Regulation of Phosphatidic Acid Phosphatase

1
Regulation of Phosphatidic Acid Phosphatase
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Phospholipid Synthesis in Yeast

• Model eukaryotic organism
• Easy to grow for biochemical studies
• Facilitate molecular/genetic studies
• Lipid synthesis is typical of higher eukaryotes

Carman Henry 2007
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Model for the PA-mediated Regulation of
Phospholipid Synthesis Genes
Carman Henry 2007
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Reaction Catalyzed by PA Phosphatase
Smith et al. 1957
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PA Phosphatases in Yeast
Type 1 Mg2-dependent (PAP1) Lipid
Synthesis Pah1p (95 kDa) - Cytosol
Membrane Unknown PAP
(peripheral) Substrate PA
Type 2 Mg2-independent (PAP2, LPP) Lipid
Signaling Dpp1p (34 kDa) - Vacuole
(integral) Lpp1p (32 kDa) - Golgi
(integral) Substrates PA, LPA, DGPP, SoP, DolP
Carman Han 2006
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Phenotypes of the PAP (pah1D) Mutant

• Increase in PA, and decreases in DAG and TAG
• Alterations in phospholipids (e.g., PC , PI )
  and neutral lipids (e.g., SE , FA )
• Derepression of phospholipid synthesis genes
  (e.g., INO1, OPI3)
• Aberrant expansion of the nuclear/ER membrane
• Temperature sensitive and respiratory deficient

Santos-Rosa et al. 2005 Han et al. 2006 Han et
al. 2007
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TAG Content of PAP Mutants
Han et al. 2006
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Effect of Growth on PAP Activity
Genetic background dpp1D lpp1D
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Effect of Growth Phase on PAH1 Expression
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Effect of Growth on PAH1 mRNA Abundance
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Effect of Growth on PAP Activity and Protein
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22
20
18
28
32
36
8
16
12
Time, h
10
14
- PAP
- PGK
Genetic background pah1D dpp1D lpp1D (PAH1)
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The Effects of MG132 and ubc Mutations on PAP
Stability
MG132 (Z-leu-leu-leu-CHO) proteasome inhibitor
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16
8
16
8
16
8
16
8
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Time, h
- PAP
WT
ubc1?
ubc4?
ubc6?
ubc8?
ubc mutations in ubiquitin conjugating enzymes
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Phosphorylation Sites of PAP
OHara et al. 2006
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PAP is a Cyclin-Dependent Kinase Substrate
Ser602 Thr723 are the major CDK sites
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Effect of CDK Phosphorylation on PAP Activity and
Location
C Cytosol M Membrane
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Other Protein Kinases That Phosphorylate PAP
Major Sites
Ser773
Ser677, Ser769, Ser788
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Summary

• PA phosphatase plays an important role in
  controlling lipid content
• Regulation of phospholipid synthesis gene
  expression
• Nuclear/ER membrane expansion
• Synthesis of TAG
• Regulation of PA phosphatase is complex
• Transcription of PAH1 is controlled by growth
  phase
• PAH1 expression may be controlled mRNA stability
• PA phosphatase stability may be controlled by
  ubiquitination
• PA phosphatase is phosphorylated by multiple
  protein kinases
• Activity is regulated by lipids and nucleotides,
  and by its location

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Regulation of Phospholipid Synthesis
PC
PMT
AdoHcy
CDP-DAG Pathway
PMT
Kennedy Pathway
PE
Inositol
CTP
CDP-DAG
PS
PI
PE, PC
PSS
PIS
PAP
PA
DAG
TAG
CDP-DAG
DGK
CTP
CDP-DAG
Opi1
OPI3 EKI1 EPT1 CKI1 CPT1
INO1 CDS1 CHO1 PSD1 CHO2
Ino4
Ino2
UASINO
Carman Han 2009
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Acknowledgments
Carman Laboratory
J. Morgan
S. Fakas
H.-S. Choi
W.-M. Su
F. Pascual
J. Casciano
A. Soto
C. Lee
M. Chae
NIH Grants GM-28140 GM-50679