Home LiteratureArticle Details
PMID: 18621733 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

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 PKCalpha C2 domain.

The Journal of biological chemistry ·Vol. 283 ·No. 38 ·2008-09-19 ·Pages 26047-58

Manna D, Bhardwaj N, Vora MS, Stahelin RV, Lu H, Cho W

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)P(2)), and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) 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 Ca(2+)-dependent C2 domain of protein kinase Calpha (PKCalpha) that is known to bind PS and phosphoinositides. In vitro membrane binding measurements by surface plasmon resonance analysis show that PKCalpha-C2 nonspecifically binds phosphoinositides, including PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3), but that PS and Ca(2+) binding is prerequisite for productive phosphoinositide binding. PtdIns(4,5)P(2) or PtdIns(3,4,5)P(3) augments the Ca(2+)- and PS-dependent membrane binding of PKCalpha-C2 by slowing its membrane dissociation. Molecular dynamics simulations also support that Ca(2+)-dependent PS binding is essential for membrane interactions of PKCalpha-C2. PtdIns(4,5)P(2) alone cannot drive the membrane attachment of the domain but further stabilizes the Ca(2+)- and PS-dependent membrane binding. When the fluorescence protein-tagged PKCalpha-C2 was expressed in NIH-3T3 cells, mutations of phosphoinositide-binding residues or depletion of PtdIns(4,5)P(2) and/or PtdIns(3,4,5)P(3) 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)P(2) or PtdIns(3,4,5)P(3) at the PM slowed membrane dissociation of PKCalpha-C2. Collectively, these studies show that PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3) augment the Ca(2+)- and PS-dependent membrane binding of PKCalpha-C2 by elongating the membrane residence of the domain but cannot drive the PM recruitment of PKCalpha-C2. These studies also suggest that effective PM recruitment of many cellular proteins may require synergistic actions of PS and phosphoinositides.

