Membrane-permeable Calmodulin Inhibitors (e.g. W-7/W-13) Bind to Membranes, Changing the Electrostatic Surface Potential
2007; Elsevier BV; Volume: 282; Issue: 11 Linguagem: Inglês
10.1074/jbc.m607211200
ISSN1083-351X
AutoresPiali Sengupta, Marı́a José Ruano, Francesc Tebar, Urszula Golebiewska, Ірина Зайцева, Carlos Enrich, Stuart McLaughlin, Antonio Villalobo,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoMembrane-permeable calmodulin inhibitors, such as the napthalenesulfonamide derivatives W-7/W-13, trifluoperazine, and calmidazolium, are used widely to investigate the role of calcium/calmodulin (Ca2+/CaM) in living cells. If two chemically different inhibitors (e.g. W-7 and trifluoperazine) produce similar effects, investigators often assume the effects are due to CaM inhibition. Zeta potential measurements, however, show that these amphipathic weak bases bind to phospholipid vesicles at the same concentrations as they inhibit Ca2+/CaM; this suggests that they also bind to the inner leaflet of the plasma membrane, reducing its negative electrostatic surface potential. This change will cause electrostatically bound clusters of basic residues on peripheral (e.g. Src and K-Ras4B) and integral (e.g. epidermal growth factor receptor (EGFR)) proteins to translocate from the membrane to the cytoplasm. We measured inhibitor-mediated translocation of a simple basic peptide corresponding to the calmodulin-binding juxtamembrane region of the EGFR on model membranes; W-7/W-13 causes translocation of this peptide from membrane to solution, suggesting that caution must be exercised when interpreting the results obtained with these inhibitors in living cells. We present evidence that they exert dual effects on autophosphorylation of EGFR; W-13 inhibits epidermal growth factor-dependent EGFR autophosphorylation under different experimental conditions, but in the absence of epidermal growth factor, W-13 stimulates autophosphorylation of the receptor in four different cell types. Our interpretation is that the former effect is due to W-13 inhibition of Ca2+/CaM, but the latter results could be due to binding of W-13 to the plasma membrane. Membrane-permeable calmodulin inhibitors, such as the napthalenesulfonamide derivatives W-7/W-13, trifluoperazine, and calmidazolium, are used widely to investigate the role of calcium/calmodulin (Ca2+/CaM) in living cells. If two chemically different inhibitors (e.g. W-7 and trifluoperazine) produce similar effects, investigators often assume the effects are due to CaM inhibition. Zeta potential measurements, however, show that these amphipathic weak bases bind to phospholipid vesicles at the same concentrations as they inhibit Ca2+/CaM; this suggests that they also bind to the inner leaflet of the plasma membrane, reducing its negative electrostatic surface potential. This change will cause electrostatically bound clusters of basic residues on peripheral (e.g. Src and K-Ras4B) and integral (e.g. epidermal growth factor receptor (EGFR)) proteins to translocate from the membrane to the cytoplasm. We measured inhibitor-mediated translocation of a simple basic peptide corresponding to the calmodulin-binding juxtamembrane region of the EGFR on model membranes; W-7/W-13 causes translocation of this peptide from membrane to solution, suggesting that caution must be exercised when interpreting the results obtained with these inhibitors in living cells. We present evidence that they exert dual effects on autophosphorylation of EGFR; W-13 inhibits epidermal growth factor-dependent EGFR autophosphorylation under different experimental conditions, but in the absence of epidermal growth factor, W-13 stimulates autophosphorylation of the receptor