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A canonical EF-loop directs Ca2+-sensitivity in phospholipase C-η2

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A canonical EF-loop directs Ca2+-sensitivity in phospholipase C-η2. / Popovics, Petra; Lu, Jin; Kamil, L Nadia; Morgan, Kevin; Millar, Robert P; Schmid, Ralf; Blindauer, Claudia A; Stewart, Alan J.

In: Journal of Cellular Biochemistry, Vol. 115, No. 3, 01.03.2014, p. 557–565.

Research output: Contribution to journalArticlepeer-review

Harvard

Popovics, P, Lu, J, Kamil, LN, Morgan, K, Millar, RP, Schmid, R, Blindauer, CA & Stewart, AJ 2014, 'A canonical EF-loop directs Ca2+-sensitivity in phospholipase C-η2', Journal of Cellular Biochemistry, vol. 115, no. 3, pp. 557–565. https://doi.org/10.1002/jcb.24690

APA

Popovics, P., Lu, J., Kamil, L. N., Morgan, K., Millar, R. P., Schmid, R., Blindauer, C. A., & Stewart, A. J. (2014). A canonical EF-loop directs Ca2+-sensitivity in phospholipase C-η2. Journal of Cellular Biochemistry, 115(3), 557–565. https://doi.org/10.1002/jcb.24690

Vancouver

Popovics P, Lu J, Kamil LN, Morgan K, Millar RP, Schmid R et al. A canonical EF-loop directs Ca2+-sensitivity in phospholipase C-η2. Journal of Cellular Biochemistry. 2014 Mar 1;115(3):557–565. https://doi.org/10.1002/jcb.24690

Author

Popovics, Petra ; Lu, Jin ; Kamil, L Nadia ; Morgan, Kevin ; Millar, Robert P ; Schmid, Ralf ; Blindauer, Claudia A ; Stewart, Alan J. / A canonical EF-loop directs Ca2+-sensitivity in phospholipase C-η2. In: Journal of Cellular Biochemistry. 2014 ; Vol. 115, No. 3. pp. 557–565.

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@article{d0cdcd1ed0ef4f1aa6dd2c01a0448915,
title = "A canonical EF-loop directs Ca2+-sensitivity in phospholipase C-η2",
abstract = "Phospholipase C-η (PLCη) enzymes are a class of phosphatidylinositol 4,5-bisphosphate-hydrolyzing enzymes involved in intracellular signaling. PLCη2 can sense Ca2+ (stimulated by ~1 μM free Ca2+) suggesting that it can amplify transient Ca2+ signals. PLCη enzymes possess an EF-hand domain composed of two EF-loops; a canonical 12-residue loop (EF-loop 1) and a non-canonical 13-residue loop (EF-loop 2). Ca2+-binding to synthetic peptides corresponding to EF-loops 1 and 2 of PLCη2 and EF-loop 1 of calmodulin (as a control) was examined by 2D-[1H,1H] TOCSY NMR. Both PLCη2 EF-loop peptides bound Ca2+ in a similar manner to that of the canonical calmodulin EF-loop 1, particularly at their N-terminus. A molecular model of the PLCη2 EF-hand domain, constructed based upon the structure of calmodulin, suggested both EF-loops may participate in Ca2+-binding. To determine whether the EF-hand is responsible for Ca2+-sensing, inositol phosphate accumulation was measured in COS7 cells transiently expressing wild-type or mutant PLCη2 proteins. Addition of 70 μM monensin (a Na+/H+ antiporter that increases intracellular Ca2+) induced a 4 to 7-fold increase in wild-type PLCη2 activity. In permeabilized cells, PLCη2 exhibited a ~4-fold increase in activity in the presence of 1 μM free Ca2+. The D256A (EF-loop1) mutant exhibited a ~10-fold reduction in Ca2+-sensitivity and was not activated by monensin, highlighting the involvement of EF-loop 1 in Ca2+-sensing. Involvement of EF-loop 2 was examined using D292A, H296A, Q297A and E304A mutants. Interestingly, the monensin responses and Ca2+-sensitivities were largely unaffected by the mutations, indicating that the non-canonical EF-loop 2 is not involved in Ca2+-sensing.",
keywords = "calcium, cell signaling, comparative modeling, EF-hand, phospholipase C, signal transduction",
author = "Petra Popovics and Jin Lu and Kamil, {L Nadia} and Kevin Morgan and Millar, {Robert P} and Ralf Schmid and Blindauer, {Claudia A} and Stewart, {Alan J.}",
note = "The authors acknowledge the University of St Andrews (PhD studentship to Petra Popovics), The Royal Society (Olga Kennard Fellowship to Claudia A. Blindauer), and the EPSRC and BP (Dorothy Hodgkin Fellowship to Jin Lu)",
year = "2014",
month = mar,
day = "1",
doi = "10.1002/jcb.24690",
language = "English",
volume = "115",
pages = "557–565",
journal = "Journal of Cellular Biochemistry",
issn = "0730-2312",
publisher = "Wiley-Liss",
number = "3",

}

RIS (suitable for import to EndNote) - Download

TY - JOUR

T1 - A canonical EF-loop directs Ca2+-sensitivity in phospholipase C-η2

AU - Popovics, Petra

AU - Lu, Jin

AU - Kamil, L Nadia

AU - Morgan, Kevin

AU - Millar, Robert P

AU - Schmid, Ralf

AU - Blindauer, Claudia A

AU - Stewart, Alan J.

