Differential roles of phosphatidylserine, PtdIns(4,5)P2, and PtdIns(3,4,5)P3 in plasma membrane targeting of C2 domains

Molecular dynamics simulation, membrane binding, and cell translocation studies of the PKCα C2 domain

Debasis Manna, Nitin Bhardwaj, Mohsin S. Vora, Robert Stahelin, Hui Lu, Wonhwa Cho

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Abstract

Many cytosolic proteins are recruited to the plasma membrane (PM) during cell signaling and other cellular processes. Recent reports have indicated that phosphatidylserine (PS), phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) that are present in the PM play important roles for their specific PM recruitment. To systematically analyze how these lipids mediate PM targeting of cellular proteins, we performed biophysical, computational, and cell studies of the Ca2+-dependent C2 domain of protein kinase Cα (PKCα) that is known to bind PS and phosphoinositides. In vitro membrane binding measurements by surface plasmon resonance analysis show that PKCα-C2 nonspecifically binds phosphoinositides, including PtdIns(4,5)P2 and PtdIns(3,4,5)P 3, but that PS and Ca2+ binding is prerequisite for productive phosphoinositide binding. PtdIns(4,5)P2 or PtdIns(3,4,5)P3 augments the Ca2+- and PS-dependent membrane binding of PKCα-C2 by slowing its membrane dissociation. Molecular dynamics simulations also support that Ca2+-dependent PS binding is essential for membrane interactions of PKCα-C2. PtdIns(4,5)P2 alone cannot drive the membrane attachment of the domain but further stabilizes the Ca2+- and PS-dependent membrane binding. When the fluorescence protein-tagged PKCα-C2 was expressed in NIH-3T3 cells, mutations of phosphoinositide-binding residues or depletion of PtdIns(4,5)P2 and/or PtdIns(3,4,5)P3 from PM did not significantly affect the PM association of the domain but accelerated its dissociation from PM. Also, local synthesis of PtdIns(4,5)P2 or PtdIns(3,4,5)P3 at the PM slowed membrane dissociation of PKCα-C2. Collectively, these studies show that PtdIns(4,5)P2 and PtdIns(3,4,5)P3 augment the Ca2+- and PS-dependent membrane binding of PKCα-C2 by elongating the membrane residence of the domain but cannot drive the PM recruitment of PKCα-C2. These studies also suggest that effective PM recruitment of many cellular proteins may require synergistic actions of PS and phosphoinositides.

Original languageEnglish (US)
Pages (from-to)26047-26058
Number of pages12
JournalJournal of Biological Chemistry
Volume283
Issue number38
DOIs
StatePublished - Sep 19 2008
Externally publishedYes

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Phosphatidylinositol 4,5-Diphosphate
Phosphatidylserines
Molecular Dynamics Simulation
Cell membranes
Protein Kinase C
Molecular dynamics
Cell Membrane
Phosphatidylinositols
Membranes
Computer simulation
Membrane Proteins
Carrier Proteins
Proteins
phosphatidylinositol 3,4,5-triphosphate
C2 Domains
Cell signaling
NIH 3T3 Cells
Surface Plasmon Resonance
Surface plasmon resonance
Protein Transport

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

@article{2aea14e0b1514ce0b5173f5bf44e2a5d,
title = "Differential roles of phosphatidylserine, PtdIns(4,5)P2, and PtdIns(3,4,5)P3 in plasma membrane targeting of C2 domains: Molecular dynamics simulation, membrane binding, and cell translocation studies of the PKCα C2 domain",
abstract = "Many cytosolic proteins are recruited to the plasma membrane (PM) during cell signaling and other cellular processes. Recent reports have indicated that phosphatidylserine (PS), phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) that are present in the PM play important roles for their specific PM recruitment. To systematically analyze how these lipids mediate PM targeting of cellular proteins, we performed biophysical, computational, and cell studies of the Ca2+-dependent C2 domain of protein kinase Cα (PKCα) that is known to bind PS and phosphoinositides. In vitro membrane binding measurements by surface plasmon resonance analysis show that PKCα-C2 nonspecifically binds phosphoinositides, including PtdIns(4,5)P2 and PtdIns(3,4,5)P 3, but that PS and Ca2+ binding is prerequisite for productive phosphoinositide binding. PtdIns(4,5)P2 or PtdIns(3,4,5)P3 augments the Ca2+- and PS-dependent membrane binding of PKCα-C2 by slowing its membrane dissociation. Molecular dynamics simulations also support that Ca2+-dependent PS binding is essential for membrane interactions of PKCα-C2. PtdIns(4,5)P2 alone cannot drive the membrane attachment of the domain but further stabilizes the Ca2+- and PS-dependent membrane binding. When the fluorescence protein-tagged PKCα-C2 was expressed in NIH-3T3 cells, mutations of phosphoinositide-binding residues or depletion of PtdIns(4,5)P2 and/or PtdIns(3,4,5)P3 from PM did not significantly affect the PM association of the domain but accelerated its dissociation from PM. Also, local synthesis of PtdIns(4,5)P2 or PtdIns(3,4,5)P3 at the PM slowed membrane dissociation of PKCα-C2. Collectively, these studies show that PtdIns(4,5)P2 and PtdIns(3,4,5)P3 augment the Ca2+- and PS-dependent membrane binding of PKCα-C2 by elongating the membrane residence of the domain but cannot drive the PM recruitment of PKCα-C2. These studies also suggest that effective PM recruitment of many cellular proteins may require synergistic actions of PS and phosphoinositides.",
author = "Debasis Manna and Nitin Bhardwaj and Vora, {Mohsin S.} and Robert Stahelin and Hui Lu and Wonhwa Cho",
year = "2008",
month = "9",
day = "19",
doi = "10.1074/jbc.M802617200",
language = "English (US)",
volume = "283",
pages = "26047--26058",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "38",

