Phosphorylation and activation of phospholipase d1 by protein kinase C in vivo: Determination of multiple phosphorylation sites

Yong Kim, Jung Min Han, Jong Bae Park, Sang Do Lee, Yong Seok Oh, Churo Chung, Taehoon G. Lee, Jae Ho Kim, Seung Kiel Park, Jong Shin Yoo, Pann Ghill Suh, Sung Ho Ryu

Research output: Contribution to journalArticle

65 Citations (Scopus)

Abstract

Protein kinase C (PKC) is an important regulator of phospholipase D1 (PLD1). Currently there is some controversy about a phosphorylation-dependent or -independent mechanism of the activation of PLD1 by PKC. To solve this problem, we examined whether PLD1 is phosphorylated by PKC in vivo. For the first time, we have now identified multiple basal phophopeptides and multiple phorbol myristate acetate (PMA) induced phosphopeptides of endogenous PLD1 in 3Y1 cells as well as of transiently expressed PLD1 in COS-7 cells. Down regulation or inhibition of PKC greatly attenuated the PMA-induced phosphorylation as well as the activation of PLD1. In the presence of PMA, purified PLD1 from rat brain was also found to be phosphorylated by PKCα in vitro at multiple sites generating seven distinct tryptic phosphopeptides. Four phosphopeptides generated in vivo and in vitro correlated well with each other, suggesting direct phosphorylation of PLD1 by PKCα in the cells. Serine 2, threonine 147, and serine 561 were identified as phosphorylation sites, and by mutation of these residues to alanine these residues were proven to be specific phosphorylation sites in vivo. Interestingly, threonine 147 is located in the PX domain and serine 561 is in the negative regulatory 'loop' region of PLD1. Mutation of serine 2, threonine 147, or serine 561 significantly reduced PMA-induced PLD1 activity. These results strongly suggest that phosphorylation plays a pivotal role in PLD1 regulation in vivo.

Original languageEnglish
Pages (from-to)10344-10351
Number of pages8
JournalBiochemistry
Volume38
Issue number32
DOIs
Publication statusPublished - 1999 Aug 10

Fingerprint

Phosphorylation
Protein Kinase C
Chemical activation
Serine
Tetradecanoylphorbol Acetate
Phosphopeptides
Threonine
phospholipase D1
Mutation
Nucleic Acid Regulatory Sequences
COS Cells
Alanine
Brain
Down-Regulation

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Kim, Yong ; Han, Jung Min ; Park, Jong Bae ; Lee, Sang Do ; Oh, Yong Seok ; Chung, Churo ; Lee, Taehoon G. ; Kim, Jae Ho ; Park, Seung Kiel ; Yoo, Jong Shin ; Suh, Pann Ghill ; Ryu, Sung Ho. / Phosphorylation and activation of phospholipase d1 by protein kinase C in vivo : Determination of multiple phosphorylation sites. In: Biochemistry. 1999 ; Vol. 38, No. 32. pp. 10344-10351.
@article{79b763f68c594ab2b21a4b51b74f3b53,
title = "Phosphorylation and activation of phospholipase d1 by protein kinase C in vivo: Determination of multiple phosphorylation sites",
abstract = "Protein kinase C (PKC) is an important regulator of phospholipase D1 (PLD1). Currently there is some controversy about a phosphorylation-dependent or -independent mechanism of the activation of PLD1 by PKC. To solve this problem, we examined whether PLD1 is phosphorylated by PKC in vivo. For the first time, we have now identified multiple basal phophopeptides and multiple phorbol myristate acetate (PMA) induced phosphopeptides of endogenous PLD1 in 3Y1 cells as well as of transiently expressed PLD1 in COS-7 cells. Down regulation or inhibition of PKC greatly attenuated the PMA-induced phosphorylation as well as the activation of PLD1. In the presence of PMA, purified PLD1 from rat brain was also found to be phosphorylated by PKCα in vitro at multiple sites generating seven distinct tryptic phosphopeptides. Four phosphopeptides generated in vivo and in vitro correlated well with each other, suggesting direct phosphorylation of PLD1 by PKCα in the cells. Serine 2, threonine 147, and serine 561 were identified as phosphorylation sites, and by mutation of these residues to alanine these residues were proven to be specific phosphorylation sites in vivo. Interestingly, threonine 147 is located in the PX domain and serine 561 is in the negative regulatory 'loop' region of PLD1. Mutation of serine 2, threonine 147, or serine 561 significantly reduced PMA-induced PLD1 activity. These results strongly suggest that phosphorylation plays a pivotal role in PLD1 regulation in vivo.",
author = "Yong Kim and Han, {Jung Min} and Park, {Jong Bae} and Lee, {Sang Do} and Oh, {Yong Seok} and Churo Chung and Lee, {Taehoon G.} and Kim, {Jae Ho} and Park, {Seung Kiel} and Yoo, {Jong Shin} and Suh, {Pann Ghill} and Ryu, {Sung Ho}",
year = "1999",
month = "8",
day = "10",
doi = "10.1021/bi990579h",
language = "English",
volume = "38",
pages = "10344--10351",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "32",

