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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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PMA induces phosphorylation of RLC at S1 and alters mobility of phospho-RLC during phos-tag SDS-PAGE.A. WBs produced by Mn2+-phos-tag separation of lysates from uterine myocytes treated with PMA (1 µM) or vehicle. The membranes were probed with an Ab directed against the C- terminus of total-RLC (CtRLC). 0pRLC, 1pRLC, and 2pRLC correspond to non-, mono-, and di-phosphorylated RLC. The bands labeled with ** exhibit unexpected mobility in the Mn2+-phos-tag acrylamide matrix. B. Traditional WBs demonstrating phosphorylation of RLC at S1 (p1RLC) in uterine myocytes treated with PMA. C. Quantification of p1RLC concentrations in uterine myocytes treated with PMA and 4-α-PMA (negative control) by the in-cell western method. In B and C, the data represent 4 independent experiments and are presented as means ± SEM.
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pone-0020903-g006: PMA induces phosphorylation of RLC at S1 and alters mobility of phospho-RLC during phos-tag SDS-PAGE.A. WBs produced by Mn2+-phos-tag separation of lysates from uterine myocytes treated with PMA (1 µM) or vehicle. The membranes were probed with an Ab directed against the C- terminus of total-RLC (CtRLC). 0pRLC, 1pRLC, and 2pRLC correspond to non-, mono-, and di-phosphorylated RLC. The bands labeled with ** exhibit unexpected mobility in the Mn2+-phos-tag acrylamide matrix. B. Traditional WBs demonstrating phosphorylation of RLC at S1 (p1RLC) in uterine myocytes treated with PMA. C. Quantification of p1RLC concentrations in uterine myocytes treated with PMA and 4-α-PMA (negative control) by the in-cell western method. In B and C, the data represent 4 independent experiments and are presented as means ± SEM.

Mentions: Figure 6A demonstrates the separation of lysates treated with PMA (1 µM, 30 min) by Mn2+-phos-tag SDS-PAGE. We noted the appearance of three bands in at positions slightly above those we previously identified as 0pRLC, 1pRLC and 2pRLC, such that the CtRLC Ab cross-reacted with six distinct bands. We hypothesized that these unidentified bands might correspond to phosphoisotypes of RLC that exhibit altered mobility in the Mn2+-phos-tag matrix. As such, these bands might represent 1pRLC, 2pRLC, and 3pRLC corresponding to phosphoisotypes modified at a site other than T18 or S19. However, without knowledge of the specific phosphoisotypes corresponding to these intermediate bands, the quantitative information provided by the preceding analysis is difficult to interpret. Thus, instead we turned our attention to identifying a candidate phosphorylation site that might account for these additional phosphoisotypes.


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)

PMA induces phosphorylation of RLC at S1 and alters mobility of phospho-RLC during phos-tag SDS-PAGE.A. WBs produced by Mn2+-phos-tag separation of lysates from uterine myocytes treated with PMA (1 µM) or vehicle. The membranes were probed with an Ab directed against the C- terminus of total-RLC (CtRLC). 0pRLC, 1pRLC, and 2pRLC correspond to non-, mono-, and di-phosphorylated RLC. The bands labeled with ** exhibit unexpected mobility in the Mn2+-phos-tag acrylamide matrix. B. Traditional WBs demonstrating phosphorylation of RLC at S1 (p1RLC) in uterine myocytes treated with PMA. C. Quantification of p1RLC concentrations in uterine myocytes treated with PMA and 4-α-PMA (negative control) by the in-cell western method. In B and C, the data represent 4 independent experiments and are presented as means ± SEM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3111472&req=5

pone-0020903-g006: PMA induces phosphorylation of RLC at S1 and alters mobility of phospho-RLC during phos-tag SDS-PAGE.A. WBs produced by Mn2+-phos-tag separation of lysates from uterine myocytes treated with PMA (1 µM) or vehicle. The membranes were probed with an Ab directed against the C- terminus of total-RLC (CtRLC). 0pRLC, 1pRLC, and 2pRLC correspond to non-, mono-, and di-phosphorylated RLC. The bands labeled with ** exhibit unexpected mobility in the Mn2+-phos-tag acrylamide matrix. B. Traditional WBs demonstrating phosphorylation of RLC at S1 (p1RLC) in uterine myocytes treated with PMA. C. Quantification of p1RLC concentrations in uterine myocytes treated with PMA and 4-α-PMA (negative control) by the in-cell western method. In B and C, the data represent 4 independent experiments and are presented as means ± SEM.
Mentions: Figure 6A demonstrates the separation of lysates treated with PMA (1 µM, 30 min) by Mn2+-phos-tag SDS-PAGE. We noted the appearance of three bands in at positions slightly above those we previously identified as 0pRLC, 1pRLC and 2pRLC, such that the CtRLC Ab cross-reacted with six distinct bands. We hypothesized that these unidentified bands might correspond to phosphoisotypes of RLC that exhibit altered mobility in the Mn2+-phos-tag matrix. As such, these bands might represent 1pRLC, 2pRLC, and 3pRLC corresponding to phosphoisotypes modified at a site other than T18 or S19. However, without knowledge of the specific phosphoisotypes corresponding to these intermediate bands, the quantitative information provided by the preceding analysis is difficult to interpret. Thus, instead we turned our attention to identifying a candidate phosphorylation site that might account for these additional phosphoisotypes.

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