PMID- 10441128
OWN - NLM
STAT- MEDLINE
DCOM- 19990908
LR  - 20131121
IS  - 0006-2960 (Print)
IS  - 0006-2960 (Linking)
VI  - 38
IP  - 32
DP  - 1999 Aug 10
TI  - Phosphorylation and activation of phospholipase D1 by protein kinase C in vivo:
      determination of multiple phosphorylation sites.
PG  - 10344-51
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 PKCalpha 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 PKCalpha 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.
FAU - Kim, Y
AU  - Kim Y
AD  - Department of Life Science, School of Environmental Engineering, Pohang
      University of Science and Technology, Korea.
FAU - Han, J M
AU  - Han JM
FAU - Park, J B
AU  - Park JB
FAU - Lee, S D
AU  - Lee SD
FAU - Oh, Y S
AU  - Oh YS
FAU - Chung, C
AU  - Chung C
FAU - Lee, T G
AU  - Lee TG
FAU - Kim, J H
AU  - Kim JH
FAU - Park, S K
AU  - Park SK
FAU - Yoo, J S
AU  - Yoo JS
FAU - Suh, P G
AU  - Suh PG
FAU - Ryu, S H
AU  - Ryu SH
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Biochemistry
JT  - Biochemistry
JID - 0370623
RN  - 0 (Peptide Fragments)
RN  - 0 (Phosphopeptides)
RN  - 2ZD004190S (Threonine)
RN  - 452VLY9402 (Serine)
RN  - 4QD397987E (Histidine)
RN  - EC 2.7.11.13 (Protein Kinase C)
RN  - EC 3.1.4.4 (Phospholipase D)
RN  - EC 3.1.4.4 (phospholipase D1)
RN  - NI40JAQ945 (Tetradecanoylphorbol Acetate)
SB  - IM
MH  - Amino Acid Sequence
MH  - Animals
MH  - Binding Sites/drug effects/genetics
MH  - Cell Line
MH  - Chromatography, High Pressure Liquid
MH  - Enzyme Activation/drug effects/genetics
MH  - Fibroblasts
MH  - Histidine/physiology
MH  - Molecular Sequence Data
MH  - Mutagenesis, Site-Directed
MH  - Peptide Fragments/physiology
MH  - Peptide Mapping
MH  - Phospholipase D/antagonists & inhibitors/genetics/*metabolism/physiology
MH  - Phosphopeptides/isolation & purification/metabolism
MH  - Phosphorylation/drug effects
MH  - Protein Kinase C/*metabolism/physiology
MH  - Rats
MH  - Serine/genetics/metabolism
MH  - Tetradecanoylphorbol Acetate/pharmacology
MH  - Threonine/genetics
EDAT- 1999/08/11 00:00
MHDA- 1999/08/11 00:01
CRDT- 1999/08/11 00:00
PHST- 1999/08/11 00:00 [pubmed]
PHST- 1999/08/11 00:01 [medline]
PHST- 1999/08/11 00:00 [entrez]
AID - 10.1021/bi990579h [doi]
AID - bi990579h [pii]
PST - ppublish
SO  - Biochemistry. 1999 Aug 10;38(32):10344-51. doi: 10.1021/bi990579h.