An Electrostatic Engine Model for Autoinhibition and Activation of the Epidermal Growth Factor Receptor (EGFR/ErbB) Family
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《普通生理学杂志》
1 Department of Physiology and Biophysics, HSC
2 Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794
3 Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794
4 Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY 10021
We propose a new mechanism to explain autoinhibition of the epidermal growth factor receptor (EGFR/ErbB) family of receptor tyrosine kinases based on a structural model that postulates both their juxtamembrane and protein tyrosine kinase domains bind electrostatically to acidic lipids in the plasma membrane, restricting access of the kinase domain to substrate tyrosines. Ligand-induced dimerization promotes partial trans autophosphorylation of ErbB1, leading to a rapid rise in intracellular [Ca2+] that can activate calmodulin. We postulate the Ca2+/calmodulin complex binds rapidly to residues 645–660 of the juxtamembrane domain, reversing its net charge from +8 to –8 and repelling it from the negatively charged inner leaflet of the membrane. The repulsion has two consequences: it releases electrostatically sequestered phosphatidylinositol 4,5-bisphosphate (PIP2), and it disengages the kinase domain from the membrane, allowing it to become fully active and phosphorylate an adjacent ErbB molecule or other substrate. We tested various aspects of the model by measuring ErbB juxtamembrane peptide binding to phospholipid vesicles using both a centrifugation assay and fluorescence correlation spectroscopy; analyzing the kinetics of interactions between ErbB peptides, membranes, and Ca2+/calmodulin using fluorescence stop flow; assessing ErbB1 activation in Cos1 cells; measuring fluorescence resonance energy transfer between ErbB peptides and PIP2; and making theoretical electrostatic calculations on atomic models of membranes and ErbB juxtamembrane and kinase domains.
Abbreviations used in this paper: Ca/CaM, calcium/calmodulin; CD, circular dichroism; EGFR, epidermal growth factor receptor; FCS, fluorescence correlation spectroscopy; FRET, fluorescence resonance energy transfer; IP3, inositiol 1,4,5-trisphosphate; JM, juxtamembrane; LUV, large unilamellar vesicle; MLV, multilamellar vesicle; NEM, [ethyl-1,2-3H]N-ethylmaleimide; PC, phosphatidylcholine; PIP2, phosphatidylinositol 4,5-bisphosphate; PS, phosphatidylserine; PTK, protein tyrosine kinase; TM, transmembrane.
INTRODUCTION
The general mechanism by which binding of a ligand to the extracellular domain of a typical receptor tyrosine kinase activates its intracellular protein tyrosine kinase (PTK) domain is well understood: binding produces dimerization (or dimer rearrangement) that leads to trans autophosphorylation of tyrosines in the "activation loop" of the PTK domain (Hubbard and Till, 2000; Schlessinger, 2000, 2003; Huse and Kuriyan, 2002; Jorissen et al., 2003; Hubbard, 2004). The ErbB tyrosine kinase family (ErbB1/HER1/EGFR, ErbB2/HER2, ErbB3/HER3, ErbB4/HER4) appears to be the major exception to this rule because their PTK domains do not require phosphorylation for catalytic competency (Gotoh et al., 1992; Jorissen et al., 2003; Hubbard, 2004). The structure of the ErbB1 PTK domain suggests why this is so: the unphosphorylated activation loop is in an active conformation and the catalytic elements are "primed and ready for phospho-transfer," suggesting "the regulation of the vital cellular processes influenced by epidermal growth factor receptor (EGFR) signaling must be exerted by control of the delivery of the COOH-terminal substrate tyrosines to the active site" (Stamos et al., 2002). Understanding this control mechanism is important because members of the ErbB family are frequently overactive in solid tumors (Blume-Jensen and Hunter, 2001; Yarden and Sliwkowski, 2001; Gschwind et al., 2004; Paez et al., 2004; Sordella et al., 2004); ErbB2, for example, is overexpressed in 25% of breast cancers, and this overexpression correlates with poor prognosis (Klapper et al., 2000).
Several groups have recently proposed models to explain autoinhibition of the ErbB family. Landau et al. (2004) developed a computational model that suggests direct contact between a positively charged face of the kinase domain and a negatively charged segment of the COOH-terminal tail region of the receptor produces autoinhibition. Alternatively, Aifa et al. (2005) proposed that this negatively charged segment, ErbB1(979–991), interacts with a cluster of basic residues in the juxtamembrane (JM) region of an adjacent ErbB molecule. The ErbB1 basic JM region also plays an important role in the structural model of autoinhibition we propose.
In our model, basic residues in the ErbB JM and PTK domains interact electrostatically with acidic lipids in the membrane, inhibiting catalytic activity in the absence of ligand. This autoinhibitory hypothesis has an obvious corollary: conditions that decrease the electrostatic binding (e.g., an increase in the cytoplasmic salt concentration, exposure to an amphipathic weak base that decreases the negative fixed charge density on the membrane) should release the JM and PTK regions from the membrane, producing ligand-independent trans autophosphorylation. We have tested this corollary by comparing data from our experiments with model membranes and peptides to data in the literature from intact cells.