N1 - The authors acknowledge the University of St Andrews (PhD studentship to Petra Popovics), The Royal Society (Olga Kennard Fellowship to Claudia A. Blindauer), and the EPSRC and BP (Dorothy Hodgkin Fellowship to Jin Lu)

PY - 2014/3/1

Y1 - 2014/3/1

N2 - Phospholipase C-η (PLCη) enzymes are a class of phosphatidylinositol 4,5-bisphosphate-hydrolyzing enzymes involved in intracellular signaling. PLCη2 can sense Ca2+ (stimulated by ~1 μM free Ca2+) suggesting that it can amplify transient Ca2+ signals. PLCη enzymes possess an EF-hand domain composed of two EF-loops; a canonical 12-residue loop (EF-loop 1) and a non-canonical 13-residue loop (EF-loop 2). Ca2+-binding to synthetic peptides corresponding to EF-loops 1 and 2 of PLCη2 and EF-loop 1 of calmodulin (as a control) was examined by 2D-[1H,1H] TOCSY NMR. Both PLCη2 EF-loop peptides bound Ca2+ in a similar manner to that of the canonical calmodulin EF-loop 1, particularly at their N-terminus. A molecular model of the PLCη2 EF-hand domain, constructed based upon the structure of calmodulin, suggested both EF-loops may participate in Ca2+-binding. To determine whether the EF-hand is responsible for Ca2+-sensing, inositol phosphate accumulation was measured in COS7 cells transiently expressing wild-type or mutant PLCη2 proteins. Addition of 70 μM monensin (a Na+/H+ antiporter that increases intracellular Ca2+) induced a 4 to 7-fold increase in wild-type PLCη2 activity. In permeabilized cells, PLCη2 exhibited a ~4-fold increase in activity in the presence of 1 μM free Ca2+. The D256A (EF-loop1) mutant exhibited a ~10-fold reduction in Ca2+-sensitivity and was not activated by monensin, highlighting the involvement of EF-loop 1 in Ca2+-sensing. Involvement of EF-loop 2 was examined using D292A, H296A, Q297A and E304A mutants. Interestingly, the monensin responses and Ca2+-sensitivities were largely unaffected by the mutations, indicating that the non-canonical EF-loop 2 is not involved in Ca2+-sensing.

AB - Phospholipase C-η (PLCη) enzymes are a class of phosphatidylinositol 4,5-bisphosphate-hydrolyzing enzymes involved in intracellular signaling. PLCη2 can sense Ca2+ (stimulated by ~1 μM free Ca2+) suggesting that it can amplify transient Ca2+ signals. PLCη enzymes possess an EF-hand domain composed of two EF-loops; a canonical 12-residue loop (EF-loop 1) and a non-canonical 13-residue loop (EF-loop 2). Ca2+-binding to synthetic peptides corresponding to EF-loops 1 and 2 of PLCη2 and EF-loop 1 of calmodulin (as a control) was examined by 2D-[1H,1H] TOCSY NMR. Both PLCη2 EF-loop peptides bound Ca2+ in a similar manner to that of the canonical calmodulin EF-loop 1, particularly at their N-terminus. A molecular model of the PLCη2 EF-hand domain, constructed based upon the structure of calmodulin, suggested both EF-loops may participate in Ca2+-binding. To determine whether the EF-hand is responsible for Ca2+-sensing, inositol phosphate accumulation was measured in COS7 cells transiently expressing wild-type or mutant PLCη2 proteins. Addition of 70 μM monensin (a Na+/H+ antiporter that increases intracellular Ca2+) induced a 4 to 7-fold increase in wild-type PLCη2 activity. In permeabilized cells, PLCη2 exhibited a ~4-fold increase in activity in the presence of 1 μM free Ca2+. The D256A (EF-loop1) mutant exhibited a ~10-fold reduction in Ca2+-sensitivity and was not activated by monensin, highlighting the involvement of EF-loop 1 in Ca2+-sensing. Involvement of EF-loop 2 was examined using D292A, H296A, Q297A and E304A mutants. Interestingly, the monensin responses and Ca2+-sensitivities were largely unaffected by the mutations, indicating that the non-canonical EF-loop 2 is not involved in Ca2+-sensing.

KW - calcium

KW - cell signaling

KW - comparative modeling

KW - EF-hand

KW - phospholipase C

KW - signal transduction

UR - http://onlinelibrary.wiley.com/doi/10.1002/jcb.24690/abstract

U2 - 10.1002/jcb.24690

DO - 10.1002/jcb.24690

M3 - Article

VL - 115

SP - 557

EP - 565

JO - Journal of Cellular Biochemistry

JF - Journal of Cellular Biochemistry

SN - 0730-2312

IS - 3

ER -

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