A Canonical EF‐Loop Directs Ca²⁺‐Sensitivity in Phospholipase C‐η2

Abstract

Phospholipase C-ɳ(PLCɳ) enzymes are a class of phosphatidylinositol 4,5-bisphosphatehydrolyzing enzymes involved in intracellular signaling. PLCɳ2 can sense Ca 2+ (stimulated bỹ 1 µM free Ca 2+ ) suggesting that it can amplify transient Ca 2+ signals. PLCɳ 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 2+ -binding to synthetic peptides corresponding to EF-loops 1 and 2 of PLCɳ2 and EF-loop 1 of calmodulin (as a control) was examined by 2D-[ 1 H, 1 H] TOCSY NMR. Both PLCɳ2 EF-loop peptides bound Ca 2+ in a similar manner to that of the canonical calmodulin EF-loop 1, particularly at their N-terminus. A molecular model of the PLCɳ2 EF-hand domain, constructed based upon the structure of calmodulin, suggested both EF-loops may participate in Ca 2+ -binding. To determine whether the EF-hand is responsible for Ca 2+ -sensing, inositol phosphate accumulation was measured in COS7 cells transiently expressing wild-type or mutant PLCɳ2 proteins. Addition of 70µM monensin (a Na + /H + antiporter that increases intracellular Ca 2+ ) induced a 4 to 7-fold increase in wild-type PLCɳ2 activity. In permeabilized cells, PLCn2 exhibited a ~4-fold increase in activity in the presence of 1 µM free Ca 2+ . The D256A (EF-loop1) mutant exhibited a ~10-fold reduction in Ca 2+ -sensitivity and was not activated by monensin, highlighting the involvement of EF-loop 1 in Ca 2+ -sensing. Involvement of EF-loop 2 was examined using D292A, H296A, Q297A and E304A mutants. Interestingly, the monensin responses and Ca 2+ -sensitivities were largely unaffected by the mutations, indicating that the non-canonical EF-loop 2 is not involved in Ca 2+ -sensing.

Chelation of Ca2+ ions by a peptide from the repeat region of the Plasmodium falciparum circumsporozoite protein

“…The magnitude of the determined Δδs is in agreement with those observed for other calcium-binding peptides [22,23]. Figure 2 indicates that calcium binding by 1 affects the signals for the protons in the C-terminal side of 1 , especially those of the D residues, which are found as participants in Ca 2+ coordination in many Ca-binding proteins [21].…”

Phospholipase C-η2 interacts with nuclear and cytoplasmic LIMK-1 during retinoic acid-stimulated neurite growth

“…Neuro2A cells stably transfected with empty vector only (EV) were used as a control. The D256A mutation has been demonstrated previously to abolish the ability of calcium to activate PLCη2 through perturbation of calcium binding at EF-loop 1 of the EF-hand domain (Popovics et al 2014 ). His460 corresponds to a key active site residue that is highly conserved within PLC isozymes (Heinz et al 1998 ), and mutation of the corresponding His residue in PLCδ1 (His356) has been shown to abolish the activity of this enzyme (Stallings et al 2005 ).…”

“…Samples were processed according to the Tokuyasu thawed frozen section method (Tokuyasu 1973 ). Briefly, Neuro2A cells stably expressing a PLCη2-targetted shRNA (PLCη2 KD) or non-target shRNA control (referred to as shRNAPLCη2-1 and shRNA control, respectively, in Popovics et al 2014 ) were grown to full confluency in a T-75 flask before fixation with 4 % p -formaldehyde, 0.05 % glutaraldehyde, buffered with 0.2 M PIPES, pH 7.2, for 15 min at ambient temperature. Cells were then scraped, collected and centrifuged at 16,000× g for 15 min to form a pellet before cryoprotection in 2.3 M sucrose in PBS (overnight at 4 °C).…”

“…This region is rich in serine and proline residues, and it is thought that this part of the protein may facilitate protein–protein interactions (Suh et al 2008 ). Both PLCη enzymes can be activated directly by mobilization of intracellular calcium (Kim et al 2011 ; Popovics et al 2011 , 2014 ). We recently reported that mutation of a putative calcium-binding residue (D256A) within EF-loop 1 of the EF-hand domain of PLCη2 reduces the sensitivity to calcium by tenfold, indicating this domain to be responsible for calcium-induced activation (Popovics et al 2014 ).…”

“…Both PLCη enzymes can be activated directly by mobilization of intracellular calcium (Kim et al 2011 ; Popovics et al 2011 , 2014 ). We recently reported that mutation of a putative calcium-binding residue (D256A) within EF-loop 1 of the EF-hand domain of PLCη2 reduces the sensitivity to calcium by tenfold, indicating this domain to be responsible for calcium-induced activation (Popovics et al 2014 ). In addition, it has also been shown that PLCη2, but not PLCη1, can be activated by Gβγ, which is released from trimeric G-protein complexes following G-protein-coupled receptor activation (Zhou et al 2005 , 2008 ).…”