Our model also suggests a novel positive feedback mechanism by which ligand-induced dimerization may contribute to activation. As discussed below, it is well established that ligand-induced dimerization of ErbB1 leads to a transient (10 min) increase in the level of free Ca2+ within a cell. We postulate that calcium/calmodulin (Ca/CaM) binds to the ErbB JM region very rapidly (100 ms when [Ca/CaM] = 1 μM), reversing its charge and repelling both it and the PTK domain from the membrane. This implies Ca/CaM binding will increase the initial rate of trans autophosphorylation over and above the rate due to the local concentration effect resulting from ligand-induced dimerization. Our postulated Ca/CaM-mediated activation mechanism will be important only when the [Ca2+] is high enough to produce a significant increase in Ca/CaM.
The model predicts that Ca/CaM can pull peptides corresponding to the ErbB JM region off a membrane rapidly and that CaM inhibitors will inhibit, but not completely block, the initial phase of EGF-mediated ErbB autophosphorylation in cells. We tested these predictions experimentally; while the results are consistent with the predictions, they neither prove that the model is correct nor rule out other potential activation mechanisms that may act in parallel (Jorissen et al., 2003; Schlessinger, 2003). For example, there is much evidence that phosphatases play an important role in controlling the trans autophosphorylation of ErbB (e.g., Reynolds et al., 2003; Tonks, 2003; Ichinos et al., 2004; Matilla et al., 2004); we return to the role of phosphatases in the concluding section of DISCUSSION.
Structural Model
We divide the JM domain into three sections (Fig. 1 C) and hypothesize that the 16-residue NH2-terminal region (Arg645 to Gln660 of ErbB1) acts as a reversible membrane anchor. Specifically, we follow the suggestions of Ullrich et al. (1984) and Hunter et al. (1984) and postulate that in a quiescent cell, i.e., when no ligands are present and the free [Ca2+] is low, the eight basic and five hydrophobic amino acids bind this region to the negatively charged inner leaflet of the bilayer. In our model, the 12-residue central region (Glu661 to Gly672 of ErbB1) acts as a flexible linker that joins the reversible membrane anchor to the COOH-terminal section of the JM domain. The flexible linker has a net charge of –2 (3 E, 1R); it should be repelled, albeit weakly, from the membrane bilayer. The 10-residue COOH-terminal region of the JM domain (Glu673 to Ile682) was identified in the structure of the ErbB1 PTK core (Stamos et al., 2002). Fig. 2 illustrates that one face of this COOH-terminal JM region + PTK structure is positively charged (blue), and thus should bind electrostatically to the negatively charged bilayer, as drawn in Fig. 1 A. Fig. 2 also shows that the PTK domain has a strong dipole character (i.e., one face is positive, while the remainder of the molecule surface is mainly negative); thus it will not be free to rotate when bound to the bilayer. A key assumption of our model is that binding of the PTK core to the membrane inhibits its activity; hindered rotation could, for example, limit its contact with, and thus ability to phosphorylate, tyrosines in the COOH-terminal tail of an ErbB family dimer or other substrate.
Under physiological conditions, a ligand such as EGF binds to the ErbB1 extracellular domain and induces dimerization. Recent structural studies of the ErbB1 extracellular domain with (Ogiso et al., 2002) and without (Ferguson et al., 2003) bound EGF indicate that ligand binding triggers a dramatic rearrangement of the four extracellular subdomains in each receptor monomer. A direct intramolecular interaction between cysteine-rich subdomains II and IV in the unactivated receptor is broken upon EGF binding, and loss of contact releases a dimerization arm on domain II that mediates direct ErbB1 monomer–monomer contacts (for reviews see Burgess et al., 2003; Ferguson, 2004).
In our model, the intracellular JM + PTK regions of each dimer pair exist in equilibrium between the membrane-bound (Fig. 1 A) and free (not shown) states, with the autoinhibited membrane-bound state predominating in the absence of Ca/CaM. Upon ErbB dimerization (not depicted in Fig. 1 for simplicity), the small fraction of dimeric receptors with membrane-free JM + PTK regions can trans autophosphorylate. Phosphorylation of tyrosine residues in the ErbB COOH-terminal tail leads to binding and activation of PLC-, hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) with concomitant production of inositiol 1,4,5-trisphosphate (IP3), and IP3-mediated release of Ca2+ from internal stores (for review see Jorissen et al., 2003). The transitory release of Ca2+ from intracellular stores mediated by IP3 is followed by a more sustained influx of Ca2+ across the plasma membrane (e.g., Pandiella et al., 1988; Cheyette and Gross, 1991; Hughes et al., 1991; Bezzerides et al., 2004; Li et al., 2004c). Adding 30 nM EGF to A431 cells, for example, increases intracellular [Ca2+] about fivefold, to 600 nM, in 5 min; [Ca2+] then declines over 10 min to a value only slightly above the basal level (Hughes et al., 1991).
