 |
kcat [PPU]a/M/Km | 0.024 ± 0.001 s−1 ml mg−1 | Figure 7 and Figure 7—figure supplement 1 | ||
[PPU]a/M | 8.9 ± 0.9 pmol/mg | Figure 8 |
Phosphatase | Substrate | Constant | Value | Source figure or (reference) |
---|---|---|---|---|
PPUb | pCPI-17 | kcat [PPU]b/M/Km | 0.0045 ± 0.0002 s−1 ml mg−1 | Figure 7 and Figure 7—figure supplement 1 |
All parameters are at 30°C except as specified.
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).
M: total extract protein concentration
Σ: Competition factor from other substrates (see Supplementary file 1)
Theoretical methods.