in four different cell types. Our interpretation is that the former effect is due to W-13 inhibition of Ca2+/CaM, but the latter results could be due to binding of W-13 to the plasma membrane. The ubiquitous second messenger Ca2+ (1Clapham D.E. Cell. 1995; 80: 259-268Abstract Full Text PDF PubMed Scopus (2272) Google Scholar, 2Berridge M.J. Bootman M.D. Roderick H.L. Nat. Rev. Mol. Cell Biol. 2003; 4: 517-529Crossref PubMed Scopus (4256) Google Scholar) exerts many of its signaling effects by binding to calmodulin (CaM) 4The abbreviations and trivial names used are: CaM, calmodulin; CDZ, calmidazolium; EGF, epidermal growth factor; EGFR, EGF receptor; FCS, fluorescence correlation spectroscopy; GF109203X, 3-4-(1H-indol-3-yl)1H-pyrrole-2,5-dione; GFP, green fluorescent protein; HPLC, high performance liquid chromatography; KN-93, 2-[N-(2-hydroxyethyl)]-N-(4-methoxyben-zenesulfonyl)] amino-N-(4-chlorocinnamyl)-N-methylbenzylamine); JM, juxtamembrane; LUV, large unilamellar vesicle; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MARCKS, myristoylated alanine-rich C kinase substrate; MOPS, 3-(N-morpholino)propanesulfonic acid; PC, phosphatidylcholine; PG, phosphatidylglycerol; PS, phosphatidylserine; TFP, trifluoperazine; W-7, N-(6-aminohexyl)-5-chloro-1-naphthale-nesulfonamide; W-12, N-(4-aminobutyl)-1-naphthalenesulfonamide; W-13, N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide; PAE, porcine aortic endothelial; MLV, multilamellar vesicle. ;Ca2+/calmodulin (Ca2+/CaM) in turn binds to and modulates the function of >100 target proteins (3Chin D. Means A.R. Trends Cell Biol. 2000; 10: 322-328Abstract Full Text Full Text PDF PubMed Scopus (1153) Google Scholar, 4Hoeflich K.P. Ikura M. Cell. 2002; 108: 739-742Abstract Full Text Full Text PDF PubMed Scopus (602) Google Scholar, 5Vetter S.W. Leclerc E. Eur. J. Biochem. 2003; 270: 404-414Crossref PubMed Scopus (287) Google Scholar, 6Yamniuk A.P. Vogel H.J. Mol. Biotechnol. 2004; 27: 33-57Crossref PubMed Google Scholar). Many investigators have used membrane-permeable calmodulin inhibitors (e.g. trifluoperazine (TFP), calmidazolium (CDZ), and the napthalenesulfonamide derivatives W-7/W-13) to sort out the multiple potential roles of calmodulin in living cells; a PubMed search for W-7 alone turns up 1,700 publications. Frequently, investigators reason that if two or more chemically distinct inhibitors (e.g. W-7 and TFP) produce similar effects on a cell, they can assume that the effects are due to specific inhibition of calmodulin. If, however, these amphipathic weak bases bind to Ca2+/CaM mainly through nonspecific hydrophobic and electrostatic interactions, they also will bind to the inner leaflet of the plasma membrane at about the same concentration at which they inhibit calmodulin. This binding reduces the net negative charge on the inner leaflet, which is due mainly to the monovalent acidic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphati-dylserine (PS). Decreasing the net negative charge on the inner leaflet of the plasma membrane can have several biological effects. For example, it could reduce the electrostatically mediated (7McLaughlin S. Murray D. Nature. 2005; 438: 605-611Crossref PubMed Scopus (715) Google Scholar, 8Yeung T. Terebiznik M. Yu L. Silvius J. Abidi W.M. Philips M. Levine T. Kapus A. Grinstein S. Science. 2006; 313: 347-351Crossref PubMed Scopus (257) Google Scholar, 9McLaughlin S. Science. 2006; 314: 1402-1403Crossref PubMed Scopus (7) Google Scholar, 10Heo W.D. Inoue T. Park W.S. Kim M.L. Park B.O. Wandless T.J. Meyer T. Science. 