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PMID: 15041659 Published · ppublish English Journal Article Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

Electrostatic sequestration of PIP2 on phospholipid membranes by basic/aromatic regions of proteins.

Biophysical journal ·Vol. 86 ·No. 4 ·2004-04-00 ·Pages 2188-207

Gambhir A, Hangyás-Mihályné G, Zaitseva I, Cafiso DS, Wang J, Murray D, Pentyala SN, Smith SO, McLaughlin S

Abstract

The basic effector domain of myristoylated alanine-rich C kinase substrate (MARCKS), a major protein kinase C substrate, binds electrostatically to acidic lipids on the inner leaflet of the plasma membrane; interaction with Ca2+/calmodulin or protein kinase C phosphorylation reverses this binding. Our working hypothesis is that the effector domain of MARCKS reversibly sequesters a significant fraction of the L-alpha-phosphatidyl-D-myo-inositol 4,5-bisphosphate (PIP2) on the plasma membrane. To test this, we utilize three techniques that measure the ability of a peptide corresponding to its effector domain, MARCKS(151-175), to sequester PIP2 in model membranes containing physiologically relevant fractions (15-30%) of the monovalent acidic lipid phosphatidylserine. First, we measure fluorescence resonance energy transfer from Bodipy-TMR-PIP2 to Texas Red MARCKS(151-175) adsorbed to large unilamellar vesicles. Second, we detect quenching of Bodipy-TMR-PIP2 in large unilamellar vesicles when unlabeled MARCKS(151-175) binds to vesicles. Third, we identify line broadening in the electron paramagnetic resonance spectra of spin-labeled PIP2 as unlabeled MARCKS(151-175) adsorbs to vesicles. Theoretical calculations (applying the Poisson-Boltzmann relation to atomic models of the peptide and bilayer) and experimental results (fluorescence resonance energy transfer and quenching at different salt concentrations) suggest that nonspecific electrostatic interactions produce this sequestration. Finally, we show that the PLC-delta1-catalyzed hydrolysis of PIP2, but not binding of its PH domain to PIP2, decreases markedly as MARCKS(151-175) sequesters most of the PIP2.

MeSH Terms
Amino Acid Sequence Cell Membrane/chemistry Fluorescence Resonance Energy Transfer Intracellular Signaling Peptides and Proteins Lipid Bilayers Lipoproteins/chemistry Membrane Proteins/chemistry Molecular Sequence Data Myristoylated Alanine-Rich C Kinase Substrate Phosphatidylcholines/chemistry Phosphatidylinositol 4,5-Diphosphate/chemistry Phosphatidylserines/chemistry Phospholipids/chemistry Type C Phospholipases/chemistry
Chemicals
Intracellular Signaling Peptides and Proteins Lipid Bilayers Lipoproteins Membrane Proteins Phosphatidylcholines Phosphatidylinositol 4,5-Diphosphate Phosphatidylserines Phospholipids Myristoylated Alanine-Rich C Kinase Substrate Type C Phospholipases
Authors & Affiliations
9 authors, click to expand affiliations / ORCID
Gambhir Alok
Department of Physics and Astronomy, SUNY Stony Brook, Stony Brook, New York 11794, USA.
Hangyás-Mihályné Gyöngyi
Zaitseva Irina
Cafiso David S
Wang Jiyao
Murray Diana
Pentyala Srinivas N
Smith Steven O
McLaughlin Stuart
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Article Info
Journal
Biophysical journal
Abbr.
Biophys J
ISSN
0006-3495
DOI
10.1016/S0006-3495(04)74278-2
Published
2004-04-00
Pages
2188-207
Language
English
Region
United States
NLM ID
0370626
PMCID
PMC1304070
Subset
IM
Grants
NIGMS NIH HHS · R37 GM024971 · United States
NIGMS NIH HHS · R01 GM069651 · United States
NIGMS NIH HHS · GM24971 · United States
NIGMS NIH HHS · GM62305 · United States
NIGMS NIH HHS · GM69651 · United States
NIGMS NIH HHS · T32 GM008444 · United States
NIGMS NIH HHS · R01 GM024971 · United States
NIGMS NIH HHS · R01 GM062305 · United States
External Links
PubMed source DOI: 10.1016/S0006-3495(04)74278-2 PMC Full Text View journal details
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