The EGF-mediated transient increase in cytoplasmic [Ca2+] activates CaM, and our data, together with previous work on peptides (Martin-Nieto and Villalobo, 1998) and native ErbB1 (Li et al., 2004a), suggest the Ca/CaM complex can bind rapidly and strongly to residues 645–660 of ErbB1, as shown in Fig. 1 B. This binding reverses the charge on the region from +8 to –8, converting its strong electrostatic attraction to the membrane into a strong electrostatic repulsion. We hypothesize that the binding energy of Ca/CaM for the reversible membrane anchor region and the electrostatic repulsion of the resulting complex from the negatively charged bilayer provide the energy to move the PTK core of an ErbB family member off the bilayer. Thus we refer to this mechanism as an "electrostatic engine" that increases both the frequency at which the JM + PTK domain moves from its autoinhibitory membrane-bound conformation (Fig. 1 A) to a freely rotating active state (Fig. 1 B) and the duration of time it spends in this active state. We discuss below experiments that suggest this putative engine may cycle rapidly (10–100 s–1).
Our fluorescence resonance energy transfer (FRET) and PLC activity measurements also show that the cluster of basic residues on the JM domain can, when bound to the membrane, electrostatically sequester PIP2. Thus ErbB may function as a scaffolding protein with its JM domain rapidly concentrating and releasing PIP2 in the vicinity of PLC- and phophoinositide 3-kinase (PI3K), enzymes that are bound to the ErbB COOH-terminal region and use PIP2 as a substrate.
MATERIALS AND METHODS
Materials
1-palmitoyl-2-oleoyl-sn-PS, 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (PC), 1-palmitoyl-2-oleoyl-sn-phosphatidylglycerol (PG), and the ammonium salt of L--phosphatidyl-D-myo-inositol 4,5-bisphosphate (PIP2) were purchased from Avanti Polar Lipids. Labeled [dioleoyl-1-14C]L--dioleoylphosphatidylcholine and [ethyl-1,2-3H]N-ethylmaleimide (NEM) were from PerkinElmer Life Sciences. 6-Acryloyl-2-dimethylaminonaphthalene (acrylodan), Alexa488, and Texas red were from Molecular Probes, Inc. Bodipy-TMR-PIP2 was purchased from Echelon. Sphingosine was from Sigma-Aldrich.
We formed multilamellar vesicles (MLVs) for zeta potential measurements, 100-nm-diameter large unilamellar vesicles (LUVs) for FRET, FCS, and stop flow fluorescence measurements, and 100-nm sucrose-loaded LUVs for centrifugation binding measurements, as described previously (Wang et al., 2002).
Methods
Measurements of Peptide Binding to LUVs.
We measured the binding of [3H]NEM-labeled peptides to sucrose-loaded PC/PS LUVs using a centrifugation technique described previously (Wang et al., 2002; Gambhir et al., 2004). In brief, we mixed sucrose-loaded LUVs and a trace concentration (5 nM) of [3H]NEM-labeled peptide, and then centrifuged the mixture at 100,000 g for 1 h. We calculated the percentage of peptide bound from counts of the radioactive peptide in the supernatant and in the pellet.
Zeta Potential of MLVs.
We measured the electrophoretic mobility (velocity/field) of single MLVs and calculated the zeta potential, the electrostatic potential at the hydrodynamic plane of shear (0.2 nm from the surface), using the Helmholtz-Smoluchowski equation (Wang et al., 2002, and references therein).
Surface Pressure Measurements.
We deposited a stock lipid–chloroform solution onto the surface of a 15-ml aqueous solution in a 5-cm-diameter Teflon trough with a magnetic stirrer at the bottom. Once the chloroform had evaporated, we measured the surface pressure of the monolayer using a square piece of filter paper and a balance from Nima Technology Ltd. We then added the peptide to the subphase and measured the change in surface pressure as described previously (Wang et al., 2002).
FRET.
We monitored FRET between a Bodipy TMR label on PIP2 and a Texas red label attached to membrane-bound ErbB1(645–660) as described previously (Gambhir et al., 2004); the membrane contained 69.7% PC, 30% PS, and 0.3% PIP2.
Circular Dichroism (CD) Spectroscopy.
We obtained the CD spectrum of ErbB1(645–660) bound to isotropic bicelles on an Olis DSM CD spectrometer (Olis Instruments) with a 0.2-mm path-length cell. The bicelles were composed of a mixture of DMPC, DMPG, and DHPC in a 10:3:13 molar ratio. The buffered bicelle solution (20 mM sodium phosphate, pH 7.0) was 10% (wt/vol) lipid and the peptide:lipid ratio was 1:100. Measurements of a bicelle solution without peptide prepared in parallel were subtracted as background. The corrected CD spectrum exhibits the strong negative ellipticity at 200 nm characteristic of random-coil structures.
Fluorescence Correlation Spectroscopy (FCS).
We used a Carl Zeiss MicroImaging, Inc. Confocor II microscope to monitor the binding of Alexa488-labeled ErbB1(645–660) to 2:1 PC/PS 100-nm LUVs and to study the ability of Ca/CaM to remove the peptide from the membrane. The experimental techniques were similar to those described in detail in Rusu et al. (2004). In brief, the correlation times of the peptide bound to 100-nm LUVs and to Ca/CaM are 1,700 μs and 100 μs, respectively; hence we could distinguish the two correlation times easily. We determined the affinity of Ca/CaM for the peptide by plotting the fraction of membrane-bound peptide against the concentration of Ca/CaM in the solution, as shown for experiments using the centrifugation technique (see Fig. 4).
