Lateral Sequestration of Phosphatidylinositol 4,5-Bisphosphate by the Basic Effector Domain of Myristoylated Alanine-rich C Kinase Substrate Is Due to Nonspecific Electrostatic Interactions
2002; Elsevier BV; Volume: 277; Issue: 37 Linguagem: Inglês
10.1074/jbc.m203954200
ISSN1083-351X
AutoresJiyao Wang, Alok Gambhir, Gyo ̈ngyi Hangyás-Mihályneá, Diana Murray, Urszula Golebiewska, Stuart McLaughlin,
Tópico(s)Protein Structure and Dynamics
ResumoA peptide corresponding to the basic (+13), unstructured effector domain of myristoylated alanine-rich C kinase substrate (MARCKS) binds strongly to membranes containing phosphatidylinositol 4,5-bisphosphate (PIP2). Although aromatic residues contribute to the binding, three experiments suggest the binding is driven mainly by nonspecific local electrostatic interactions. First, peptides with 13 basic residues, Lys-13 and Arg-13, bind to PIP2-containing vesicles with the same high affinity as the effector domain peptide. Second, removing basic residues from the effector domain peptide reduces the binding energy by an amount that correlates with the number of charges removed. Third, peptides corresponding to a basic region in GAP43 and MARCKS effector domain-like regions in other proteins (e.g. MacMARCKS, adducin, Drosophila A kinase anchor protein 200, and N-methyl-d-aspartate receptor) also bind with an energy that correlates with the number of basic residues. Kinetic measurements suggest the effector domain binds to several PIP2. Theoretical calculations show the effector domain produces a local positive potential, even when bound to a bilayer with 33% monovalent acidic lipids, and should thus sequester PIP2 laterally. This electrostatic sequestration was observed experimentally using a phospholipase C assay. Our results are consistent with the hypothesis that MARCKS could reversibly sequester much of the PIP2 in the plasma membrane. A peptide corresponding to the basic (+13), unstructured effector domain of myristoylated alanine-rich C kinase substrate (MARCKS) binds strongly to membranes containing phosphatidylinositol 4,5-bisphosphate (PIP2). Although aromatic residues contribute to the binding, three experiments suggest the binding is driven mainly by nonspecific local electrostatic interactions. First, peptides with 13 basic residues, Lys-13 and Arg-13, bind to PIP2-containing vesicles with the same high affinity as the effector domain peptide. Second, removing basic residues from the effector domain peptide reduces the binding energy by an amount that correlates with the number of charges removed. Third, peptides corresponding to a basic region in GAP43 and MARCKS effector domain-like regions in other proteins (e.g. MacMARCKS, adducin, Drosophila A kinase anchor protein 200, and N-methyl-d-aspartate receptor) also bind with an energy that correlates with the number of basic residues. Kinetic measurements suggest the effector domain binds to several PIP2. Theoretical calculations show the effector domain produces a local positive potential, even when bound to a bilayer with 33% monovalent acidic lipids, and should thus sequester PIP2 laterally. This electrostatic sequestration was observed experimentally using a phospholipase C assay. Our results are consistent with the hypothesis that MARCKS could reversibly sequester much of the PIP2 in the plasma membrane. 