Ceramide-1-phosphate binds group IVA cytosolic phospholipase a2 via a novel site in the C2 domain

Robert V. Stahelin, Preeti Subramanian, Mohsin Vora, Wonhwa Cho, Charles E. Chalfant

Research output: Contribution to journalArticle

83 Citations (Scopus)

Abstract

Previously, ceramide-1-phosphate (C1P) was demonstrated to be a potent and specific activator of group IV cytosolic phospholipase A2α (cPLA2α) via interaction with the C2 domain. In this study, we hypothesized that the specific interaction site for C1P was localized to the cationic β-groove (Arg57, Lys58, Arg59) of the C2 domain of cPLA2α. In this regard, mutants of this region of cPLA2α were generated (R57A/K58A/R59A, R57A/R59A, K58A/R59A, R57A/K58A, R57A, K58A, and R59A) and examined for C1P affinity by surface plasmon resonance. The triple mutants (R57A/K58A/ R59A), the double mutants (R57A/R59A, K58A/R59A, and R57A/K58A), and the single mutant (R59A) demonstrated significantly reduced affinity for C1P-containing vesicles as compared with wild-type cPLA2α. Examining these mutants for enzymatic activity demonstrated that these five mutants of cPLA 2α also showed a significant reduction in the ability of C1P to: 1) increase the Vmax of the reaction; and 2) significantly decrease the dissociation constant (Ks A) of the reaction as compared with the wild-type enzyme. The mutational effect was specific for C1P as all of the cationic mutants of cPLA2α demonstrated normal basal activity as well as normal affinities for phosphatidylcholine and phosphatidylinositol-4,5-bisphosphate as compared with wild-type cPLA 2α. This study, for the first time, demonstrates a novel C1P interaction site mapped to the cationic β-groove of the C2 domain of cPLA2α.

Original languageEnglish (US)
Pages (from-to)20467-20474
Number of pages8
JournalJournal of Biological Chemistry
Volume282
Issue number28
DOIs
StatePublished - Jul 13 2007

Fingerprint

Group IV Phospholipases A2
Cytosolic Phospholipases A2
Surface Plasmon Resonance
Surface plasmon resonance
Phosphatidylinositols
ceramide 1-phosphate
C2 Domains
Phosphatidylcholines

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Ceramide-1-phosphate binds group IVA cytosolic phospholipase a2 via a novel site in the C2 domain. / Stahelin, Robert V.; Subramanian, Preeti; Vora, Mohsin; Cho, Wonhwa; Chalfant, Charles E.

In: Journal of Biological Chemistry, Vol. 282, No. 28, 13.07.2007, p. 20467-20474.

Research output: Contribution to journalArticle

Stahelin, Robert V. ; Subramanian, Preeti ; Vora, Mohsin ; Cho, Wonhwa ; Chalfant, Charles E. / Ceramide-1-phosphate binds group IVA cytosolic phospholipase a2 via a novel site in the C2 domain. In: Journal of Biological Chemistry. 2007 ; Vol. 282, No. 28. pp. 20467-20474.
@article{817566fd66544154991006fb594c792a,
title = "Ceramide-1-phosphate binds group IVA cytosolic phospholipase a2 via a novel site in the C2 domain",
abstract = "Previously, ceramide-1-phosphate (C1P) was demonstrated to be a potent and specific activator of group IV cytosolic phospholipase A2α (cPLA2α) via interaction with the C2 domain. In this study, we hypothesized that the specific interaction site for C1P was localized to the cationic β-groove (Arg57, Lys58, Arg59) of the C2 domain of cPLA2α. In this regard, mutants of this region of cPLA2α were generated (R57A/K58A/R59A, R57A/R59A, K58A/R59A, R57A/K58A, R57A, K58A, and R59A) and examined for C1P affinity by surface plasmon resonance. The triple mutants (R57A/K58A/ R59A), the double mutants (R57A/R59A, K58A/R59A, and R57A/K58A), and the single mutant (R59A) demonstrated significantly reduced affinity for C1P-containing vesicles as compared with wild-type cPLA2α. Examining these mutants for enzymatic activity demonstrated that these five mutants of cPLA 2α also showed a significant reduction in the ability of C1P to: 1) increase the Vmax of the reaction; and 2) significantly decrease the dissociation constant (Ks A) of the reaction as compared with the wild-type enzyme. The mutational effect was specific for C1P as all of the cationic mutants of cPLA2α demonstrated normal basal activity as well as normal affinities for phosphatidylcholine and phosphatidylinositol-4,5-bisphosphate as compared with wild-type cPLA 2α. This study, for the first time, demonstrates a novel C1P interaction site mapped to the cationic β-groove of the C2 domain of cPLA2α.",
author = "Stahelin, {Robert V.} and Preeti Subramanian and Mohsin Vora and Wonhwa Cho and Chalfant, {Charles E.}",
year = "2007",
month = "7",
day = "13",
doi = "10.1074/jbc.M701396200",
language = "English (US)",
volume = "282",
pages = "20467--20474",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "28",