Stop Flow Kinetics.
We made fluorescence stop flow kinetic measurements to determine the rate at which Ca/CaM removes membrane-bound acrylodan-labeled ErbB1(645–660) from PC/PS vesicles; adding Ca/CaM increased the fluorescence approximately fourfold as the peptide moved from vesicle to Ca/CaM. Specifically, one solution contained 200 or 400 nM acrylodan-labeled ErbB1(645–660) bound to 100-nm 85:14:1 PC/PS/NBD-PS vesicles (100 μM accessible lipid; the 1% NBD-PS in these membranes quenches the acrylodan fluorescence), and the other solution contained 0.5, 1, 2, 4, or 7 μM CaM, and 50 μM CaCl2. Both solutions contained 100 mM KCl buffered to pH 7.0 with 1 mM MOPS. We measured the time constants of the exponential increase in fluorescence, , and determined the slope of 1/ vs [CaM]. This slope is equal to the transfer rate constant. The two peptide concentrations produced identical time constants, as expected. We repeated the stop flow measurements with vesicles containing 10, 12, and 18% PS. The results and experimental details are similar to those shown in Fig. 7 of Arbuzova et al. (1997) for a different basic/hydrophobic acrylodan-labeled peptide.
Calculation of Electrostatic Potentials.
We built atomic models of the 2:1 PC/PS bilayer (Wang et al., 2002) and ErbB1(645–660) in an extended conformation using the Insight Biopolymer and Discover modules (Accelrys); the atomic radii and partial charges assigned to the peptide were taken from the CHARMM forcefield. We solved the nonlinear Poisson-Boltzmann equation for atomic models of these systems in 100 mM KCl. The resulting potential maps, as well as the atomic coordinates for the peptide/membrane/CaM models, were displayed using GRASP.
Online Supplemental Material
RESULTS
ErbB1(645–660) Binds Strongly to Phospholipid Vesicles through Nonspecific Electrostatic Interactions
We first tested the postulate that the ErbB1 reversible membrane anchor region binds to the plasma membrane by determining if a peptide corresponding to this region, ErbB1(645–660), binds to phospholipid vesicles. The data in Fig. 3 show this is indeed the case: ErbB1(645–660) binds strongly to vesicles containing physiological (15–30%) mole fractions of the monovalent acidic lipid PS. We describe the binding using Eq. 1 (Arbuzova et al., 2000):
(1)
ErbB1(645–660) Laterally Sequesters PIP2
Biological Experiments Consistent with our Autoinhibition Hypothesis
Ca/CaM Removes ErbB JM Peptides from Membranes Rapidly
The mechanism shown in Fig. 1 B, i.e., Ca/CaM binds to the membrane anchor region and removes it and the PTK domain to facilitate EGF-mediated activation, is admittedly speculative, but peptide experiments provide evidence that it is feasible. We first tested whether Ca/CaM competes with phospholipid membranes for binding of ErbB1(645–660); Fig. 4 shows peptide binding to 2:1 PC/PS vesicles in the presence of increasing [Ca/CaM]. In the absence of Ca/CaM, 90% of the peptide binds to the vesicles, as expected from both theoretical calculations (unpublished data; see Murray et al., 2002, for methods) and the binding results in Fig. 3. The open circles in Fig. 4 illustrate the effect of increasing [Ca/CaM]: adding 0.1 μM Ca/CaM reduces binding by 50% (total [CaM] 10–100 μM in a mammalian cell). Adding CaM in the absence of free Ca2+ does not affect ErbB1(645–660) binding to the vesicles (filled circles). These data indicate that Ca/CaM binds to ErbB1(645–660) with high affinity (Kd = 10 nM), preventing the peptide from binding to a phospholipid vesicle. We used a modified version of Eq. 1 to describe the effect of Ca/CaM on ErbB1(645–660) membrane binding, incorporating the assumption that Ca/CaM and the membrane compete for the peptide:
(2)
where K is the molar partition coefficient of the peptide onto the 2:1 PC/PS vesicles (106 M–1 from Fig. 3) and KCaM is the association constant of the peptide with Ca/CaM (KCaM = 108 M–1 from the fit of Eq. 2 to data in Fig. 4).
We repeated the measurements shown in Fig. 4 using 3:1 rather than 2:1 PC/PS vesicles and obtained a similar value for KCaM. We also used an independent method, fluorescence correlation spectroscopy (FCS), to validate the centrifugation technique used to obtain the measurements shown in Fig. 4. FCS measures the correlation time (inversely proportional to diffusion constant) of a fluorescent label, in this case Alexa488 attached to ErbB1(645–660). The labeled peptide diffuses slowly when bound to the large (100 nm) vesicles and rapidly when bound to the smaller Ca/CaM; thus we can determine the fraction of membrane-bound peptide as a function of the concentration of Ca/CaM. Both centrifugation and FCS measurements with Alexa488-labeled ErbB1(645–660) indicate KCaM = 107 M–1 (U. Golebiewska, personal communication; unpublished data). Thus, we conclude that the FCS measurements validate the centrifugation technique. As expected from electrostatics, the negatively charged Alexa probe decreases the affinity of the peptide for Ca/CaM (and vesicles) 10-fold.