5)P2, phosphatidylinositol 4,5-bisphosphate 4)P2, phosphatidylinositol 3,4-bisphosphate inositol 1,4,5-trisphosphate phosphatidylcholine phosphatidylserine myristoylated alanine-rich C kinase substrate a peptide corresponding to residues 151−175 of bovine MARCKS a peptide lacking 3 Lys residues at the C terminus of MARCKS-(151–175) a peptide lacking 5 Lys residues at the N terminus of MARCKS-(151–175) a peptide lacking 3 Lys residues at the C terminus and 5 Lys residues at the N-terminus of MARCKS-(151–175) a peptide with all 5 Phe replaced by Ala in MARCKS-(151–175) macrophage-enriched myristoylated alanine-rich C kinase substrate Drosophila A kinase anchor protein 200 N-methyl-d-aspartate receptor growth-associated protein of Mr 43,000 cortical cytoskeleton-associated protein of approximate molecular mass 23 kDa neuronal Wiskott-Aldrich Syndrome protein secretory carrier membrane protein 2 A-kinase anchoring protein 79 phosphatidylcholine-specific phospholipase D phosphoinositide-specific phospholipase C pleckstrin homology PH domain of PLC-δ1 protein kinase C N-ethylmaleimide large unilamellar vesicle multilamellar vesicle nonlinear Poisson-Boltzmann matrix-assisted laser desorption ionization N,N-dimethylformamide 4-morpholinepropanesulfonic acid nonlinear Poisson-Boltzmann Phosphatidylinositol 4,5-bisphosphate (PIP2)1 plays many important roles in cells (reviewed in Refs. 1Cremona O. 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The membrane-bound basic effector domain produces a significant positive electrostatic potential that can act as a basin of attraction for multivalent acidic lipids such as PIP2 (8McLaughlin S. Wang J. Gambhir A. Murray D. Annu. Rev. Biophys. Biomol. Struct. 2002; 31: 151-175Crossref PubMed Scopus (662) Google Scholar). The electrostatic sequestration of PIP2 can be reversed either by the binding of calcium/calmodulin to the effector domain or by the PKC phosphorylation of the effector domain, which decreases the positive electrostatic potential (33Murray D. Arbuzova A. Honig B. McLaughlin S. Curr. Top. Membr. 2002; 52: 271-302Google Scholar). MARCKS has an extended conformation in solution (34Matsubara M. Yamauchi E. Hayashi N. Taniguchi H. FEBS Lett. 1998; 421: 203-207Crossref PubMed Scopus (36) Google Scholar, 35Manenti S. Sorokine O. Van Dorsselaer A. Taniguchi H. J. Biol. 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Morris A. Rebecchi M. Scarlata S.F. Runnels L.W. Prestwich G.D. Chen J. Aderem A. Ahn J. McLaughlin S. J. Biol. Chem. 1996; 271: 26187-26193Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar) inhibit the PLC-catalyzed hydrolysis of PIP2. In the work reported here, we investigated whether the binding of MARCKS-(151–175) to PIP2 is due to nonspecific electrostatic interactions. We used three approaches to determine whether the binding depends on specific residues or just on the number of basic residues. We measured the binding of truncated versions of MARCKS-(151–175) (i.e. peptides missing basic residues from the N- and/or C-terminal regions) to PC/PIP2 vesicles. We also measured the binding of peptides corresponding to basic regions in other proteins (macrophage-enriched myristoylated alanine-rich C kinase substrate (MacMARCKS), adducin, Drosophila A kinase anchor protein 200 (DAKAP200), N-methyl-d-aspartate (NMDA) receptor, and growth-associated protein ofMr 43,000 (GAP43)) to PC/PIP2vesicles to determine whether the binding correlates with the number of basic residues. We compared the binding of peptides with 13 Lys or 13 Arg residues to investigate whether the chemical nature of the basic residues affects the interaction with PIP2. We further tested the hypothesis that several PIP2 diffuse together to form a binding site for MARCKS-(151–175) (8McLaughlin S. Wang J. Gambhir A. Murray D. Annu. Rev. Biophys. Biomol. Struct. 