A Canonical EF‐Loop Directs Ca2+‐Sensitivity in Phospholipase C‐η2. Issue 3 (March 2014)

• Record Type: Journal Article
• Authors: Popovics, Petra; Lu, Jin; Nadia Kamil, L.; Morgan, Kevin; Millar, Robert P.; Schmid, Ralf; Blindauer, Claudia A.; Stewart, Alan J.
• Is Part Of: Journal of cellular biochemistry. Volume 115:Issue 3(2014:Mar.)
• Page Start: 557
• Page End: 565
• Publication Date: 2014-03
• Subjects: Cytochemistry -- Periodicals; 572
• DOI: 10.1002/jcb.24690
• Languages: English
• ISSNs: 0730-2312

Abstract

<abstract abstract-type="main" xml:lang="en"> <title>ABSTRACT</title> <sec id="jcb24690-sec-0001" sec-type="section"> <p>Phospholipase C‐η (PLCη) enzymes are a class of phosphatidylinositol 4, 5‐bisphosphate‐hydrolyzing enzymes involved in intracellular signaling. PLCη2 can sense Ca<sup>2+</sup> (stimulated by ∼1 µM free Ca<sup>2+</sup>) suggesting that it can amplify transient Ca<sup>2+</sup> 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). Ca<sup>2+</sup>‐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‐[<sup>1</sup>H, <sup>1</sup>H] TOCSY NMR. Both PLCη2 EF‐loop peptides bound Ca<sup>2+</sup> 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 Ca<sup>2+</sup>‐binding. To determine whether the EF‐hand is responsible for Ca<sup>2+</sup>‐sensing, inositol phosphate accumulation was measured in COS7 cells transiently expressing wild‐type or mutant PLCη2 proteins. Addition of 70 µM monensin (a Na<sup>+</sup>/H<sup>+</sup> antiporter that increases intracellular Ca<sup>2+</sup>) 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 Ca<sup>2+</sup>. The D256A (EF‐loop1) mutant exhibited a ∼10‐fold reduction in Ca<sup>2+</sup>‐sensitivity and was not activated by monensin, highlighting the involvement of EF‐loop 1 in Ca<sup>2+</sup>‐sensing. Involvement of EF‐loop 2 was examined using D292A, H296A, Q297A, and E304A mutants. Interestingly, the monensin responses and Ca<sup>2+</sup>‐sensitivities were largely unaffected by the mutations, indicating that the non‐canonical EF‐loop 2 is not involved in Ca<sup>2+</sup>‐sensing. J. Cell. Biochem. 115: 557–565, 2014. © 2013 Wiley Periodicals, Inc.</p> </sec> </abstract>