Phospholipase D1 in caveolae: Regulation by protein kinase Cα and caveolin-1

Jae Ho Kim, Jung Min Han, Sukmook Lee, Yong Kim, Taehoon G. Lee, Jong Bae Park, Sang Do Lee, Pann Ghill Suh, Sung Ho Ryu

Research output: Contribution to journalArticle

56 Citations (Scopus)

Abstract

Caveolae are small plasma membrane invaginations that have been implicated in cell signaling, and caveolin is a principal structural component of the caveolar membrane. Previously we have demonstrated that protein kinase Cα (PKCα) directly interacts with phospholipase D1 (PLD1), activating the enzymatic activity of PLD1 in the presence of phorbol 12- myristate 13-acetate (PMA) [Lee, T. G., et al. (1997) Biochim. Biophys. Acta 1347, 199-204]. In this study, using a detergent-free procedure for the purification of a caveolin-enriched membrane fraction (CEM) and immunoblot analysis, we show that PLD1 is enriched in the CEMs of 3Y1 rat fibroblasts. Purified PLD1 directly bound to a glutathione S-transferase-caveolin-1 fusion protein in in vitro binding assays. The association of PLD1 with caveolin-1 could be completely eliminated by preincubation of PLD1 with an oligopeptide corresponding to the scaffolding domain (amino acids 82-101) of caveolin-1, indicating that caveolin-1 interacts with PLD1 through the scaffolding domain. The peptide also inhibited PKCα-stimulated PLD1 activity and the interaction between PLD1 and PKCα with an IC50 of 0.5 μM. PMA elicits translocation of PKCα to the CEMs, inducing PLD activation through the interaction of PKCα with PLD1 in the CEMs. Caveolin-1 also coimmunoprecipitated with PLD1 in the absence of PMA, and the amounts of coimmunoprecipitated caveolin-1 decreased in response to treatment with PMA. Taken together, our results suggest a new mechanism for the regulation of the PKCα-dependent PLD activity through the molecular interaction between PLD1, PKCα, and caveolin-1 in caveolae.

Original languageEnglish
Pages (from-to)3763-3769
Number of pages7
JournalBiochemistry
Volume38
Issue number12
DOIs
Publication statusPublished - 1999 Mar 23

Fingerprint

Caveolin 1
Caveolae
Protein Kinase C
Acetates
Caveolins
phospholipase D1
Cell signaling
Membranes
Oligopeptides
Molecular interactions
Fibroblasts
Cell membranes
Glutathione Transferase
Detergents
Inhibitory Concentration 50
Purification
Rats
Assays
Fusion reactions

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Kim, J. H., Han, J. M., Lee, S., Kim, Y., Lee, T. G., Park, J. B., ... Ryu, S. H. (1999). Phospholipase D1 in caveolae: Regulation by protein kinase Cα and caveolin-1. Biochemistry, 38(12), 3763-3769. https://doi.org/10.1021/bi982478+
Kim, Jae Ho ; Han, Jung Min ; Lee, Sukmook ; Kim, Yong ; Lee, Taehoon G. ; Park, Jong Bae ; Lee, Sang Do ; Suh, Pann Ghill ; Ryu, Sung Ho. / Phospholipase D1 in caveolae : Regulation by protein kinase Cα and caveolin-1. In: Biochemistry. 1999 ; Vol. 38, No. 12. pp. 3763-3769.
@article{f938e82fb16740f1b36848cafd259405,
title = "Phospholipase D1 in caveolae: Regulation by protein kinase Cα and caveolin-1",
abstract = "Caveolae are small plasma membrane invaginations that have been implicated in cell signaling, and caveolin is a principal structural component of the caveolar membrane. Previously we have demonstrated that protein kinase Cα (PKCα) directly interacts with phospholipase D1 (PLD1), activating the enzymatic activity of PLD1 in the presence of phorbol 12- myristate 13-acetate (PMA) [Lee, T. G., et al. (1997) Biochim. Biophys. Acta 1347, 199-204]. In this study, using a detergent-free procedure for the purification of a caveolin-enriched membrane fraction (CEM) and immunoblot analysis, we show that PLD1 is enriched in the CEMs of 3Y1 rat fibroblasts. Purified PLD1 directly bound to a glutathione S-transferase-caveolin-1 fusion protein in in vitro binding assays. The association of PLD1 with caveolin-1 could be completely eliminated by preincubation of PLD1 with an oligopeptide corresponding to the scaffolding domain (amino acids 82-101) of caveolin-1, indicating that caveolin-1 interacts with PLD1 through the scaffolding domain. The peptide also inhibited PKCα-stimulated PLD1 activity and the interaction between PLD1 and PKCα with an IC50 of 0.5 μM. PMA elicits translocation of PKCα to the CEMs, inducing PLD activation through the interaction of PKCα with PLD1 in the CEMs. Caveolin-1 also coimmunoprecipitated with PLD1 in the absence of PMA, and the amounts of coimmunoprecipitated caveolin-1 decreased in response to treatment with PMA. Taken together, our results suggest a new mechanism for the regulation of the PKCα-dependent PLD activity through the molecular interaction between PLD1, PKCα, and caveolin-1 in caveolae.",
author = "Kim, {Jae Ho} and Han, {Jung Min} and Sukmook Lee and Yong Kim and Lee, {Taehoon G.} and Park, {Jong Bae} and Lee, {Sang Do} and Suh, {Pann Ghill} and Ryu, {Sung Ho}",
year = "1999",
month = "3",
day = "23",
doi = "10.1021/bi982478+",
language = "English",
volume = "38",
pages = "3763--3769",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "12",