2002; 31: 151-175Crossref PubMed Scopus (662) Google Scholar, 25Wang J. Arbuzova A. Hangyas-Mihalyne G. McLaughlin S. J. Biol. 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The ammonium salt ofl-α-phosphatidyl-d-myo-inositol 4,5-bisphosphate (PIP2) was purchased either from Avanti Polar Lipids (Alabaster, AL) or Roche Molecular Biochemicals or purified from bovine brain extract (Sigma) as described elsewhere (42Morris A.J. Rudge S.A. Mahlum C.E. Jenco J.M. Mol. Pharmacol. 1995; 48: 532-539PubMed Google Scholar). Labeled [dioleoyl-1-14C]l-α-dioleoylphosphatidylcholine ([14C]DOPC), [inositol-2-3H]l-α-phosphatidyl-d-myoinositol 4,5-bisphosphate ([3H]PIP2), and [ethyl-1,2-3H]N-ethylmaleimide ([3H]NEM) were from PerkinElmer Life Sciences. Non-radioactive N-ethylmaleimide (NEM) was from Sigma. 6-Acryloyl-2-dimethylaminonaphthalene (acrylodan) was from Molecular Probes, Inc. (Eugene, OR). Recombinant human PLC-δ1 was purified from Escherichia coli as described elsewhere (43Tall E. Dorman G. Garcia P. Runnels L. Shah S. Chen J. Profit A., Gu, Q.M. Chaudhary A. Prestwich G.D. Rebecchi M.J. Biochemistry. 1997; 36: 7239-7248Crossref PubMed Scopus (60) Google Scholar). Unless specified, all peptides (sequences listed in Table I) were obtained from American Peptide Co., Inc. (Sunnyvale, CA), and were determined to be >80% pure by reverse phase-high pressure liquid chromatography and MALDI-time-of-flight mass spectroscopy. Each peptide was blocked with an acetyl group at its N terminus and an amide group at its C terminus. A peptide corresponding to a basic region in secretory carrier membrane protein 2 (SCAMP2-(201–211)) was a generous gift from Prof. David Cafiso; its N terminus is unblocked, introducing an extra positive charge.Table ISequences of peptidesPeptideSequenceRef.MARCKS peptides MARCKS-(151–175)CKKKKKRFSFKKSFKLSGFSFKKNKK26Aderem A. Cell. 1992; 71: 713-716Abstract Full Text PDF PubMed Scopus (423) Google Scholar,27Blackshear P.J. J. Biol. Chem. 1993; 268: 1501-1504Abstract Full Text PDF PubMed Google Scholar ΔC-MARCKSCKKKKKRFSFKKSFKLSGFSFK ΔN-MARCKSCRFSFKKSFKLSGFSFKKNKK ΔNΔC-MARCKSCRFSFKKSFKLSGFSFK FA-MARCKSCKKKKKRASAKKSAKLSGASAKKNKKPoly-Lys/Arg peptides Arg-13CRRRRRRRRRRRRR Lys-13CKKKKKKKKKKKKK Lys-10CKKKKKKKKKK Lys-7CKKKKKKKMARCKS-like peptides MacMARCKS-(87–110)CKKKKKFSFKKPFKLSGLSFKRNRK86Verghese G.M. Johnson J.D. Vasulka C. Haupt D.M. Stumpo D.J. Blackshear P.J. J. Biol. Chem. 1994; 269: 9361-9367Abstract Full Text PDF PubMed Google Scholar,111Blackshear P.J. Verghese G.M. Johnson J.D. Haupt D.M. Stumpo D.J. J. Biol. Chem. 1992; 267: 13540-13546Abstract Full Text PDF PubMed Google Scholar Adducin-(717–734)CKKKKKFRTPSFLKKSKKK88Matsuoka Y. Hughes C.A. Bennett V. J. Biol. Chem. 1996; 271: 25157-25166Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar DAKAP200-(119–141)CKSKSKKDKVKKKWSFRSISFGKK81Rossi E.A., Li, Z. Feng H. Rubin C.S. J. Biol. Chem. 1999; 274: 27201-27210Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar NMDA-NR1-(875–898)CKKKATFRAITSTLASSFKRRRSSK89Ehlers M.D. Zhang S. Bernhadt J.P. Huganir R.L. Cell. 1996; 84: 745-755Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar,90Tingley W.G. Ehlers M.D. Kameyama K. Doherty C. Ptak J.B. Riley C.T. Huganir R.L. J. Biol. Chem. 1997; 272: 5157-5166Abstract Full Text Full Text PDF PubMed Scopus (433) Google ScholarGMC family peptides GAP43-(30–56)CKAHKAATKIQASFRGHITRKKLKGEKK93Skene J.H. Annu. Rev. Neurosci. 