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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 RLC phospho-states in human uterine myocyte lysates.A. WBs produced by separation of proteins in lysates from unstimulated uterine myocytes by traditional SDS-PAGE. Single lanes were loaded with ∼25 µg of protein/lane. Abs directed against the C- terminus of total-RLC (CtRLC), phospho-S19-RLC (p19RLC), and diphospho-T18/S19-RLC (p18p19RLC) identify a single prominent band of 20 kDa. B. WBs produced after Mn2+-phos-tag SDS-PAGE. CtRLC, p19RLC, and p18p19RLC Abs identified three, two, and one specific band(s), respectively. The lower, middle, and upper bands in these blots correspond to non-, mono-, and di-phosphorylated RLC, denoted as 0pRLC, 1pRLC, and 2pRLC.
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pone-0020903-g001: Demonstration of RLC phospho-states in human uterine myocyte lysates.A. WBs produced by separation of proteins in lysates from unstimulated uterine myocytes by traditional SDS-PAGE. Single lanes were loaded with ∼25 µg of protein/lane. Abs directed against the C- terminus of total-RLC (CtRLC), phospho-S19-RLC (p19RLC), and diphospho-T18/S19-RLC (p18p19RLC) identify a single prominent band of 20 kDa. B. WBs produced after Mn2+-phos-tag SDS-PAGE. CtRLC, p19RLC, and p18p19RLC Abs identified three, two, and one specific band(s), respectively. The lower, middle, and upper bands in these blots correspond to non-, mono-, and di-phosphorylated RLC, denoted as 0pRLC, 1pRLC, and 2pRLC.

Mentions: Several studies have shown that three distinct phospho-states of RLC are detectable in SM preparations [5], [18]–[25]. These three phosphoisotypes most likely correspond to unphosphorylated, mono-phosphorylated (S19), and diphosphorylated (T18/S19) RLC. To ensure preservation of phospho-modifications during cell lysis, we harvested total cellular proteins by trichloroacetate precipitation (see methods). After traditional SDS-PAGE separation, Abs directed toward the C-terminus of total-RLC (CtRLC, no phospho-specificity), phospho-S19-RLC (p19RLC), and phospho-T18/S19-RLC (p18p19RLC) recognize only a single band of 20 kDa on WB replica membranes (Figure 1A). In contrast, when uterine myocyte protein lysates are separated by Mn2+-phos-tag SDS-PAGE these three Abs yield distinct banding patterns. The CtRLC Ab (Figure 1B) produces three distinct bands in lysates from unstimulated uterine myocytes. These data do not provide information regarding the phosphoisotypes contained within each band. Thus, we have chosen ‘0pRLC’, ‘1pRLC’, and ‘2pRLC’ as the terminology to emphasize phosphorylation stoichiometry irrespective of the phosphoisotypes represented.


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 RLC phospho-states in human uterine myocyte lysates.A. WBs produced by separation of proteins in lysates from unstimulated uterine myocytes by traditional SDS-PAGE. Single lanes were loaded with ∼25 µg of protein/lane. Abs directed against the C- terminus of total-RLC (CtRLC), phospho-S19-RLC (p19RLC), and diphospho-T18/S19-RLC (p18p19RLC) identify a single prominent band of 20 kDa. B. WBs produced after Mn2+-phos-tag SDS-PAGE. CtRLC, p19RLC, and p18p19RLC Abs identified three, two, and one specific band(s), respectively. The lower, middle, and upper bands in these blots correspond to non-, mono-, and di-phosphorylated RLC, denoted as 0pRLC, 1pRLC, and 2pRLC.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3111472&req=5

pone-0020903-g001: Demonstration of RLC phospho-states in human uterine myocyte lysates.A. WBs produced by separation of proteins in lysates from unstimulated uterine myocytes by traditional SDS-PAGE. Single lanes were loaded with ∼25 µg of protein/lane. Abs directed against the C- terminus of total-RLC (CtRLC), phospho-S19-RLC (p19RLC), and diphospho-T18/S19-RLC (p18p19RLC) identify a single prominent band of 20 kDa. B. WBs produced after Mn2+-phos-tag SDS-PAGE. CtRLC, p19RLC, and p18p19RLC Abs identified three, two, and one specific band(s), respectively. The lower, middle, and upper bands in these blots correspond to non-, mono-, and di-phosphorylated RLC, denoted as 0pRLC, 1pRLC, and 2pRLC.
Mentions: Several studies have shown that three distinct phospho-states of RLC are detectable in SM preparations [5], [18]–[25]. These three phosphoisotypes most likely correspond to unphosphorylated, mono-phosphorylated (S19), and diphosphorylated (T18/S19) RLC. To ensure preservation of phospho-modifications during cell lysis, we harvested total cellular proteins by trichloroacetate precipitation (see methods). After traditional SDS-PAGE separation, Abs directed toward the C-terminus of total-RLC (CtRLC, no phospho-specificity), phospho-S19-RLC (p19RLC), and phospho-T18/S19-RLC (p18p19RLC) recognize only a single band of 20 kDa on WB replica membranes (Figure 1A). In contrast, when uterine myocyte protein lysates are separated by Mn2+-phos-tag SDS-PAGE these three Abs yield distinct banding patterns. The CtRLC Ab (Figure 1B) produces three distinct bands in lysates from unstimulated uterine myocytes. These data do not provide information regarding the phosphoisotypes contained within each band. Thus, we have chosen ‘0pRLC’, ‘1pRLC’, and ‘2pRLC’ as the terminology to emphasize phosphorylation stoichiometry irrespective of the phosphoisotypes represented.

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