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A canonical EF-Loop directs Ca2+-sensitivity in phospholipase C-η2
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Popovics, Petra, Lu, Jin, Nadia Kamil, L., Morgan, Kevin, Millar, Robert P., Schmid, Ralf, Blindauer, Claudia A. and Stewart, Alan J. (2014) A canonical EF-Loop directs Ca2+-sensitivity in phospholipase C-η2. Journal of Cellular Biochemistry , Volume 115 (Number 3). pp. 557-565. doi:10.1002/jcb.24690 ISSN 0730-2312.
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Official URL: http://dx.doi.org/10.1002/jcb.24690
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. J. Cell. Biochem. 115: 557–565, 2014. © 2013 Wiley Periodicals, Inc.
Item Type: | Journal Article | ||||||||||
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Divisions: | Faculty of Science, Engineering and Medicine > Science > Chemistry | ||||||||||
Journal or Publication Title: | Journal of Cellular Biochemistry | ||||||||||
Publisher: | Wiley-Blackwell Publishing Ltd. | ||||||||||
ISSN: | 0730-2312 | ||||||||||
Official Date: | March 2014 | ||||||||||
Dates: |
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Volume: | Volume 115 | ||||||||||
Number: | Number 3 | ||||||||||
Page Range: | pp. 557-565 | ||||||||||
DOI: | 10.1002/jcb.24690 | ||||||||||
Status: | Peer Reviewed | ||||||||||
Publication Status: | Published | ||||||||||
Access rights to Published version: | Restricted or Subscription Access |
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