Phosphorylation of Thr654 also decreases the affinity of ErbB1(645–660) for Ca/CaM. Experiments similar to those shown in Fig. 4, but conducted with an ErbB1(645–660) peptide with a phosphorylated Thr654, show that phosphorylation reduces KCaM 20-fold: removal of 50% of the phosphopeptide from the vesicles requires 20-fold more Ca/CaM than shown in Fig. 4 under similar conditions, i.e., when 90% of the phosphopeptide is bound initially. Phosphorylation also decreases the membrane binding of the peptide 10-fold. These results agree well with previous measurements showing that phosphorylation "drastically hampers" (Martin-Nieto and Villalobo, 1998) or "totally inhibits" (Aifa et al., 2002) the ability of Ca/CaM to bind to this region of ErbB1. The model shown in Fig. 1 thus predicts that phosphorylation of Thr654 by PKC should significantly attenuate at least the early phase of ErbB1 autophosphorylation, a prediction consistent with experiment (Couchet et al., 1984; Hunter et al., 1984; Countaway et al., 1990; Welsh et al., 1991). However, phosphorylation of Thr654 also produces a less robust inhibition of ErbB1 autophosphorylation in model systems, such as A431 cell membranes (e.g., Couchet et al., 1984), which must be due to some other phenomenon.
The kinetics results are perhaps surprising: the simplest interpretation of our equilibrium measurements (Fig. 4) is that Ca/CaM acts as a passive peptide buffer. That is, adding it to a solution containing peptides bound to vesicles should merely decrease the equilibrium concentration of free peptide in the bulk aqueous phase, allowing peptide to desorb from the vesicles at its spontaneous rate until a new equilibrium is attained. The stop flow measurements, however, reveal that Ca/CaM increases the rate at which ErbB1(645–660) desorbs from the vesicles, presumably by ripping the peptide directly from the surface; we have discussed this mechanism elsewhere (Arbuzova et al., 1997, 1998).
CaM Inhibitors Reduce the Initial Rate of EGF-mediated Autophosphorylation in Cos1 Cells
A key prediction of the mechanism shown in Fig. 1 B is that exposing cells to membrane-permeable CaM inhibitors such as W-7 should inhibit the initial rate of EGF-mediated ErbB1 autophosphorylation. According to our model, CaM inhibitors should affect autophosphorylation only for a short time (<15 min) after EGF stimulation because the concomitant increase in cytoplasmic Ca2+ to values that activate CaM significantly is only transient. For example, Hughes et al. (1991) reported the intracellular [Ca2+] in A431 cells increases to 600 nM in 5 min, but falls to 150 nM by 15 min. Nojiri and Hoek (2000) show the intracellular [Ca2+] in hepatocytes increases to 450 nM in 1–2 min, and then falls to 150 nM in the next 2–3 min. Fig. 5 shows treating Cos1 cells with W-7 produces dose-dependent inhibition of ErbB1 tyrosine phosphorylation measured 10 min after addition of EGF; the concentration range used, 20–50 μM, was selected on the basis of the inhibitor's affinity for Ca/CaM (Osawa et al., 1998). EGF-stimulated ErbB1 autophosphorylation is maximal 10 min after adding EGF to Cos1 cells under our conditions (unpublished data). Recent detailed studies in two other cell types showed that the CaM inhibitors W-7, W-12, and W-13 inhibit the initial peak of ErbB1 autophosphorylation, but not the steady-state value observed for times >20 min (Li et al., 2004a), as expected from our model. Li et al. (2004a) report an important control experiment: W-7, W-12, and W-13 do not inhibit the tyrosine kinase activity of a purified ErbB1 preparation.
DISCUSSION
Membrane Binding of ErbB Intracellular Regions and Postulated Role of Ca/CaM
Our peptide experiments and/or theoretical calculations support the hypothesis that both the JM region and PTK domain of ErbB family members bind electrostatically to the inner leaflet of the plasma membrane. If our postulate that this binding produces autoinhibition is correct, factors that reduce these electrostatic interactions should stimulate cellular ErbB autophosphorylation in the absence of ligand. Specifically, we postulate that autophosphorylation depends on both the rate at which ErbB monomers collide in the plasma membrane and the probability the JM + PTK domain is in a membrane-dissociated active conformation. Our model membrane experiments allowed us to measure quantitatively the levels of salt, divalent cations, or the amphipathic weak base sphingosine required to disengage a peptide corresponding to the membrane anchor region from a lipid bilayer; when we compared the values to those reported for ligand-independent activation of ErbB1 in cells or broken membrane preparations (Carpenter et al., 1979; Davis et al., 1988; King et al., 1989), there was quantitative agreement in each case. Thus the mechanism illustrated in Fig. 1 A provides a plausible and experimentally testable explanation for autoinhibition, but it does not address the specific mechanism by which membrane binding inhibits tyrosine kinase activity. Possible mechanisms could include restricted rotation of the PTK domain that prevents interaction of the catalytic site and substrate, or structural effects on the activation loop within the PTK domain (Stamos et al., 2002; Wood et al., 2004); several recent reviews discuss autoinhibitory mechanisms for receptor tyrosine kinases in some detail (Jorissen et al., 2003; Schlessinger, 2003; Hubbard, 2004).