MeSH Terms
Animals Biophysics/methods Cell Membrane/metabolism Cell Movement Gene Expression Regulation, Enzymologic Mice Mutation NIH 3T3 Cells Phosphatidylinositol 4,5-Diphosphate/metabolism Phosphatidylinositol Phosphates/metabolism Phosphatidylserines/chemistry Protein Binding Protein Kinase C-alpha/chemistry,metabolism Protein Structure, Tertiary Protein Transport
Chemicals
Phosphatidylinositol 4,5-Diphosphate Phosphatidylinositol Phosphates Phosphatidylserines phosphatidylinositol 3,4,5-triphosphate Protein Kinase C-alpha
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Manna Debasis
Department of Bioengineering, University of Illinois, Chicago, Illinois 60607, USA.
Bhardwaj Nitin
Vora Mohsin S
Stahelin Robert V
Lu Hui
Cho Wonhwa
References (49)
49 references, click to expand
  1. Mechanism of specific membrane targeting by C2 domains: localized pools of target lipids enhance Ca2+ affinity.
    Biochemistry. 2007 Apr 10;46(14):4322-36 PMID: 17367165
  2. Identification of the phosphatidylserine binding site in the C2 domain that is important for PKC alpha activation and in vivo cell localization.
    Biochemistry. 2001 Nov 20;40(46):13898-905 PMID: 11705379
  3. PI(3,4,5)P3 and PI(4,5)P2 lipids target proteins with polybasic clusters to the plasma membrane.
    Science. 2006 Dec 1;314(5804):1458-61 PMID: 17095657
  4. Membrane binding assays for peripheral proteins.
    Anal Biochem. 2001 Sep 15;296(2):153-61 PMID: 11554709
  5. An inducible translocation strategy to rapidly activate and inhibit small GTPase signaling pathways.
    Nat Methods. 2005 Jun;2(6):415-8 PMID: 15908919
  6. Membrane phosphatidylserine regulates surface charge and protein localization.
    Science. 2008 Jan 11;319(5860):210-3 PMID: 18187657
  7. The mechanism of membrane targeting of human sphingosine kinase 1.
    J Biol Chem. 2005 Dec 30;280(52):43030-8 PMID: 16243846
  8. The C2 domain of PKCalpha is a Ca2+ -dependent PtdIns(4,5)P2 sensing domain: a new insight into an old pathway.
    J Mol Biol. 2006 Oct 6;362(5):901-14 PMID: 16949603
  9. The Amber biomolecular simulation programs.
    J Comput Chem. 2005 Dec;26(16):1668-88 PMID: 16200636
  10. Development and testing of a general amber force field.
    J Comput Chem. 2004 Jul 15;25(9):1157-74 PMID: 15116359
  11. Specific translocation of protein kinase Calpha to the plasma membrane requires both Ca2+ and PIP2 recognition by its C2 domain.
    Mol Biol Cell. 2006 Jan;17(1):56-66 PMID: 16236797
  12. Molecular dynamics simulation of lipid reorientation at bilayer edges.
    Biophys J. 2004 Jun;86(6):3744-9 PMID: 15189870
  13. Molecular dynamics simulation of proton transport near the surface of a phospholipid membrane.
    Biophys J. 2002 Mar;82(3):1460-8 PMID: 11867461
  14. Contrasting membrane interaction mechanisms of AP180 N-terminal homology (ANTH) and epsin N-terminal homology (ENTH) domains.
    J Biol Chem. 2003 Aug 1;278(31):28993-9 PMID: 12740367
  15. Ca(2+) bridges the C2 membrane-binding domain of protein kinase Calpha directly to phosphatidylserine.
    EMBO J. 1999 Nov 15;18(22):6329-38 PMID: 10562545
  16. VMD: visual molecular dynamics.
    J Mol Graph. 1996 Feb;14(1):33-8, 27-8 PMID: 8744570
  17. Differential roles of ionic, aliphatic, and aromatic residues in membrane-protein interactions: a surface plasmon resonance study on phospholipases A2.
    Biochemistry. 2001 Apr 17;40(15):4672-8 PMID: 11294634
  18. Metabolism and functions of phosphatidylserine.
    Prog Lipid Res. 2005 Jul;44(4):207-34 PMID: 15979148
  19. Roles of calcium ions in the membrane binding of C2 domains.
    Biochem J. 2001 Nov 1;359(Pt 3):679-85 PMID: 11672443
  20. Mutagenesis of the C2 domain of protein kinase C-alpha. Differential roles of Ca2+ ligands and membrane binding residues.
    J Biol Chem. 1998 Jul 10;273(28):17544-52 PMID: 9651347
  21. Molecular dynamics study of a gelsolin-derived peptide binding to a lipid bilayer containing phosphatidylinositol 4,5-bisphosphate.
    Biopolymers. 2003;71(1):49-70 PMID: 12712500
  22. Molecular dynamics simulation of a palmitoyl-oleoyl phosphatidylserine bilayer with Na+ counterions and NaCl.
    Biophys J. 2004 Mar;86(3):1601-9 PMID: 14990486
  23. Membrane binding and subcellular targeting of C2 domains.
    Biochim Biophys Acta. 2006 Aug;1761(8):838-49 PMID: 16945584
  24. The molecular basis of differential subcellular localization of C2 domains of protein kinase C-alpha and group IVa cytosolic phospholipase A2.
    J Biol Chem. 2003 Apr 4;278(14):12452-60 PMID: 12531893
