Deletion and Site-Directed Mutagenesis of EF-Hand Domain of Phospholipase C-δ1: Effects on Its Activity

Nakashima, Shigeru; Banno, Yutaka; Watanabe, Teruhisa; Nakamura, Yoshikazu; Mizutani, Taku; Sakai, Hiroshi; Zhao, Yutong; Sugimoto, Yoshimi; Nozawa, Yoshinori

doi:10.1006/bbrc.1995.1822

Cited by 39 publications

(24 citation statements)

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“…The two N-terminal EF-hand domains, therefore, are considered to play a structural role to form the active conformation rather than a Ca 2ϩ -binding site for activation of the catalytic activity. Similarly, in PLC␦1, deletion of the N-terminal EF-hand domain markedly reduces the PLC activity, but point mutation of EF1 at the x, z, and Ϫz positions does not affect the PLC activity and the Ca 2ϩ sensitivity (24).…”

Section: The Role Of Ef-hand Domains Of Plc In Regulation Of Its Camentioning

confidence: 97%

The Role of EF-hand Domains and C2 Domain in Regulation of Enzymatic Activity of Phospholipase Cζ

Kouchi

Shikano

Nakamura

et al. 2005

Journal of Biological Chemistry

110

111

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). In the present structure-function analysis, deletion of EF1 and EF2 of the N-terminal four EF-hand domains caused marked reduction of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 )-hydrolyzing activity in vitro and loss of Ca 2؉ oscillation-inducing activity in mouse eggs after injection of RNA encoding the mutant. However, deletion of EF1 and EF2 or mutation of EF1 or EF2 at the x and z positions of the putative Ca 2؉ -binding loop little affected the Ca 2؉ sensitivity of the PLC activity, whereas deletion of EF1 to EF3 caused 12-fold elevation of the EC 50 of Ca 2؉ concentration. Thus, EF1 and EF2 are important for the PLC activity, and EF3 is responsible for its high Ca 2؉ sensitivity. Deletion of four EF-hand domains or the C-terminal C2 domain caused complete loss of PLC activity, indicating that both regions are prerequisites for PLC activity. Screening of interactions between the C2 domain and phosphoinositides revealed that C2 has substantial affinity to PI(3)P and, to the lesser extent, to PI(5)P but not to PI(4,5)P 2 or acidic phospholipids. PI(3)P and PI(5)P reduced PLC activity in vitro, suggesting that the interaction could play a role for negative regulation of PLC.

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Section: The Role Of Ef-hand Domains Of Plc In Regulation Of Its Camentioning

confidence: 97%

The Role of EF-hand Domains and C2 Domain in Regulation of Enzymatic Activity of Phospholipase Cζ

Kouchi

Shikano

Nakamura

et al. 2005

Journal of Biological Chemistry

110

111

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“…EF-hand motifs are helix-turnhelix structural domains which bind Ca 2＋ ions. It was shown that the deletion of EF-hand motifs of PLC-δ1 resulted in a decrease in PLC activity in a Ca 2＋ -independent manner (17,18). Although the EF-hand motif of PLC isozyme may have an important regulatory function, currently there is no strong evidence to support the notion that EF-hand motifs bind to metal ions.…”

Section: C2 Domain and Ef-handsmentioning

confidence: 99%

Multiple roles of phosphoinositide-specific phospholipase C isozymes

Suh¹,

Park²,

Manzoli³

et al. 2008

BMB Reports

447

482

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“…Althoughthe loop regions of EF-3 and EF-4 do not have side chains that would act as typical calcium ligands, EF-1 and EF-2 do (7). However, the mutations of these putative calcium-binding residues (Asp-153, Asp-157, and Glu-164) do not affect enzyme activity (12), suggesting that EF-hand domain binding of calcium, even if it occurs, is not connected to the enzymatic reaction of PLC ␦1. In fact, in all the known crystal structures of PLC ␦1, no calcium ion is found associated with the EF-hand domain, even though the enzyme is soaked in calcium or its analogs (7,(25)(26)(27).…”

Section: Discussionmentioning

confidence: 98%

“…This is not surprising, given that other domains of the PLC ␦1 can contribute to membrane tethering of the enzyme. However, the fact that PLC ␦1 loses enzymatic activity with membrane substrates when the EF-hand domain is deleted (11,12) argues that the EF-hand domain carries significance in the regulation of the enzymatic activity of the PLC ␦1.…”

Section: Discussionmentioning

confidence: 99%

“…The EFhand domain of PLC ␦1 consists of four consecutive EF-hand motifs, pairwise distributed in two lobes, exhibiting a characteristic helix-loop-helix topology (7). The EF-hand motifs, found in all PLC isoforms, are necessary to express full PLC activity, as evidenced by the abolition of PLC activity resulting from the deletion of EF-hand residues (11,12). Based on the structural similarities between the PLC ␦1 EF-hand and other calcium-binding EF-hand proteins such as calmodulin, it has been suggested that the EF-hand domain of PLC ␦1 might serve a regulatory role through calcium binding.…”

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confidence: 99%

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Ca2+-independent Binding of Anionic Phospholipids by Phospholipase C δ1 EF-hand Domain

Cai

Guo

Lomasney

et al. 2013

Journal of Biological Chemistry

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Background:The conserved EF-hand (EF) domain is necessary for active phospholipase. Results: EF binds to anionic phospholipid-containing vesicles; EF mutations introduced into PLC ␦1 reduce activity not recoverable with added PIP 2 . Conclusion: EF-hand domain aids substrate binding in the active site when the protein is membrane-anchored. Significance: This may be the function of the EF-hand domain in other PLC enzymes as well.

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Deletion and Site-Directed Mutagenesis of EF-Hand Domain of Phospholipase C-δ1: Effects on Its Activity

Cited by 39 publications

References 0 publications

The Role of EF-hand Domains and C2 Domain in Regulation of Enzymatic Activity of Phospholipase Cζ

The Role of EF-hand Domains and C2 Domain in Regulation of Enzymatic Activity of Phospholipase Cζ

Multiple roles of phosphoinositide-specific phospholipase C isozymes

Ca2+-independent Binding of Anionic Phospholipids by Phospholipase C δ1 EF-hand Domain

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