}

Kim, Y, Han, JM, Park, JB, Lee, SD, Oh, YS, Chung, C, Lee, TG, Kim, JH, Park, SK, Yoo, JS, Suh, PG & Ryu, SH 1999, 'Phosphorylation and activation of phospholipase d1 by protein kinase C in vivo: Determination of multiple phosphorylation sites', Biochemistry, vol. 38, no. 32, pp. 10344-10351. https://doi.org/10.1021/bi990579h

Phosphorylation and activation of phospholipase d1 by protein kinase C in vivo : Determination of multiple phosphorylation sites. / Kim, Yong; Han, Jung Min; Park, Jong Bae; Lee, Sang Do; Oh, Yong Seok; Chung, Churo; Lee, Taehoon G.; Kim, Jae Ho; Park, Seung Kiel; Yoo, Jong Shin; Suh, Pann Ghill; Ryu, Sung Ho.

In: Biochemistry, Vol. 38, No. 32, 10.08.1999, p. 10344-10351.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Phosphorylation and activation of phospholipase d1 by protein kinase C in vivo

T2 - Determination of multiple phosphorylation sites

AU - Kim, Yong

AU - Han, Jung Min

AU - Park, Jong Bae

AU - Lee, Sang Do

AU - Oh, Yong Seok

AU - Chung, Churo

AU - Lee, Taehoon G.

AU - Kim, Jae Ho

AU - Park, Seung Kiel

AU - Yoo, Jong Shin

AU - Suh, Pann Ghill

AU - Ryu, Sung Ho

PY - 1999/8/10

Y1 - 1999/8/10

N2 - Protein kinase C (PKC) is an important regulator of phospholipase D1 (PLD1). Currently there is some controversy about a phosphorylation-dependent or -independent mechanism of the activation of PLD1 by PKC. To solve this problem, we examined whether PLD1 is phosphorylated by PKC in vivo. For the first time, we have now identified multiple basal phophopeptides and multiple phorbol myristate acetate (PMA) induced phosphopeptides of endogenous PLD1 in 3Y1 cells as well as of transiently expressed PLD1 in COS-7 cells. Down regulation or inhibition of PKC greatly attenuated the PMA-induced phosphorylation as well as the activation of PLD1. In the presence of PMA, purified PLD1 from rat brain was also found to be phosphorylated by PKCα in vitro at multiple sites generating seven distinct tryptic phosphopeptides. Four phosphopeptides generated in vivo and in vitro correlated well with each other, suggesting direct phosphorylation of PLD1 by PKCα in the cells. Serine 2, threonine 147, and serine 561 were identified as phosphorylation sites, and by mutation of these residues to alanine these residues were proven to be specific phosphorylation sites in vivo. Interestingly, threonine 147 is located in the PX domain and serine 561 is in the negative regulatory 'loop' region of PLD1. Mutation of serine 2, threonine 147, or serine 561 significantly reduced PMA-induced PLD1 activity. These results strongly suggest that phosphorylation plays a pivotal role in PLD1 regulation in vivo.

AB - Protein kinase C (PKC) is an important regulator of phospholipase D1 (PLD1). Currently there is some controversy about a phosphorylation-dependent or -independent mechanism of the activation of PLD1 by PKC. To solve this problem, we examined whether PLD1 is phosphorylated by PKC in vivo. For the first time, we have now identified multiple basal phophopeptides and multiple phorbol myristate acetate (PMA) induced phosphopeptides of endogenous PLD1 in 3Y1 cells as well as of transiently expressed PLD1 in COS-7 cells. Down regulation or inhibition of PKC greatly attenuated the PMA-induced phosphorylation as well as the activation of PLD1. In the presence of PMA, purified PLD1 from rat brain was also found to be phosphorylated by PKCα in vitro at multiple sites generating seven distinct tryptic phosphopeptides. Four phosphopeptides generated in vivo and in vitro correlated well with each other, suggesting direct phosphorylation of PLD1 by PKCα in the cells. Serine 2, threonine 147, and serine 561 were identified as phosphorylation sites, and by mutation of these residues to alanine these residues were proven to be specific phosphorylation sites in vivo. Interestingly, threonine 147 is located in the PX domain and serine 561 is in the negative regulatory 'loop' region of PLD1. Mutation of serine 2, threonine 147, or serine 561 significantly reduced PMA-induced PLD1 activity. These results strongly suggest that phosphorylation plays a pivotal role in PLD1 regulation in vivo.

UR - http://www.scopus.com/inward/record.url?scp=0040737600&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0040737600&partnerID=8YFLogxK

U2 - 10.1021/bi990579h

DO - 10.1021/bi990579h

M3 - Article

C2 - 10441128

AN - SCOPUS:0040737600

VL - 38

SP - 10344

EP - 10351

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 32

ER -