2006; 314: 1458-1461Crossref PubMed Scopus (546) Google Scholar) membrane binding of clusters of basic residues on numerous peripheral (e.g. K-Ras4B, Src, myristoylated alaninerich C kinase substrate (MARCKS), and gravin) and integral proteins (e.g. receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR)). We investigated whether commonly used membrane-permeable calmodulin inhibitors bind to phospholipid vesicles formed with the same mole fraction of PS present in the inner leaflet of a typical mammalian plasma membrane. They do. For example, ∼10 μm W-7 or W-13 reduces the negative zeta potential of a phospholipid vesicle significantly and induces membrane-bound basic peptide translocation from membrane to solution, as determined by both centrifugation and fluorescence correlation spectroscopy (FCS) measurements; the same inhibitor concentration binds to and inhibits Ca2+/CaM. Our results suggest that these inhibitors bind both to Ca2+/CaM and to membranes in cells. We investigated the dual effects of Ca2+/CaM inhibitors on an important cellular process, EGFR activation. Exposing cells to EGF produces receptor dimerization (11Ferguson K.M. Biochem. Soc. Trans. 2004; 32: 742-745Crossref PubMed Scopus (57) Google Scholar) and a transient increase in the cytoplasmic free concentration of Ca2+, [Ca2+]i, and consequently in Ca2+/CaM (12Pandiella A. Magni M. Lovisolo D. Meldolesi J. J. Biol. Chem. 1989; 264: 12914-12921Abstract Full Text PDF PubMed Google Scholar, 13Cheyette T.E. Gross D.J. Cell Regul. 1991; 2: 827-840Crossref PubMed Scopus (28) Google Scholar, 14Hughes A.R. Bird G.S. Obie J.F. Thastrup O. Putney Jr., J.W. Mol. Pharmacol. 1991; 40: 254-262PubMed Google Scholar, 15Bezzerides V.J. Ramsey I.S. Kotecha S. Greka A. Clapham D.E. Nat. Cell Biol. 2004; 6: 709-720Crossref PubMed Scopus (455) Google Scholar, 16Li W.P. Tsiokas L. Sansom S.C. Ma R. J. Biol. Chem. 2004; 279: 4570-4577Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 17Uyemura T. Takagi H. Yanagida T. Sako Y. Biophys. J. 2005; 88: 3720-3730Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). We hypothesize that this transient increase in Ca2+/CaM could be important in EGFR activation, because Ca2+/CaM binds both to the EGFR (18Li H. Villalobo A. Biochem. J. 2002; 362: 499-505Crossref PubMed Scopus (41) Google Scholar, 19Martin-Nieto J. Villalobo A. Biochemistry. 1998; 37: 227-236Crossref PubMed Scopus (100) Google Scholar, 20Li H. Ruano M.J. Villalobo A. FEBS Lett. 2004; 559: 175-180Crossref PubMed Scopus (37) Google Scholar) and to peptides corresponding to its cytoplasmic juxtamembrane (JM; residues 645-660) region with a sufficiently high affinity (Kd ∼ 10 nm for the peptide) (21McLaughlin S. Smith S.O. Hayman M.J. Murray D. J. Gen. Physiol. 2005; 126: 41-53Crossref PubMed Scopus (103) Google Scholar) to suggest that this interaction could be biologically relevant. A recent report provides important evidence that ligand-mediated activation of EGFR involves dimer formation between two kinase domains, which stimulates rearrangement of the activation loop within the kinase domain by an allosteric mechanism (22Zhang X. Gureasko J. Shen K. Cole P.A. Kuriyan J. Cell. 2006; 125: 1137-1149Abstract Full Text Full Text PDF PubMed Scopus (1191) Google Scholar, 23Hubbard S.R. Cell. 2006; 125: 1029-1031Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 24Pellicena P. Kuriyan J. Curr. Opin. Struct. Biol. 2006; 16: 702-709Crossref PubMed Scopus (87) Google Scholar). Ca2+/CaM, for example, could facilitate formation of the kinase domain dimers by enhancing desorption of the JM plus kinase domains from the cytoplasmic leaflet (7McLaughlin S. Murray D. Nature. 2005; 438: 605-611Crossref PubMed Scopus (715) Google Scholar, 25Sato T. Pallavi P. Golebiewska U. McLaughlin S. Smith S.O. Biochemistry. 