  25. Electrostatic control of the membrane targeting of C2 domains.
    Mol Cell. 2002 Jan;9(1):145-54 PMID: 11804593
  26. Scalable molecular dynamics with NAMD.
    J Comput Chem. 2005 Dec;26(16):1781-802 PMID: 16222654
  27. Molecular dynamics simulation of melittin in a dimyristoylphosphatidylcholine bilayer membrane.
    Biophys J. 1998 Oct;75(4):1603-18 PMID: 9746504
  28. The C2 domains of synaptotagmin--partners in exocytosis.
    Trends Biochem Sci. 2004 Mar;29(3):143-51 PMID: 15003272
  29. Membrane targeting by C1 and C2 domains.
    J Biol Chem. 2001 Aug 31;276(35):32407-10 PMID: 11432875
  30. Fast, efficient generation of high-quality atomic charges. AM1-BCC model: II. Parameterization and validation.
    J Comput Chem. 2002 Dec;23(16):1623-41 PMID: 12395429
  31. Mechanistic basis of differential cellular responses of phosphatidylinositol 3,4-bisphosphate- and phosphatidylinositol 3,4,5-trisphosphate-binding pleckstrin homology domains.
    J Biol Chem. 2007 Nov 2;282(44):32093-105 PMID: 17823121
  32. Recruitment and regulation of phosphatidylinositol phosphate kinase type 1 gamma by the FERM domain of talin.
    Nature. 2002 Nov 7;420(6911):85-9 PMID: 12422219
  33. Membrane-protein interactions in cell signaling and membrane trafficking.
    Annu Rev Biophys Biomol Struct. 2005;34:119-51 PMID: 15869386
  34. Molecular dynamics simulation study of the interaction of trehalose with lipid membranes.
    Langmuir. 2004 Aug 31;20(18):7844-51 PMID: 15323539
  35. Subcellular targeting by membrane lipids.
    Curr Opin Cell Biol. 2001 Apr;13(2):146-52 PMID: 11248547
  36. The C2 domains of classical PKCs are specific PtdIns(4,5)P2-sensing domains with different affinities for membrane binding.
    J Mol Biol. 2007 Aug 17;371(3):608-21 PMID: 17586528
  37. Plasma membrane phosphoinositide organization by protein electrostatics.
    Nature. 2005 Dec 1;438(7068):605-11 PMID: 16319880
  38. C2 domain of protein kinase C alpha: elucidation of the membrane docking surface by site-directed fluorescence and spin labeling.
    Biochemistry. 2003 Feb 11;42(5):1254-65 PMID: 12564928
  39. Mechanism of diacylglycerol-induced membrane targeting and activation of protein kinase Cdelta.
    J Biol Chem. 2004 Jul 9;279(28):29501-12 PMID: 15105418
  40. Use of EPR power saturation to analyze the membrane-docking geometries of peripheral proteins: applications to C2 domains.
    Annu Rev Biophys Biomol Struct. 2005;34:71-90 PMID: 15869384
  41. Automatic atom type and bond type perception in molecular mechanical calculations.
    J Mol Graph Model. 2006 Oct;25(2):247-60 PMID: 16458552
  42. MeTaDoR: a comprehensive resource for membrane targeting domains and their host proteins.
    Bioinformatics. 2007 Nov 15;23(22):3110-2 PMID: 17720983
  43. Atomic detail peptide-membrane interactions: molecular dynamics simulation of gramicidin S in a DMPC bilayer.
    Biophys J. 2000 Oct;79(4):1718-30 PMID: 11023880
  44. Building signaling complexes at the membrane.
    Sci STKE. 2006 Feb 07;2006(321):pe7 PMID: 16467194
  45. Translocation and reversible localization of signaling proteins: a dynamic future for signal transduction.
    Cell. 2000 Oct 13;103(2):181-4 PMID: 11057890
  46. PIP(2) and proteins: interactions, organization, and information flow.
    Annu Rev Biophys Biomol Struct. 2002;31:151-75 PMID: 11988466
  47. Molecular dynamics simulation of dipalmitoylphosphatidylcholine membrane with cholesterol sulfate.
    Biophys J. 2000 Apr;78(4):1672-80 PMID: 10733950
  48. Direct observation of Bin/amphiphysin/Rvs (BAR) domain-induced membrane curvature by means of molecular dynamics simulations.
    Proc Natl Acad Sci U S A. 2006 Oct 10;103(41):15068-72 PMID: 17008407
  49. A new phosphatidylinositol 4,5-bisphosphate-binding site located in the C2 domain of protein kinase Calpha.
    J Biol Chem. 2003 Feb 14;278(7):4972-80 PMID: 12426311
Article Info
Journal
The Journal of biological chemistry
Abbr.
J Biol Chem
ISSN
0021-9258
DOI
10.1074/jbc.M802617200
Published
2008-09-19
Epub
2008-00-11
Pages
26047-58
Language
English
Region
United States
NLM ID
2985121R
PMCID
PMC2533782
Subset
IM
Grants
NIGMS NIH HHS · GM52598 · United States
NIGMS NIH HHS · GM68849 · United States
NIGMS NIH HHS · GM76581 · United States
NIAID NIH HHS · P01AI060915 · United States
External Links
PubMed source DOI: 10.1074/jbc.M802617200 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: product@genelibs.com

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2