}

TY - JOUR

T1 - Ceramide-1-phosphate binds group IVA cytosolic phospholipase a2 via a novel site in the C2 domain

AU - Stahelin, Robert V.

AU - Subramanian, Preeti

AU - Vora, Mohsin

AU - Cho, Wonhwa

AU - Chalfant, Charles E.

PY - 2007/7/13

Y1 - 2007/7/13

N2 - Previously, ceramide-1-phosphate (C1P) was demonstrated to be a potent and specific activator of group IV cytosolic phospholipase A2α (cPLA2α) via interaction with the C2 domain. In this study, we hypothesized that the specific interaction site for C1P was localized to the cationic β-groove (Arg57, Lys58, Arg59) of the C2 domain of cPLA2α. In this regard, mutants of this region of cPLA2α were generated (R57A/K58A/R59A, R57A/R59A, K58A/R59A, R57A/K58A, R57A, K58A, and R59A) and examined for C1P affinity by surface plasmon resonance. The triple mutants (R57A/K58A/ R59A), the double mutants (R57A/R59A, K58A/R59A, and R57A/K58A), and the single mutant (R59A) demonstrated significantly reduced affinity for C1P-containing vesicles as compared with wild-type cPLA2α. Examining these mutants for enzymatic activity demonstrated that these five mutants of cPLA 2α also showed a significant reduction in the ability of C1P to: 1) increase the Vmax of the reaction; and 2) significantly decrease the dissociation constant (Ks A) of the reaction as compared with the wild-type enzyme. The mutational effect was specific for C1P as all of the cationic mutants of cPLA2α demonstrated normal basal activity as well as normal affinities for phosphatidylcholine and phosphatidylinositol-4,5-bisphosphate as compared with wild-type cPLA 2α. This study, for the first time, demonstrates a novel C1P interaction site mapped to the cationic β-groove of the C2 domain of cPLA2α.

AB - Previously, ceramide-1-phosphate (C1P) was demonstrated to be a potent and specific activator of group IV cytosolic phospholipase A2α (cPLA2α) via interaction with the C2 domain. In this study, we hypothesized that the specific interaction site for C1P was localized to the cationic β-groove (Arg57, Lys58, Arg59) of the C2 domain of cPLA2α. In this regard, mutants of this region of cPLA2α were generated (R57A/K58A/R59A, R57A/R59A, K58A/R59A, R57A/K58A, R57A, K58A, and R59A) and examined for C1P affinity by surface plasmon resonance. The triple mutants (R57A/K58A/ R59A), the double mutants (R57A/R59A, K58A/R59A, and R57A/K58A), and the single mutant (R59A) demonstrated significantly reduced affinity for C1P-containing vesicles as compared with wild-type cPLA2α. Examining these mutants for enzymatic activity demonstrated that these five mutants of cPLA 2α also showed a significant reduction in the ability of C1P to: 1) increase the Vmax of the reaction; and 2) significantly decrease the dissociation constant (Ks A) of the reaction as compared with the wild-type enzyme. The mutational effect was specific for C1P as all of the cationic mutants of cPLA2α demonstrated normal basal activity as well as normal affinities for phosphatidylcholine and phosphatidylinositol-4,5-bisphosphate as compared with wild-type cPLA 2α. This study, for the first time, demonstrates a novel C1P interaction site mapped to the cationic β-groove of the C2 domain of cPLA2α.

UR - http://www.scopus.com/inward/record.url?scp=34547122922&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=34547122922&partnerID=8YFLogxK

U2 - 10.1074/jbc.M701396200

DO - 10.1074/jbc.M701396200

M3 - Article

C2 - 17472963

AN - SCOPUS:34547122922

VL - 282

SP - 20467

EP - 20474

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 28

ER -