2006; 45: 12704-12714Crossref PubMed Scopus (42) Google Scholar). Previous published (20Li H. Ruano M.J. Villalobo A. FEBS Lett. 2004; 559: 175-180Crossref PubMed Scopus (37) Google Scholar, 21McLaughlin S. Smith S.O. Hayman M.J. Murray D. J. Gen. Physiol. 2005; 126: 41-53Crossref PubMed Scopus (103) Google Scholar) and unpublished work showed that treating cells with appropriate concentrations of W-7, W-13, and the less potent W-12 inhibits the initial transient phase of EGF-stimulated intermolecular EGFR autophosphorylation (hereafter referred to as autophosphorylation) in five different cell types. We report new experiments that provide additional evidence that W-13 inhibits this phase of EGF-mediated autophosphorylation by inhibiting Ca2+/CaM in cells exposed to a calcium ionophore. These results support the hypothesis that Ca2+/CaM is involved in the initial phase of ligand-mediated EGFR activation in several cell types. Introducing a mutation that impedes Ca2+/CaM binding to the EGFR JM domain abrogates W-13-mediated inhibition of EGFR autophosphorylation, which suggests that Ca2+/CaM interacts directly with the EGFR. W-7 and W-13, however, exert an opposing effect that appears not to be restricted to their ability to inhibit Ca2+/CaM; measurements on four different cell types show that W-13 stimulates autophosphorylation of EGFR in the absence of EGF, confirming and extending earlier work (21McLaughlin S. Smith S.O. Hayman M.J. Murray D. J. Gen. Physiol. 2005; 126: 41-53Crossref PubMed Scopus (103) Google Scholar, 26Tebar F. Villalonga P. Sorkina T. Agell N. Sorkin A. Enrich C. Mol. Biol. Cell. 2002; 13: 2057-2068Crossref PubMed Scopus (70) Google Scholar, 27Tebar F. Llado A. Enrich C. FEBS Lett. 2002; 517: 206-210Crossref PubMed Scopus (38) Google Scholar). We discuss how the membrane binding of W-7 and W-13 could produce this effect. Electrostatic Potentials Adjacent to Membranes and Determination of the Zeta Potential—Fixed negative charges on the inner leaflet of a typical mammalian plasma membrane are due mainly to the presence of monovalent acidic lipids, such as PS, which accounts for 20-35% of these phospholipids (28Holthuis J.C. Levine T.P. Nat. Rev. Mol. Cell Biol. 2005; 6: 209-220Crossref PubMed Scopus (413) Google Scholar). Most other lipids are neutral (e.g. cholesterol) or zwitterionic (e.g. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (PC) and phosphatidylethanolamine); positive lipids are rare or absent. The fixed charges on PS (red circles at left in Fig. 1A) attract ions of opposite charge, K+ (blue circles), from the aqueous phase; these counterions form an ion atmosphere, as shown in Fig. 1A. Helmholtz in 1879 (29Helmholtz H.L. Ann. Phys. Leipzig. 1879; 7: 337-382Crossref Scopus (534) Google Scholar) first recognized that the fixed charges and counterions "form an electrical double layer... that has an extraordinarily small, but not disappearing thickness"; double layer theory is discussed in detail in an excellent modern physical chemical text (30Dill K. Bromberg S. Molecular Driving Forces, Garland Science, New York. 2003; : 369-447Google Scholar) and reviews of membrane electrostatics (e.g. Refs. 31McLaughlin S. Curr. Top. Membr. Transp. 