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Phos-tag-based analysis of myosin regulatory light chain phosphorylation in human uterine myocytes.

Aguilar HN, Tracey CN, Tsang SC, McGinnis JM, Mitchell BF - PLoS ONE (2011)

Bottom Line: The method incorporates corrections for lane-to-lane loading variability and for the effects of drug vehicles thus enabling the comparison of multiple treatments by using the untreated cellular set-point as a reference.This analysis is useful for assessing effects of putative agonists and antagonists where all phospho-states are represented in control and experimental samples.We also demonstrated that phosphorylation of RLC at S1 is inducible in intact uterine myocytes, though the signal in the resting samples was not sufficiently abundant to allow quantification by the approach used here.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.

ABSTRACT

Background: The 'phosphate-binding tag' (phos-tag) reagent enables separation of phospho-proteins during SDS-PAGE by impeding migration proportional to their phosphorylation stoichiometry. Western blotting can then be used to detect and quantify the bands corresponding to the phospho-states of a target protein. We present a method for quantification of data regarding phospho-states derived from phos-tag SDS-PAGE. The method incorporates corrections for lane-to-lane loading variability and for the effects of drug vehicles thus enabling the comparison of multiple treatments by using the untreated cellular set-point as a reference. This method is exemplified by quantifying the phosphorylation of myosin regulatory light chain (RLC) in cultured human uterine myocytes.

Methodology/principal findings: We have evaluated and validated the concept that, when using an antibody (Ab) against the total-protein, the sum of all phosphorylation states in a single lane represents a 'closed system' since all possible phospho-states and phosphoisotypes are detected. Using this approach, we demonstrate that oxytocin (OT) and calpeptin (Calp) induce RLC kinase (MLCK)- and rho-kinase (ROK)-dependent enhancements in phosphorylation of RLC at T18 and S19. Treatment of myocytes with a phorbol ester (PMA) induced phosphorylation of S1-RLC, which caused a mobility shift in the phos-tag matrices distinct from phosphorylation at S19.

Conclusion/significance: We have presented a method for analysis of phospho-state data that facilitates quantitative comparison to a reference control without the use of a traditional 'loading' or 'reference' standard. This analysis is useful for assessing effects of putative agonists and antagonists where all phospho-states are represented in control and experimental samples. We also demonstrated that phosphorylation of RLC at S1 is inducible in intact uterine myocytes, though the signal in the resting samples was not sufficiently abundant to allow quantification by the approach used here.

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Demonstration of Phospho-S19-RLC phospho-state distribution in uterine myocytes.A. Detection of 0pRLC, 1pRLC and 2pRLC separated by Mn2+-phos-tag SDS-PAGE with an Ab toward the C-terminus of RLC (CtRLC) and two Abs (mouse MonoclonalAb [MMAb] and rabbit PolyclonalAb [RPAb]) directed toward phospho-S19-RLC. B. Representative WB demonstrating RLC phospho-states separated by Mn2+-phos-tag SDS-PAGE and probed using PRAb from panel A. Uterine myocytes were lysed and total protein was harvested after the following treatments: untreated, or treated with g-H, Calp, OT, or with their corresponding vehicles. C. Vehicle-corrected distribution data for 1pRLC19 and 2pRLC19 obtained by normalizing the data in panel A to the untreated group distribution. g-H (1 µM, n = 5). Calp (0.5 mU/mL, n = 10). OT (100 nM, n = 10). All data are shown as means ± SEMs. The corresponding numerical data are compiled in Table 2.
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pone-0020903-g005: Demonstration of Phospho-S19-RLC phospho-state distribution in uterine myocytes.A. Detection of 0pRLC, 1pRLC and 2pRLC separated by Mn2+-phos-tag SDS-PAGE with an Ab toward the C-terminus of RLC (CtRLC) and two Abs (mouse MonoclonalAb [MMAb] and rabbit PolyclonalAb [RPAb]) directed toward phospho-S19-RLC. B. Representative WB demonstrating RLC phospho-states separated by Mn2+-phos-tag SDS-PAGE and probed using PRAb from panel A. Uterine myocytes were lysed and total protein was harvested after the following treatments: untreated, or treated with g-H, Calp, OT, or with their corresponding vehicles. C. Vehicle-corrected distribution data for 1pRLC19 and 2pRLC19 obtained by normalizing the data in panel A to the untreated group distribution. g-H (1 µM, n = 5). Calp (0.5 mU/mL, n = 10). OT (100 nM, n = 10). All data are shown as means ± SEMs. The corresponding numerical data are compiled in Table 2.

Mentions: We next focused attention on the phosphoisotype composition of each of the phospho-states following Mn2+-phos-tag separation. In preliminary experiments, we validated the specificity of two anti-phospho-S19-RLC Abs by WB after Mn2+-phos-tag SDS-PAGE separation (Figure 5A). These Abs were derived from mice (monoclonal – ‘p19RLC (MMAb)’), or rabbits (polyclonal – ‘p19RLC (RPAb)’). The p19RLC (MMAb) was unable to detect the 2pRLC band present in blots probed with the CtRLC Ab and the p19RLC (RPAb). The most likely explanation for this finding is obstructed binding of the Ab in the presence of phospho-T18-RLC. Still, this Ab was useful for quantifying monophosphorylation of RLC specifically, as was demonstrated in Figure 2. Anti-total-protein Abs might also suffer from obstructed binding. Here, we selected the CtRLC Ab on the basis of published evidence suggesting phosphorylation of RLC at sites concentrated in the first 20 amino acids of the N-terminal region and the possibility of such interference was minimized [18]–[20]. We utilized the p19RLC (RPAb) to quantify phosphorylation of RLC at S19 in subsequent experiments.