1989; 12: 127-156Crossref PubMed Google Scholar CAP23-(1–13)CGKLSKKKKGYSV112Widmer F. Caroni P. J. Cell Biol. 1990; 111: 3035-3047Crossref PubMed Scopus (56) Google ScholarOther peptides PLD2-(554–575)CRDLARHFIQRWNFTKTTKARYK113Sciorra V.A. Rudge S.A. Prestwich G.D. Frohman M.A. Engebrecht J. Morris A.J. EMBO J. 1999; 18: 5911-5921Crossref PubMed Scopus (145) Google Scholar SCAMP2-(201–211)CWYRPIYKAFR114Singleton D.R., Wu, T.T. Castle J.D. J. Cell Sci. 1997; 110: 2099-2107Crossref PubMed Google Scholar N-WASP-(181–197)CNISHTKEKKKGKAKKKR115Miki H. Miura K. Takenawa T. EMBO J. 1996; 15: 5326-5335Crossref PubMed Google Scholar, 116Prehoda K.E. Scott J.A. Dyche Mullins R. Lim W.A. Science. 2000; 290: 801-806Crossref PubMed Scopus (395) Google Scholar, 117Rohatgi R., Ho, H.Y. Kirschner M.W. J. Cell Biol. 2000; 150: 1299-1310Crossref PubMed Scopus (471) Google Scholar WASP-(223–232)CADKKRSGKKK115Miki H. Miura K. Takenawa T. EMBO J. 1996; 15: 5326-5335Crossref PubMed Google Scholar,118Higgs H.N. Pollard T.D. J. Cell Biol. 2000; 150: 1311-1320Crossref PubMed Scopus (398) Google Scholar Syndecan-4-(188–194)CKKPIYKK95Horowitz A. Murakami M. Gao Y. Simons M. Biochemistry. 1999; 38: 15871-15877Crossref PubMed Scopus (75) Google ScholarBasic residues Lys and Arg are in bold. Acidic residues Asp and Glu are in italics. Aromatic residues Phe, Trp, and Tyr are underlined. Unless specified, the N terminus of each peptide is blocked with acetyl and the C terminus is blocked with amide. The exception is that the N terminus of SCAMP2-(201–211) is unblocked, introducing an extra positive charge. The sequences of the basic regions in these proteins are documented in the references indicated. Open table in a new tab Basic residues Lys and Arg are in bold. Acidic residues Asp and Glu are in italics. Aromatic residues Phe, Trp, and Tyr are underlined. Unless specified, the N terminus of each peptide is blocked with acetyl and the C terminus is blocked with amide. The exception is that the N terminus of SCAMP2-(201–211) is unblocked, introducing an extra positive charge. The sequences of the basic regions in these proteins are documented in the references indicated. In the binding and kinetic measurements, we used peptides with an extra Cys at the N terminus, which permitted covalent attachment of either a radioactive ([3H]NEM) or a fluorescent (acrylodan) label. In the ζ potential measurements, we used MARCKS-(151–175) and Lys-13 without the extra N-terminal Cys. In the PLC assays, we used MARCKS-(151–175) and a peptide corresponding to a basic region in phosphatidylcholine-specific phospholipase D 2 (PLD2-(554–575)) without the extra N-terminal Cys, but GAP43-(30–56), ΔNΔC-MARCKS, Lys-7, and SCAMP2-(201–211) had the extra N-terminal Cys. We added 1 mm dithiothreitol to the buffer to avoid the formation of disulfide bonds when the peptides contain the extra Cys. MARCKS-(151–175) with or without the extra Cys had the same effect on the PLC-catalyzed hydrolysis of PIP2. We used a protocol modified from Molecular Probes (Conjugation with Thiol-reactive Probes) to label peptides with a thiol-reactive fluorescent acrylodan probe, as described in detail elsewhere (44Arbuzova A. Wang J. Murray D. Jacob J. Cafiso D.S. McLaughlin S. J. Biol. Chem. 1997; 272: 