1977; 19: 71-144Crossref Scopus (777) Google Scholar and 32McLaughlin S. Annu. Rev. Biophys. Biophys. Chem. 1989; 18: 113-136Crossref PubMed Scopus (894) Google Scholar and references therein). The essential features are summarized very briefly below and in Fig. 1. The thickness of the ion atmosphere adjacent to the inner leaflet is about 1 nm = 10 Aå. The counterions do not stick to the membrane surface for the same reason the gas molecules in the earth's atmosphere do not fall to the ground; they diffuse away from a region of high concentration. Gouy and Chapman used the Poisson equation to describe the electrostatic attraction of the counterions for the charged surface and the Boltzmann equation to describe the statistical distribution of the counter- and co-ions in the aqueous diffuse double layer; Fig. 1, B and C, illustrates two key predictions of the Gouy-Chapman theory. When the surface potential, ψ(0), is small, it is proportional to both the surface charge density, σ (number of fixed charges per unit area of surface), and the Debye length, ψ(0)=σ/ɛ1ɛ0κEq. 1 where ɛr is the dielectric constant or relative permittivity (∼80), ɛo is the permittivity of free space, and 1/κ is the Debye length (∼1 nm for 0.1 m salt; ∼10 nm for 0.001 m salt). Note that this limiting form of the Gouy equation also describes the potential difference between two capacitor plates with charge density σ separated by a distance 1/κ (see Fig. 1C). Equation 1 and the simple capacitor model illustrate the following two important features of double layer theory. The surface potential increases with increasing density of fixed charge (i.e. fraction of acidic lipids increases) and/or Debye length (i.e. salt concentration decreases). Fig. 1B shows the dependence of potential on distance. When the potential in the aqueous phase is small, it falls with distance x from the surface according to Equation 2. ψ(x)=ψ(0)exp(-κx)Eq. 2 Numerous experimental studies have shown that this theory provides a surprisingly accurate description of the potential adjacent to a phospholipid bilayer membrane (32McLaughlin S. Annu. Rev. Biophys. Biophys. Chem. 1989; 18: 113-136Crossref PubMed Scopus (894) Google Scholar). The Helmholtz-Smoluchowski equation gives the relationship between the measured electrophoretic mobility, μ (velocity of a multilamellar vesicle in a unit electric field), and the zeta potential, ζ, ζ=μη/(ɛ1ɛo)Eq. 3 where η is the viscosity of the aqueous solution, ɛr is the dielectric constant (∼80) and ɛo is the permittivity of free space. By definition, ζ is the potential at the hydrodynamic plane of shear, which experiments suggest is located about 0.2 nm from the surface (see Fig. 1B) in a 100 mm salt solution (32McLaughlin S. Annu. Rev. Biophys. Biophys. Chem. 1989; 18: 113-136Crossref PubMed Scopus (894) Google Scholar). The experimental aspects of determining the electrophoretic mobility are considered in detail elsewhere (33Cafiso D. McLaughlin A. McLaughlin S. Winiski A. Methods Enzymol. 1989; 171: 342-364Crossref PubMed Scopus (38) Google Scholar). For the experiments shown in Figs. 3 and 4, we added the inhibitor in excess concentration relative to the lipids ([lipid] < 1 μm), so the binding of inhibitor to the vesicles does not significantly decrease its free concentration in solution.FIGURE 4Effect of TFP and CDZ on the zeta potentials of phospholipid vesicles. TFP binds equally well to 2:1 PC/PS (black diamonds) and 2:1 PC/PG (white diamonds) vesicles through a combination of nonspecific hydrophobic and electrostatic interactions. It binds to PC vesicles (gray diamonds) through hydrophobic interactions only. CDZ binds strongly to the 2:1 PC/PS vesicles (white triangles); 1 μm CDZ reverses the charge on 2:1 PC/PS vesicles and produces a positive zeta potential (ζ =+17 mV).