Our data show that Ca/CaM binds strongly to peptides corresponding to the membrane anchor region of ErbB1 (Fig. 4) and the corresponding JM regions in other ErbB family members (Table I). The results agree qualitatively with previous reports that Ca/CaM binds strongly to these regions of ErbB1 (Martin-Nieto and Villalobo, 1998; Li et al., 2004a) and ErbB2 (Li et al., 2004b). We note that Martin-Nieto and Villalobo (1998) reported KCaM = 3 x 106 M–1, but their Ca/CaM binding measurements employed 0.3 x 10–6 M of a GST-ErbB1(645–660) construct; thus we interpret their measurements to mean KCaM > 3 x 106 M–1, which agrees with our estimate. These data suggest that the activation mechanism illustrated in Fig. 1 B is feasible and could enhance the activation produced by ligand-induced dimerization, which presumably acts through a local concentration effect (Schlessinger, 2000).
The Electrostatic Engine Mechanism in ErbB Activation
Our model suggests the following process for EGF-mediated trans autophosphorylation. When EGF-stimulated dimerization occurs, the level of intracellular [Ca2+], and thus Ca/CaM, is initially low. The ErbB JM + PTK regions, however, move off the membrane spontaneously, albeit at a low rate (e.g., stop flow measurements demonstrate acrylodan-labeled ErbB1(645–660) moves off a 85:15 PC/PS vesicle at a rate of 3 s–1). The small fraction of receptors with dissociated JM + PTK domains will produce a low level of trans autophosphorylation even in the absence of Ca/CaM. When PLC- binds to a phosphorylated ErbB and hydrolyzes its substrate PIP2, it produces IP3, which in turn releases Ca2+ from internal stores, increasing the concentration of Ca/CaM (for review see Jorissen et al., 2003). Our measurements show that Ca/CaM increases both the rate at which peptides corresponding to the ErbB1 membrane anchor region leave the membrane and the fraction of membrane-dissociated peptide (Fig. 4). As the JM and kinase domains of an ErbB move off the membrane, the latter becomes catalytically active in our model, as illustrated for one member of the ErbB1 dimer in Fig. 1 B. Thus we postulate that Ca/CaM drives a positive feedback mechanism that produces maximal activation of ErbB. The mechanism functions only when the local [Ca2+] is sufficiently elevated to provide a source of Ca/CaM; intracellular [Ca2+] measurements indicate this occurs only for times <15 min after exposure to EGF (Hughes et al., 1991; Nojiri and Hoek, 2000). This electrostatic engine model predicts that CaM inhibitors (or agents that increase the intracellular Ca2+ buffering capacity) will diminish the transient increase in trans autophosphorylation observed within 15 min of EGF stimulation, but have little effect on the steady-state level of activity measured at later times. CaM inhibitors do indeed inhibit peak level of EGF-mediated ErbB1 autophosphorylation in Cos1 (Fig. 5) and two other cell types (Li et al., 2004a), but have no effect on the steady-state level of autophosphorylation (Li et al., 2004a). Agents that deplete intracellular stores of Ca2+, such as thapsigargin, should and do inhibit receptor autophosphorylation in A431 cells measured 5 min after addition of EGF (Friedman et al., 1989).
Less direct evidence in support of this hypothesis comes from studies of Ca2+-induced transactivation of ErbB1 (i.e., activation that occurs without addition of a ligand that binds directly to ErbB1). Transactivation can occur in response to activation of Gq-coupled receptors, opening of ion channels selective for Ca2+, or addition of Ca2+ ionophores (for review see Zwick et al., 1999). Much recent work on the transactivation of ErbB1 has focused on the unique triple-membrane-passing signal (Prenzel et al., 1999; for reviews see Gschwind et al., 2001; Blobel, 2005), but our model provides a clue as to how an increase in [Ca2+] and Ca/CaM could initiate this interesting phenomenon. If Ca2+ helps initiate transactivation by the mechanism shown in Fig. 1 B, both CaM inhibitors and Ca2+ buffers should inhibit transactivation. Murasawa et al. (1998) reported both the CaM inhibitor W-7 and the Ca2+ buffer BAPTA-AM inhibit angiotensin II–stimulated ErbB1 transactivation in cardiac fibroblasts.
Rigorous testing of the model will require extensive molecular and cell biological experiments as well as biophysical measurements on larger peptides corresponding to the ErbB TM + JM domains reconstituted into vesicles, which are in progress. If additional work supports our postulate that Ca/CaM may act in concert with dimerization to stimulate ErbB1 activation, the model shown in Fig. 1 can probably be extrapolated to the ErbB2 and ErbB4 (Carpenter, 2003) family members: peptides corresponding to their JM regions also bind with high affinity to both membranes and Ca/CaM (Table I).
