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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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Advanced phos-tag analysis of phosphorylated RLC in PMA-treated uterine myocytes.A and B. WBs produced by Zn2+-phos-tag (40 µM) separation of lysates from untreated samples, or samples treated with PMA (1 µM), CalyculinA (20 nM), or with both. In both A and B, the red signal corresponds to a mouse monoclonal Ab (MMAb) targeting p19RLC that is detected independently of the green signals (CtRLC in panel A, and p1RLC in panel B). A yellow signal indicates overlap of the individual green and red signals. 0pRLC, 1pRLC, 2pRLC and 3pRLC denote non-, mono-, di-, and tri-phosphorylated RLC. The superscripts denote the position of the phospho-modification: 1: S1, 18: T18, 19: S19. The unlabeled arrow below 3pRLC1/18/19 corresponds to 2pRLC18/19 that is particularly prominent in lysates treated with CalyculinA alone.
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pone-0020903-g007: Advanced phos-tag analysis of phosphorylated RLC in PMA-treated uterine myocytes.A and B. WBs produced by Zn2+-phos-tag (40 µM) separation of lysates from untreated samples, or samples treated with PMA (1 µM), CalyculinA (20 nM), or with both. In both A and B, the red signal corresponds to a mouse monoclonal Ab (MMAb) targeting p19RLC that is detected independently of the green signals (CtRLC in panel A, and p1RLC in panel B). A yellow signal indicates overlap of the individual green and red signals. 0pRLC, 1pRLC, 2pRLC and 3pRLC denote non-, mono-, di-, and tri-phosphorylated RLC. The superscripts denote the position of the phospho-modification: 1: S1, 18: T18, 19: S19. The unlabeled arrow below 3pRLC1/18/19 corresponds to 2pRLC18/19 that is particularly prominent in lysates treated with CalyculinA alone.

Mentions: Thus far, we have focused on the alkaline Mn2+-phos-tag method. Subsequent attempts to improve resolution of the unidentified bands from 0pRLC, 1pRLC and 2pRLC by Mn2+-phos-tag were inconsistent. Recently, an improved method of separation using Zn2+ instead of Mn2+ ions in the phos-tag matrix and an alternate buffer set for electrophoresis was developed [42]. We used this advanced Zn2+-phos-tag method to study the PMA-induced changes in the RLC phospho-states. In order to detect as many phospho-states as possible, we studied the effects of PMA and of the protein phosphatase 1 and 2A inhibitor, calyculin A. To elucidate the phosphoisotypes contained within each phospho-state, we used the MMAb directed toward p19RLC since it is detected independently using a secondary Ab directed towards murine primary Ab whereas the CtRLC and p1RLC (both RPAbs) are detected using a secondary Ab directed towards rabbit polyclonal Abs. Figure 7A demonstrates that the CtRLC Ab recognizes more than 3 bands in the presence of PMA, whereas phosphatase inhibition with calyculin A enhances 1pRLC and 2pRLC, but does not create any additional phospho-states. When uterine myocytes are challenged with both PMA and calyculin A, a band corresponding to a 3pRLC state can be discerned, and most likely corresponds to RLC phosphorylated at S1, T18, and S19. Figure 7B shows that the intermediate bands produced by PMA with or without calyculin A contain p1RLC, and suggests that phosphoisotypes of RLC containing phospho-S1 move more rapidly through the phos-tag matrices than those containing phospho-S19 with equivalent phosphate stoichiometries. They also suggest that there exists a 3pRLC state corresponding to phospho-S1/T18/S19-RLC that is inducible with PMA treatment. Thus, it appears that S1, T18, and T19 can exist simultaneously, rather than in mutual exclusivity to one another. However, we cannot exclude the possibility that sites other than S1, T18, and S19 have been induced by PMA, in the absence of other phospho-site-specific Abs. To clarify this possibility, future studies might benefit from the use of phos-tag SDS-PAGE in combination with other techniques such as mass spectrometry [43].