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Reagents—Fig. 2 shows the chemical structures of five commonly used membrane-permeable calmodulin inhibitors. The naphthalenesulfonamide derivatives (W-7, W-12, and W-13), trifluoperazine (TFP), and calmidazolium (CDZ) are all amphipathic weak bases. (As discussed in the supplemental materials, the neutral form of these weak bases equilibrates between the bathing solution and cytoplasm.) W-7, W-13, and W-12 were purchased from both Sigma and Calbiochem. Similar results (zeta potential and peptide binding to large unilamellar vesicles (LUVs)) were obtained with both samples. CDZ, TFP, and rhodamine green were purchased from Sigma. The phospholipids PC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (PG) and PS were from Avanti Polar Lipids (Alabaster, AL). Peptides corresponding to the JM region of EGFR (EGFR-(645-660)), with and without a Cys residue at the N terminus, were purchased from American Peptide Co. (Sunnyvale, CA). Alexa-488 maleimide was purchased from Invitrogen. Radioactively labeled [dioleoyl-1-14C]l-α-dio-leoylphosphatidylcholine and [ethyl-1, 2-3H]N-ethylmaleimide were from PerkinElmer Life Sciences. Cell culture media and sera were purchased from Invitrogen or Biological Industries (Beit Haemek, Israel). Mouse monoclonal anti-phosphotyrosine RC20 antibody conjugated to horseradish peroxidase was from Transduction Laboratories/BD Biosciences. A23187, ionomycin, and KN-93 were from Calbiochem. GF109203X and Fast Green FCF were from Sigma. The enhanced chemiluminescent Luminol (ECL™) reagents were from Amersham Biosciences. Polyvinylidene difluoride membranes were from Pall Gelman Laboratory (Ann Arbor, MI), and Immobilon-P filters were from Millipore Corp. (Billerica, MA). Vesicle Preparation—We used 100-nm diameter LUVs for the FCS and centrifugation-binding experiments, as described in detail elsewhere (34Wang J. Arbuzova A. Hangyas-Mihalyne G. McLaughlin S. J. Biol. Chem. 2001; 276: 5012-5019Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Briefly, we added a mixture of solutions of PC and PS in chloroform to a 50-ml round-bottom flask, which was then immersed in a 30-35 °C water bath and attached to a rotary evaporator. The flask was rotated without vacuum for ∼5 min to warm the flask and solution. We then evaporated most of the solvent rapidly by applying the maximum vacuum that does not boil the chloroform. The flask was kept under full vacuum for 30 min to remove all traces of chloroform. A solution (typically containing 100 mm KCl, 10 mm MOPS, pH 7.0) was added to form multilamellar vesicles (MLVs), followed by five cycles of rapid freezing and thawing. We formed LUVs by extruding the MLVs through 100-nm diameter polycarbonate filters. (A solution containing 176 mm sucrose, 10 mm MOPS, pH 7.0, was used to obtain sucrose-loaded LUVs for centrifugation binding measurements. The solution bathing the LUVs was then exchanged for 100 mm KCl, 10 mm MOPS, pH 7.) We used MLVs, which typically had a diameter of 10-20 μm for electrophoretic mobility/zeta potential measurements. Peptide Labeling and Purification—Labeled or unlabeled peptides used for experiments were determined to be >95% pure by HPLC and MALDI-TOF mass spectroscopy. We used a protocol modified from Ref. 36Golebiewska U.P. Gambhir A. Hangyas-Mihalyne G. Zaitseva I. Raedler J. McLaughlin S. Biophys. J. 2006; 91: 588-599Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar to label the peptides with the thiol-reactive fluorescent probes. In brief, we mixed 1 ml of 1mm peptide in 10 mm K2HPO4/KH2PO4, pH 7.0, with the probe dissolved in N,N-dimethyl formamide (probe/peptide molar ratio 1:1) for 1 h. We purified the labeled peptide using HPLC and checked that it has the correct molecular weight using MALDI-TOF mass spectrometry (Proteomics Center, State University of New York at Stony Brook). We labeled the peptides with [ethyl-1,2-3H]N-ethylmaleimide as described previously (34Wang J. Arbuzova A. Hangyas-Mihalyne G. McLaughlin S. J. Biol. Chem. 2001; 276: 5012-5019Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 35Rusu L. Gambhir A. McLaughlin S. Radler J. Biophys. J. 2004; 87: 1044-1053Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 36Golebiewska U.P. Gambhir A. Hangyas-Mihalyne G. Zaitseva I. Raedler J. McLaughlin S. Biophys. J. 2006; 91: 588-599Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Centrifugation Measurements—We measured the binding of EGFR-(645-660) peptides to sucrose-loaded PC/PS LUVs using a centrifugation technique described previously (21McLaughlin S. Smith S.O. Hayman M.J. Murray D. J. Gen. Physiol. 