27167-27177Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Briefly, we mixed 1 ml of ∼1 mm peptide in 10 mmK2HPO4/KH2PO4, pH 7.0, with acrylodan probe dissolved in DMF (mole ratio of 1.5:1 acrylodan/peptide) for 1 h, purified the labeled peptide using high pressure liquid chromatography, and checked its purity with MALDI mass spectrometry (CASM, State University of New York, Stony Brook). We labeled peptides with radioactive [3H]NEM as described previously (45Arbuzova A. Wang L. Wang J. Hangyas-Mihalyne G. Murray D. Honig B. McLaughlin S. Biochemistry. 2000; 39: 10330-10339Crossref PubMed Scopus (151) Google Scholar). Briefly, we placed 250 μCi of [3H]NEM in pentane on top of 20 μl of DMF, evaporated the pentane with argon gas, and then mixed the [3H]NEM in DMF with 1 ml of an ∼1 mm peptide solution; this procedure labeled ∼1% of the peptide. We added to the solution containing the labeled peptide an excess of non-radioactive NEM (mole ratio of 1.5:1 NEM/peptide) to block the unlabeled Cys. We used multilamellar vesicles (MLVs) for ζ potential measurements, 100 nm diameter large unilamellar vesicles (LUVs) for fluorescence measurements, and sucrose-loaded LUVs for centrifugation experiments as described in detail elsewhere (25Wang J. Arbuzova A. Hangyas-Mihalyne G. McLaughlin S. J. Biol. Chem. 2001; 276: 5012-5019Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar,46Buser C.A. McLaughlin S. Methods Mol. Biol. 1998; 84: 267-281PubMed Google Scholar). In our experience, the protocol for preparing MLVs must be followed carefully to ensure a uniform distribution of PIP2in PC/PIP2 (99:1) vesicles (either LUV or MLV). We measure the ζ potential of several individual vesicles to assess the uniformity of the MLV preparations. The protocol described below consistently produces a population of vesicles that move with similar velocity in an electric field, i.e. the ζ potentials due to the negatively charged PIP2 are similar. The critical step is to produce a dried lipid film in which PC and PIP2 are mixed uniformly. We add solutions of PIP2 (it is important to use the ammonium salt, which is more soluble in chloroform than the sodium salt (47Toner M. Vaio G. McLaughlin A. McLaughlin S. Biochemistry. 1988; 27: 7435-7443Crossref PubMed Google Scholar, 48Gabev E. Kasianowicz J. Abbott T. McLaughlin S. Biochim. Biophys. Acta. 1989; 979: 105-112Crossref PubMed Google Scholar)) and PC in chloroform (typically 500 μl total volume) to a 50-ml round-bottom flask, which is then well immersed in a 30–35 °C water bath and attached to a rotary evaporator. The flask is rotated without vacuum for 5 min to warm the solution and speed the subsequent evaporation under vacuum. Deleting this warming step can produce a strikingly nonuniform distribution of PIP2 in the MLVs (as indicated by ζ potential measurements), presumably because PIP2 is less soluble in chloroform than PC and, if the chloroform does not evaporate rapidly, forms a layer on the bottom of the flask. We next applied the maximum vacuum that does not boil the chloroform to evaporate most of the solvent (∼1 min) and then applied full vacuum for 30 min to remove all traces of chloroform. Electrophoresis measurements indicated that most of the MLVs produced using this protocol do indeed contain 1% PIP2; specifically, the ζ potentials of the PC/PIP2 MLVs made in this way had low standard deviation (see e.g. Fig. 7 for 98:2 PC/PIP2 MLVs). The PC/PIP2 LUVs, which are made by extrusion of MLVs, presumably also have a