}

TY - JOUR

T1 - Differential roles of phosphatidylserine, PtdIns(4,5)P2, and PtdIns(3,4,5)P3 in plasma membrane targeting of C2 domains

T2 - Molecular dynamics simulation, membrane binding, and cell translocation studies of the PKCα C2 domain

AU - Manna, Debasis

AU - Bhardwaj, Nitin

AU - Vora, Mohsin S.

AU - Stahelin, Robert

AU - Lu, Hui

AU - Cho, Wonhwa

PY - 2008/9/19

Y1 - 2008/9/19

N2 - Many cytosolic proteins are recruited to the plasma membrane (PM) during cell signaling and other cellular processes. Recent reports have indicated that phosphatidylserine (PS), phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) that are present in the PM play important roles for their specific PM recruitment. To systematically analyze how these lipids mediate PM targeting of cellular proteins, we performed biophysical, computational, and cell studies of the Ca2+-dependent C2 domain of protein kinase Cα (PKCα) that is known to bind PS and phosphoinositides. In vitro membrane binding measurements by surface plasmon resonance analysis show that PKCα-C2 nonspecifically binds phosphoinositides, including PtdIns(4,5)P2 and PtdIns(3,4,5)P 3, but that PS and Ca2+ binding is prerequisite for productive phosphoinositide binding. PtdIns(4,5)P2 or PtdIns(3,4,5)P3 augments the Ca2+- and PS-dependent membrane binding of PKCα-C2 by slowing its membrane dissociation. Molecular dynamics simulations also support that Ca2+-dependent PS binding is essential for membrane interactions of PKCα-C2. PtdIns(4,5)P2 alone cannot drive the membrane attachment of the domain but further stabilizes the Ca2+- and PS-dependent membrane binding. When the fluorescence protein-tagged PKCα-C2 was expressed in NIH-3T3 cells, mutations of phosphoinositide-binding residues or depletion of PtdIns(4,5)P2 and/or PtdIns(3,4,5)P3 from PM did not significantly affect the PM association of the domain but accelerated its dissociation from PM. Also, local synthesis of PtdIns(4,5)P2 or PtdIns(3,4,5)P3 at the PM slowed membrane dissociation of PKCα-C2. Collectively, these studies show that PtdIns(4,5)P2 and PtdIns(3,4,5)P3 augment the Ca2+- and PS-dependent membrane binding of PKCα-C2 by elongating the membrane residence of the domain but cannot drive the PM recruitment of PKCα-C2. These studies also suggest that effective PM recruitment of many cellular proteins may require synergistic actions of PS and phosphoinositides.

AB - Many cytosolic proteins are recruited to the plasma membrane (PM) during cell signaling and other cellular processes. Recent reports have indicated that phosphatidylserine (PS), phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) that are present in the PM play important roles for their specific PM recruitment. To systematically analyze how these lipids mediate PM targeting of cellular proteins, we performed biophysical, computational, and cell studies of the Ca2+-dependent C2 domain of protein kinase Cα (PKCα) that is known to bind PS and phosphoinositides. In vitro membrane binding measurements by surface plasmon resonance analysis show that PKCα-C2 nonspecifically binds phosphoinositides, including PtdIns(4,5)P2 and PtdIns(3,4,5)P 3, but that PS and Ca2+ binding is prerequisite for productive phosphoinositide binding. PtdIns(4,5)P2 or PtdIns(3,4,5)P3 augments the Ca2+- and PS-dependent membrane binding of PKCα-C2 by slowing its membrane dissociation. Molecular dynamics simulations also support that Ca2+-dependent PS binding is essential for membrane interactions of PKCα-C2. PtdIns(4,5)P2 alone cannot drive the membrane attachment of the domain but further stabilizes the Ca2+- and PS-dependent membrane binding. When the fluorescence protein-tagged PKCα-C2 was expressed in NIH-3T3 cells, mutations of phosphoinositide-binding residues or depletion of PtdIns(4,5)P2 and/or PtdIns(3,4,5)P3 from PM did not significantly affect the PM association of the domain but accelerated its dissociation from PM. Also, local synthesis of PtdIns(4,5)P2 or PtdIns(3,4,5)P3 at the PM slowed membrane dissociation of PKCα-C2. Collectively, these studies show that PtdIns(4,5)P2 and PtdIns(3,4,5)P3 augment the Ca2+- and PS-dependent membrane binding of PKCα-C2 by elongating the membrane residence of the domain but cannot drive the PM recruitment of PKCα-C2. These studies also suggest that effective PM recruitment of many cellular proteins may require synergistic actions of PS and phosphoinositides.

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U2 - 10.1074/jbc.M802617200

DO - 10.1074/jbc.M802617200

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JF - Journal of Biological Chemistry

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