ErbB as a Scaffolding Protein
How rapidly can Ca/CaM bind to the JM region and release the electrostatically sequestered PIP2 Our stop flow experiments reveal that 2 μM Ca/CaM can remove acrylodan-labeled ErbB1(645–660) peptides from PC/PS vesicles at rates of 10 and 100 s–1 for vesicles containing 15 and 10% PS, respectively. It is difficult to extrapolate these results using a model system to a living cell for several reasons. For example, ErbB1 may be in noncaveolar cholesterol- and PIP2-enriched "rafts" that have a different lipid composition than the bulk plasma membrane (e.g., Chen and Resh, 2002; Roepstorff et al., 2002; Westhover et al., 2003; Simons and Vaz, 2004, and references therein). We can state that if the Ca/CaM level rises to 1 μM in a cell, the maximum (diffusion limited) rate at which Ca/CaM could bind to the 645–660 JM region of ErbB1 and rip it off the plasma membrane is 100 s–1.
How long will the Ca/CaM remain bound to the JM region The lifetime of Ca/CaM bound to a solubilized ErbB1 molecule is probably 1 s (KCaM/kon = 108 M–1/108 M–1s–1), but theoretical considerations suggest the lifetime of Ca/CaM bound to ErbB1 in a membrane will decrease significantly (possibly to 0.01 s) as the mole fraction of acidic lipid in the membrane increases. This is because the acidic lipids repel the negatively charged Ca/CaM bound to the JM region.
Thus the electrostatic engine shown in Fig. 1 (A and B) could cycle 10–100 times a second when the [Ca/CaM] increases to 1 μM. Even if the JM region remains bound to the bilayer for only 0.01 s, this provides more than sufficient time, t, for PIP2 to equilibrate with the basic cluster through diffusion (from the Einstein relation, t = x2/4D, where x is the distance PIP2 must diffuse in the plasma membrane and D is its diffusion constant). FCS measurements show that the diffusion constant of Bodipy-PIP2 in a fluid phase PC phospholipid membrane has the expected value of D = 3 x 10–8 cm2s–1 (Golebiewska, U., personal communication). In a plasma membrane, D could be 10-fold lower because cholesterol increases the viscosity, and as much as 90% of the PIP2 could be sequestered such that x = distance between PIP2 free to diffuse = 30 nm; even under these conditions the diffusion time is only 0.001 s. The maximal rate at which PLCs can hydrolyze PIP2 is 102 s–1, so the JM region of ErbB can potentially cycle on and off the membrane at a frequency that could facilitate the hydrolysis of PIP2 by an adjacent PLC.
One caveat concerning this electrostatic engine mechanism: the free [Ca/CaM] in cytoplasm may be significantly lower than the total cellular [CaM] of 10–100 μM. Recent measurements suggest that much of the Ca/CaM in cells may be bound to target proteins (Persechini and Stemmer, 2002; Black et al., 2004; Kim et al., 2004; Rakhilin et al., 2004).
Predictions of the Model
ACKNOWLEDGMENTS
We thank B. Kholodenko, T. Miller, J. Pessin, and J. Staros for helpful discussions and G. Hangyás-Mihályné, I. Zaitseva, and I. Ischenko for excellent technical assistance.
This work was supported by National Institutes of Health grants GM24971 (to S. McLaughlin), GM69651 (to S.O. Smith), CA28146 (to M. Hayman), National Science Foundation/Molecular and Cellular Biosciences 0212362 (to D. Murray), and a Carol M. Baldwin Foundation Breast Cancer Research Award (to S. McLaughlin).
Olaf S. Andersen served as editor.
Submitted: 11 February 2005
Accepted: 19 May 2005
REFERENCES
Aifa, S., K. Johansen, U.K. Nilsson, B. Liedberg, I. Lundstrom, and S.P.S. Svensson. 2002. Interactions between the juxtamembrane domain of the EGFR and calmodulin measured by surface plasmon resonance. Cell. Signal. 14:1005–1013.
Aifa, S., J. Aydin, G. Nordvall, I. Lundstrom, S.P.S. Svensson, and O. Hermanson. 2005. A basic peptide within the juxtamembrane region is required for EGF receptor dimerization. Exp. Cell Res. 302:108–114.
Arbuzova, A., J. Wang, D. Murray, J. Jacob, D.S. Cafiso, and S. McLaughlin. 1997. Kinetics of interaction of the myristoylated alanine-rich C kinase substrate, membranes, and calmodulin. J. Biol. Chem. 272:27167–27177.
Arbuzova, A., D. Murray, and S. McLaughlin. 1998. MARCKS, membranes and calmodulin: kinetics of their interaction. Biochim. Biophys. Acta. 1376:369–379.
Arbuzova, A., L. Wang, J. Wang, G. Hangyàs-Mihàlynè, D. Murray, B. Honig, and S. McLaughlin. 2000. Membrane binding of peptides containing both basic and aromatic residues. Biochemistry. 39:10330–10339.
Bezzerides, V.J., I.S. Ramsey, S. Kotecha, A. Greka, and D.E. Clapham. 2004. Rapid vesicular translocation and insertion of TRP channels. Nat. Cell Biol. 6:709–720.
Black, D.J., Q.-K. Tran, and A. Persechini. 2004. Monitoring the total available calmodulin concentration in intact cells over the physiological range in free Ca2+. Cell Calcium. 35:415–425.
Blobel, C.P. 2005. Adams: key components in EGFR signaling and development. Nat. Rev. Mol. Cell Biol. 6:32–43.
Blume-Jensen, P., and T. Hunter. 2001. Oncogenic kinase signalling. Nature. 411:355–365.