2005; 126: 41-53Crossref PubMed Scopus (103) Google Scholar, 34Wang J. Arbuzova A. Hangyas-Mihalyne G. McLaughlin S. J. Biol. Chem. 2001; 276: 5012-5019Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 37Buser C.A. McLaughlin S. Methods Mol. Biol. 1998; 84: 267-281PubMed Google Scholar). Briefly, sucrose-loaded PC/PS LUVs (20 μm lipid for data in Fig. 5) were mixed with trace concentrations of [ethyl-1,2-3H]N-ethylmaleimide-labeled peptides (typically 2-10 nm). The mixture was centrifuged at 100,000 × g for 1 h. We calculated the percentage of peptide bound by measuring the radio-activity of the peptide in the supernatant and in the pellet. FCS Measurements and Data Analysis/Interpretation—Information about the size of a particle (e.g. free versus vesicle-bound peptide) can be obtained by measuring its diffusion constant D and using the Stokes-Einstein relationship, D=kT/(6πηR)Eq. 1 where k is the Boltzmann constant, T is the temperature, η is the viscosity of the medium, and R is the hydrodynamic radius of the diffusing particle. We can determine D by measuring the residence time τD of a particle diffusing through a small observation volume of radius r and using the Einstein relation for diffusion, r2=4DτDEq. 5 FCS measures the residence time, τD of molecules diffusing through a small, optically defined, open probe volume using sensitive fluorescence detection (38Magde D. Elson E.L. Webb W.W. Biopolymers. 1974; 13: 29-61Crossref PubMed Scopus (1114) Google Scholar, 39Maiti S. Haupts U. Webb W.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11753-11757Crossref PubMed Scopus (299) Google Scholar, 40Sengupta P. Balaji J. Maiti S. Methods. 2002; 27: 374-387Crossref PubMed Scopus (83) Google Scholar, 41Elson E.L. J. Biomed. Opt. 2004; 9: 857-864Crossref PubMed Scopus (87) Google Scholar). Fig. 6 illustrates the principle of FCS and how this technique can be used to measure the binding of fluorescently labeled EGFR-(645-660) to negatively charged phospholipid vesicles (35Rusu L. Gambhir A. McLaughlin S. Radler J. Biophys. J. 2004; 87: 1044-1053Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Fig. 6B shows a schematic diagram of free and LUV-bound peptides undergoing Brownian motion in the laser focus volume; the fluorescence signal from this probe volume fluctuates as the molecules diffuse in and out of it. This fluorescence fluctuation is recorded with an avalanche photodiode detector with good temporal resolution and then analyzed by a digital temporal correlator to compute the autocorrelation function G(τ), the measured variable for FCS experiments, as described in the supplemental materials. Fig. 6, C and D, shows the fluorescence time traces and the autocorrelation functions calculated from those time traces, respectively, using the same color coding. We can extract the characteristic residence time, τD, of a molecule (i.e. the average time the molecule takes to diffuse in and out of the ∼300-nm diameter probe volume) by fitting the autocorrelation functions to the appropriate equation. Because the LUV-bound peptides diffuse more slowly than free peptides, the autocorrelation function decays on a longer time scale, and the correlation time is ∼25-fold longer. It is easy to distinguish the bound and free species when τD(bound) is ≫τD(free). Cell Cultures—N7XHERc fibroblasts, a stable transfected cell line expressing the wild type human EGFR, was donated by Axel Ullrich from the Max-Planck-Institut fuör Biochemie (Martinsried, Germany), and RI1 fibroblasts, a stable transfected cell line expressing a human EGFR mutant with an insertion of a highly acidic 23-amino acid sequence between Arg647 and His648, which divides the CaM-binding domain into two segments (42Sorokin A. Oncogene. 