}

Kim, JH, Han, JM, Lee, S, Kim, Y, Lee, TG, Park, JB, Lee, SD, Suh, PG & Ryu, SH 1999, 'Phospholipase D1 in caveolae: Regulation by protein kinase Cα and caveolin-1', Biochemistry, vol. 38, no. 12, pp. 3763-3769. https://doi.org/10.1021/bi982478+

Phospholipase D1 in caveolae : Regulation by protein kinase Cα and caveolin-1. / Kim, Jae Ho; Han, Jung Min; Lee, Sukmook; Kim, Yong; Lee, Taehoon G.; Park, Jong Bae; Lee, Sang Do; Suh, Pann Ghill; Ryu, Sung Ho.

In: Biochemistry, Vol. 38, No. 12, 23.03.1999, p. 3763-3769.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Phospholipase D1 in caveolae

T2 - Regulation by protein kinase Cα and caveolin-1

AU - Kim, Jae Ho

AU - Han, Jung Min

AU - Lee, Sukmook

AU - Kim, Yong

AU - Lee, Taehoon G.

AU - Park, Jong Bae

AU - Lee, Sang Do

AU - Suh, Pann Ghill

AU - Ryu, Sung Ho

PY - 1999/3/23

Y1 - 1999/3/23

N2 - Caveolae are small plasma membrane invaginations that have been implicated in cell signaling, and caveolin is a principal structural component of the caveolar membrane. Previously we have demonstrated that protein kinase Cα (PKCα) directly interacts with phospholipase D1 (PLD1), activating the enzymatic activity of PLD1 in the presence of phorbol 12- myristate 13-acetate (PMA) [Lee, T. G., et al. (1997) Biochim. Biophys. Acta 1347, 199-204]. In this study, using a detergent-free procedure for the purification of a caveolin-enriched membrane fraction (CEM) and immunoblot analysis, we show that PLD1 is enriched in the CEMs of 3Y1 rat fibroblasts. Purified PLD1 directly bound to a glutathione S-transferase-caveolin-1 fusion protein in in vitro binding assays. The association of PLD1 with caveolin-1 could be completely eliminated by preincubation of PLD1 with an oligopeptide corresponding to the scaffolding domain (amino acids 82-101) of caveolin-1, indicating that caveolin-1 interacts with PLD1 through the scaffolding domain. The peptide also inhibited PKCα-stimulated PLD1 activity and the interaction between PLD1 and PKCα with an IC50 of 0.5 μM. PMA elicits translocation of PKCα to the CEMs, inducing PLD activation through the interaction of PKCα with PLD1 in the CEMs. Caveolin-1 also coimmunoprecipitated with PLD1 in the absence of PMA, and the amounts of coimmunoprecipitated caveolin-1 decreased in response to treatment with PMA. Taken together, our results suggest a new mechanism for the regulation of the PKCα-dependent PLD activity through the molecular interaction between PLD1, PKCα, and caveolin-1 in caveolae.

AB - Caveolae are small plasma membrane invaginations that have been implicated in cell signaling, and caveolin is a principal structural component of the caveolar membrane. Previously we have demonstrated that protein kinase Cα (PKCα) directly interacts with phospholipase D1 (PLD1), activating the enzymatic activity of PLD1 in the presence of phorbol 12- myristate 13-acetate (PMA) [Lee, T. G., et al. (1997) Biochim. Biophys. Acta 1347, 199-204]. In this study, using a detergent-free procedure for the purification of a caveolin-enriched membrane fraction (CEM) and immunoblot analysis, we show that PLD1 is enriched in the CEMs of 3Y1 rat fibroblasts. Purified PLD1 directly bound to a glutathione S-transferase-caveolin-1 fusion protein in in vitro binding assays. The association of PLD1 with caveolin-1 could be completely eliminated by preincubation of PLD1 with an oligopeptide corresponding to the scaffolding domain (amino acids 82-101) of caveolin-1, indicating that caveolin-1 interacts with PLD1 through the scaffolding domain. The peptide also inhibited PKCα-stimulated PLD1 activity and the interaction between PLD1 and PKCα with an IC50 of 0.5 μM. PMA elicits translocation of PKCα to the CEMs, inducing PLD activation through the interaction of PKCα with PLD1 in the CEMs. Caveolin-1 also coimmunoprecipitated with PLD1 in the absence of PMA, and the amounts of coimmunoprecipitated caveolin-1 decreased in response to treatment with PMA. Taken together, our results suggest a new mechanism for the regulation of the PKCα-dependent PLD activity through the molecular interaction between PLD1, PKCα, and caveolin-1 in caveolae.

UR - http://www.scopus.com/inward/record.url?scp=0000784989&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0000784989&partnerID=8YFLogxK

U2 - 10.1021/bi982478+

DO - 10.1021/bi982478+

M3 - Article

C2 - 10090765

AN - SCOPUS:0000784989

VL - 38

SP - 3763

EP - 3769

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 12

ER -