Phos-tag-based analysis of myosin regulatory light chain phosphorylation in human uterine myocytes.

Aguilar HN, Tracey CN, Tsang SC, McGinnis JM, Mitchell BF - PLoS ONE (2011)

Demonstration of Phospho-S19-RLC phospho-state distribution in uterine myocytes.A. Detection of 0pRLC, 1pRLC and 2pRLC separated by Mn2+-phos-tag SDS-PAGE with an Ab toward the C-terminus of RLC (CtRLC) and two Abs (mouse MonoclonalAb [MMAb] and rabbit PolyclonalAb [RPAb]) directed toward phospho-S19-RLC. B. Representative WB demonstrating RLC phospho-states separated by Mn2+-phos-tag SDS-PAGE and probed using PRAb from panel A. Uterine myocytes were lysed and total protein was harvested after the following treatments: untreated, or treated with g-H, Calp, OT, or with their corresponding vehicles. C. Vehicle-corrected distribution data for 1pRLC19 and 2pRLC19 obtained by normalizing the data in panel A to the untreated group distribution. g-H (1 µM, n = 5). Calp (0.5 mU/mL, n = 10). OT (100 nM, n = 10). All data are shown as means ± SEMs. The corresponding numerical data are compiled in Table 2.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3111472&req=5

pone-0020903-g005: Demonstration of Phospho-S19-RLC phospho-state distribution in uterine myocytes.A. Detection of 0pRLC, 1pRLC and 2pRLC separated by Mn2+-phos-tag SDS-PAGE with an Ab toward the C-terminus of RLC (CtRLC) and two Abs (mouse MonoclonalAb [MMAb] and rabbit PolyclonalAb [RPAb]) directed toward phospho-S19-RLC. B. Representative WB demonstrating RLC phospho-states separated by Mn2+-phos-tag SDS-PAGE and probed using PRAb from panel A. Uterine myocytes were lysed and total protein was harvested after the following treatments: untreated, or treated with g-H, Calp, OT, or with their corresponding vehicles. C. Vehicle-corrected distribution data for 1pRLC19 and 2pRLC19 obtained by normalizing the data in panel A to the untreated group distribution. g-H (1 µM, n = 5). Calp (0.5 mU/mL, n = 10). OT (100 nM, n = 10). All data are shown as means ± SEMs. The corresponding numerical data are compiled in Table 2.
Mentions: We next focused attention on the phosphoisotype composition of each of the phospho-states following Mn2+-phos-tag separation. In preliminary experiments, we validated the specificity of two anti-phospho-S19-RLC Abs by WB after Mn2+-phos-tag SDS-PAGE separation (Figure 5A). These Abs were derived from mice (monoclonal – ‘p19RLC (MMAb)’), or rabbits (polyclonal – ‘p19RLC (RPAb)’). The p19RLC (MMAb) was unable to detect the 2pRLC band present in blots probed with the CtRLC Ab and the p19RLC (RPAb). The most likely explanation for this finding is obstructed binding of the Ab in the presence of phospho-T18-RLC. Still, this Ab was useful for quantifying monophosphorylation of RLC specifically, as was demonstrated in Figure 2. Anti-total-protein Abs might also suffer from obstructed binding. Here, we selected the CtRLC Ab on the basis of published evidence suggesting phosphorylation of RLC at sites concentrated in the first 20 amino acids of the N-terminal region and the possibility of such interference was minimized [18]–[20]. We utilized the p19RLC (RPAb) to quantify phosphorylation of RLC at S19 in subsequent experiments.

Bottom Line: The method incorporates corrections for lane-to-lane loading variability and for the effects of drug vehicles thus enabling the comparison of multiple treatments by using the untreated cellular set-point as a reference.This analysis is useful for assessing effects of putative agonists and antagonists where all phospho-states are represented in control and experimental samples.We also demonstrated that phosphorylation of RLC at S1 is inducible in intact uterine myocytes, though the signal in the resting samples was not sufficiently abundant to allow quantification by the approach used here.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.

ABSTRACT

Background: The 'phosphate-binding tag' (phos-tag) reagent enables separation of phospho-proteins during SDS-PAGE by impeding migration proportional to their phosphorylation stoichiometry. Western blotting can then be used to detect and quantify the bands corresponding to the phospho-states of a target protein. We present a method for quantification of data regarding phospho-states derived from phos-tag SDS-PAGE. The method incorporates corrections for lane-to-lane loading variability and for the effects of drug vehicles thus enabling the comparison of multiple treatments by using the untreated cellular set-point as a reference. This method is exemplified by quantifying the phosphorylation of myosin regulatory light chain (RLC) in cultured human uterine myocytes.

Methodology/principal findings: We have evaluated and validated the concept that, when using an antibody (Ab) against the total-protein, the sum of all phosphorylation states in a single lane represents a 'closed system' since all possible phospho-states and phosphoisotypes are detected. Using this approach, we demonstrate that oxytocin (OT) and calpeptin (Calp) induce RLC kinase (MLCK)- and rho-kinase (ROK)-dependent enhancements in phosphorylation of RLC at T18 and S19. Treatment of myocytes with a phorbol ester (PMA) induced phosphorylation of S1-RLC, which caused a mobility shift in the phos-tag matrices distinct from phosphorylation at S19.

Conclusion/significance: We have presented a method for analysis of phospho-state data that facilitates quantitative comparison to a reference control without the use of a traditional 'loading' or 'reference' standard. This analysis is useful for assessing effects of putative agonists and antagonists where all phospho-states are represented in control and experimental samples. We also demonstrated that phosphorylation of RLC at S1 is inducible in intact uterine myocytes, though the signal in the resting samples was not sufficiently abundant to allow quantification by the approach used here.

Show MeSH
Related in: MedlinePlus