1995; 11: 1531-1540PubMed Google Scholar), was a kind gift of Andrey Sorokin from the Medical College of Wisconsin (Milwaukee, WI). N7XHERc, N7XHERc/654A, RI1, EGFR-T17 (stably transfected mouse fibroblasts overexpressing human EGFR), and green monkey kidney COS-1 cells (expressing 4 × 105 EGFR/cell) were grown in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum, 5 mm pyruvate, 2 mm l-glutamine, and 40 μg/ml gentamicin in a humidified atmosphere of 5% (v/v) CO2 at 37 °C. Mouse NIH3T3 (clone WT8) fibroblasts (expressing 4 × 105 EGFRs/cell) (43Sorkin A. Mazzotti M. Sorkina T. Scotto L. Beguinot L. J. Biol. Chem. 1996; 271: 13377-13384Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar) were grown as above except that 10% (v/v) donor calf serum was used. Porcine aortic endothelial (PAE/EGFR-GFP) cells, a stable transfected cell line expressing a human EGFR-green fluorescent protein (GFP) chimera (2 × 105 EGFR-GFP/cell) (44Carter R.E. Sorkin A. J. Biol. Chem. 1998; 273: 35000-35007Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar), were grown in F-12 medium with the supplements indicated above. Cells were counted using a Neubauer's chamber after detachment from the culture dishes and seeded in 6-well plates (1.4 × 105 cells/well) in the media indicated above and grown to ∼90% confluence for 24 h. Thereafter, the cells were washed twice with phosphate-buffered saline (137 mm NaCl, 2.7 mm KCl, 1.8 mm KH2PO4, and 10 mm Na2HPO4 at pH 7.4) and maintained overnight in a serum-free medium before performing the experiments. Measurement of EGFR Autophosphorylation—EGF-dependent EGFR autophosphorylation was measured upon the addition of 10 nm EGF to serum-starved cells in multiwell plates containing 2 ml of medium. Depending on the experiment (see legends to Figs. 7 and S1), the medium also contained a calcium/calmodulin-activated kinase II inhibitor, a protein kinase C inhibitor, the CaM inhibitor W-13, and a Ca2+ ionophore. We added 1 ml of ice-cold 30% trichloroacetic acid to stop the reaction and prepared a total cell lysate using Laemmli sample buffer, boiling the sample for 5 min as described previously (20Li H. Ruano M.J. Villalobo A. FEBS Lett. 2004; 559: 175-180Crossref PubMed Scopus (37) Google Scholar). We measured EGF-independent EGFR autophosphorylation after adding W-13 to serum-starved cells (see legend to Fig. 8), preparing the cell lysate as described previously (26Tebar F. Villalonga P. Sorkina T. Agell N. Sorkin A. Enrich C. Mol. Biol. Cell. 2002; 13: 2057-2068Crossref PubMed Scopus (70) Google Scholar).FIGURE 8W-13 increases tyrosine phosphorylation of EGFR in different cell lines. Four different cell lines were serum-starved overnight and then incubated with 10 μg/ml (29 μm) W-13 for 10 min at 37 °C. Lysates normalized to an equal amount of protein were subjected to electrophoresis and then analyzed by Western blot with an antiphosphotyrosine (P-Tyr) antibody (RC20); the arrow indicates phosphorylated EGFR.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Electrophoresis and Western Blot Analysis—Slab gel linear gradient (5-20% (w/v) polyacrylamide and 0.1% (w/v) SDS at pH 8.3) electrophoresis was p
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