uniform fraction of PIP2. We measured the binding of [3H]NEM-labeled peptides (Table I) to sucrose-loaded PC/PIP2 LUVs using the centrifugation technique described previously (25Wang J. Arbuzova A. Hangyas-Mihalyne G. McLaughlin S. J. Biol. Chem. 2001; 276: 5012-5019Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 46Buser C.A. McLaughlin S. Methods Mol. Biol. 1998; 84: 267-281PubMed Google Scholar). Briefly, sucrose-loaded PC/PIP2 LUVs were mixed with trace concentrations of [3H]NEM-labeled peptides (typically 2–10 nm). The mixture was centrifuged at 100,000 × g for 1 h. We calculated the percentage of peptide bound from the radioactivity of the peptide in the supernatant and in the pellet. We use a molar partition coefficient K (49, 50) to describe the binding of the peptide to lipid vesicles without making assumptions about the absorption mechanism. The molar partition coefficientK is defined by the equation: [P]m/[L] = K[P], where [P]m is the molar concentration of peptide partitioned onto the membrane, [P] is the molar concentration of free peptide in the bulk aqueous phase, and [L] is the molar concentration of lipid accessible to the peptide. Under our conditions, [L] ≫ [P]m. Thus [L] does not change significantly after the peptide binds and is approximately one-half of the total lipid concentration for the LUVs because the peptide interacts only with the outer leaflet of the bilayer (the peptides are added to a solution of preformed vesicles). Combining the definition ofK with the equation [P]tot = [P]m + [P], we get Equation 1.[P]m[P]tot=K[L]1+K[L]Equation 1 Note that this equation for the molar partition coefficientK has the same form as the equation for the association constant if we assume (incorrectly) that the peptide forms a 1:1 complex with a lipid (51Ben-Tal N. Honig B. Peitzsch R.M. Denisov G. McLaughlin S. Biophys. J. 1996; 71: 561-575Abstract Full Text PDF PubMed Google Scholar) (for different definitions of partition coefficients, see Refs. 49Peitzsch R.M. McLaughlin S. Biochemistry. 1993; 32: 10436-10443Crossref PubMed Scopus (454) Google Scholar and 52White S.H. Wimley W.C. Annu. Rev. Biophys. Biomol. Struct. 1999; 28: 319-365Crossref PubMed Scopus (1426) Google Scholar). In the biochemical literature it is conventional (e.g. see page 186 in Ref. 53Stryer L. Biochemistry. W. H. Freeman & Co., New York1995Google Scholar) to define the binding free energy ΔG0 = −RTlnK where the standard state is one in which all reactants have a concentration of 1 m. As discussed in detail elsewhere (49Peitzsch R.M. McLaughlin S. Biochemistry. 1993; 32: 10436-10443Crossref PubMed Scopus (454) Google Scholar, 52White S.H. Wimley W.C. Annu. Rev. Biophys. Biomol. Struct. 1999; 28: 319-365Crossref PubMed Scopus (1426) Google Scholar), the relationship between the partition coefficient and the binding energy depends on the units used for the concentration and the definition of the standard state. To facilitate comparison with other studies where different standard states may be used and the free energy may differ by a cratic term (e.g. see Refs. 49Peitzsch R.M. McLaughlin S. Biochemistry. 1993; 32: 10436-10443Crossref PubMed Scopus (454) Google Scholar, 50Ben-Tal N. Honig B. Miller C. McLaughlin S. Biophys. J. 1997; 73: 1717-1727Abstract Full Text PDF PubMed Google Scholar and 52White S.H. Wimley W.C. Annu. Rev. Biophys. Biomol. Struct. 1999; 28: 319-365Crossref PubMed Scopus
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