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)

Advanced phos-tag analysis of phosphorylated RLC in PMA-treated uterine myocytes.A and B. WBs produced by Zn2+-phos-tag (40 µM) separation of lysates from untreated samples, or samples treated with PMA (1 µM), CalyculinA (20 nM), or with both. In both A and B, the red signal corresponds to a mouse monoclonal Ab (MMAb) targeting p19RLC that is detected independently of the green signals (CtRLC in panel A, and p1RLC in panel B). A yellow signal indicates overlap of the individual green and red signals. 0pRLC, 1pRLC, 2pRLC and 3pRLC denote non-, mono-, di-, and tri-phosphorylated RLC. The superscripts denote the position of the phospho-modification: 1: S1, 18: T18, 19: S19. The unlabeled arrow below 3pRLC1/18/19 corresponds to 2pRLC18/19 that is particularly prominent in lysates treated with CalyculinA alone.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3111472&req=5

pone-0020903-g007: Advanced phos-tag analysis of phosphorylated RLC in PMA-treated uterine myocytes.A and B. WBs produced by Zn2+-phos-tag (40 µM) separation of lysates from untreated samples, or samples treated with PMA (1 µM), CalyculinA (20 nM), or with both. In both A and B, the red signal corresponds to a mouse monoclonal Ab (MMAb) targeting p19RLC that is detected independently of the green signals (CtRLC in panel A, and p1RLC in panel B). A yellow signal indicates overlap of the individual green and red signals. 0pRLC, 1pRLC, 2pRLC and 3pRLC denote non-, mono-, di-, and tri-phosphorylated RLC. The superscripts denote the position of the phospho-modification: 1: S1, 18: T18, 19: S19. The unlabeled arrow below 3pRLC1/18/19 corresponds to 2pRLC18/19 that is particularly prominent in lysates treated with CalyculinA alone.
Mentions: Thus far, we have focused on the alkaline Mn2+-phos-tag method. Subsequent attempts to improve resolution of the unidentified bands from 0pRLC, 1pRLC and 2pRLC by Mn2+-phos-tag were inconsistent. Recently, an improved method of separation using Zn2+ instead of Mn2+ ions in the phos-tag matrix and an alternate buffer set for electrophoresis was developed [42]. We used this advanced Zn2+-phos-tag method to study the PMA-induced changes in the RLC phospho-states. In order to detect as many phospho-states as possible, we studied the effects of PMA and of the protein phosphatase 1 and 2A inhibitor, calyculin A. To elucidate the phosphoisotypes contained within each phospho-state, we used the MMAb directed toward p19RLC since it is detected independently using a secondary Ab directed towards murine primary Ab whereas the CtRLC and p1RLC (both RPAbs) are detected using a secondary Ab directed towards rabbit polyclonal Abs. Figure 7A demonstrates that the CtRLC Ab recognizes more than 3 bands in the presence of PMA, whereas phosphatase inhibition with calyculin A enhances 1pRLC and 2pRLC, but does not create any additional phospho-states. When uterine myocytes are challenged with both PMA and calyculin A, a band corresponding to a 3pRLC state can be discerned, and most likely corresponds to RLC phosphorylated at S1, T18, and S19. Figure 7B shows that the intermediate bands produced by PMA with or without calyculin A contain p1RLC, and suggests that phosphoisotypes of RLC containing phospho-S1 move more rapidly through the phos-tag matrices than those containing phospho-S19 with equivalent phosphate stoichiometries. They also suggest that there exists a 3pRLC state corresponding to phospho-S1/T18/S19-RLC that is inducible with PMA treatment. Thus, it appears that S1, T18, and T19 can exist simultaneously, rather than in mutual exclusivity to one another. However, we cannot exclude the possibility that sites other than S1, T18, and S19 have been induced by PMA, in the absence of other phospho-site-specific Abs. To clarify this possibility, future studies might benefit from the use of phos-tag SDS-PAGE in combination with other techniques such as mass spectrometry [43].

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