Burgess, A.W., H.-S. Cho, C. Eigenbrot, K.M. Ferguson, T.P.J. Garrett, D.J. Leahy, M.A. Lemmon, M.X. Sliwkowski, C.W. Ward, and S. Yokoyama. 2003. An open-and-shut case Recent insights into the activation of EGF/ErbB receptors. Mol. Cell. 12:541–552.
Carpenter, G. 2003. ErbB-4: mechanism of action and biology. Exp. Cell Res. 284:66–77.
Carpenter, G., L. King Jr., and S. Cohen. 1979. Rapid enhancement of protein phosphorylation in A-431 cell membrane preparations by epidermal growth factor. J. Biol. Chem. 254:4884–4891.
Chen, X., and M.D. Resh. 2002. Cholesterol depletion from the plasma membrane triggers ligand-independent activation of the epidermal growth factor receptor. J. Biol. Chem. 277:49631–49637.
Cheyette, T.E., and D.J. Gross. 1991. Epidermal growth factor-stimulated calcium ion transients in individual A431 cells: initiation kinetics and ligand concentration dependence. Cell Regul. 2:827–840.
Couchet, C., G.N. Gill, J. Meisenhelder, J.A. Cooper, and T. Hunter. 1984. C-kinase phosphorylates the epidermal growth factor receptor and reduces its epidermal growth factor-stimulated tyrosine protein kinase activity. J. Biol. Chem. 259:2553–2558.
Countaway, J.L., P. McQuilkin, N. Girones, and R.J. Davis. 1990. Multisite phosphorylation of the epidermal growth factor receptor. J. Biol. Chem. 265:3407–3416.
Davis, R.J., N. Gironès, and M. Faucher. 1988. Two alternative mechanisms control the interconversion of functional states of the epidermal growth factor receptor. J. Biol. Chem. 263:5373–5379.
Ferguson, K.M. 2004. Active and inactive conformations of the epidermal growth factor receptor. Biochem. Soc. Trans. 32:742–745.
Ferguson, K.M., M.B. Berger, J.M. Mendrola, H.S. Cho, D.J. Leahy, and M.A. Lemmon. 2003. EGF activates its receptor by removing interactions that autoinhibit ectodomain dimerization. Mol. Cell. 11:507–517.
Friedman, B., J. van Amsterdam, H. Fujiki, and M.R. Rosner. 1989. Phosphorylation at threonine-654 is not required for negative regulation of the epidermal growth factor receptor by non-phorbol tumor promoters. Proc. Natl. Acad. Sci. USA. 86:812–816.
Gambhir, A., G. Hangyás-Mihályné, I. Zaitseva, D.S. Cafiso, J. Wang, D. Murray, S. Pentyala, S.O. Smith, and S. McLaughlin. 2004. Electrostatic sequestration of PIP2 on phospholipid membranes by basic/aromatic regions of proteins. Biophys. J. 86:2188–2207.
Gotoh, N., A. Tojo, M. Hino, Y. Yazaki, and M. Shibuya. 1992. A highly conserved tyrosine residue at codon 845 within the kinase domain is not required for the transforming activity of human epidermal growth factor receptor. Biochem. Biophys. Res. Commun. 186:768–774.
Gschwind, A., E. Zwick, N. Prenzel, M. Leserer, and A. Ullrich. 2001. Cell communication networks: epidermal growth factor receptor transactivation as the paradigm for interreceptor signal transmission. Oncogene. 20:1594–1600.
Gschwind, A., O.M. Fischer, and A. Ullrich. 2004. The discovery of receptor tyrosine kinases: targets for cancer therapy. Nat. Rev. Cancer. 4:361–370.
Hendriks, B.S., G. Orr, A. Well, H.S. Wiley, and D.A. Lauffenburger. 2005. Parsing ERK activation reveals quantitatively equivalent contributions from epidermal growth factor receptor and HER2 in human mammary epithelial cells. J. Biol. Chem. 280:6157–6169.
Holthuis, J.C.M., and T.P. Levine. 2005. Lipid traffic: floppy drives and a superhighway. Nat. Rev. Mol. Cell Biol. 6:209–220.
Hubbard, S.R. 2004. Juxtamembrane autoinhibition in receptor tyrosine kinases. Nat. Rev. Mol. Cell Biol. 5:464–470.
Hubbard, S.R., and J.H. Till. 2000. Protein tyrosine kinase structure and function. Annu. Rev. Biochem. 69:373–398.
Hughes, A.R., G.S. Bird, J.F. Obie, O. Thastrup, and J.W. Putney Jr. 1991. Role of inositol (1,4,5)trisphosphate in epidermal growth factor-induced Ca2+ signaling in A431 cells. Mol. Pharmacol. 40:254–262.
Hunter, T., N. Ling, and J.A. Cooper. 1984. Protein kinase C phosphorylation of the EGF receptor at a threonine residue close to the cytoplasmic face of the plasma membrane. Nature. 311:480–483.
Huse, M., and J. Kuriyan. 2002. The conformational plasticity of protein kinases. Cell. 109:275–282.
Ichinos, J., M. Murata, T. Yanagida, and Y. Sako. 2004. EGF signaling amplification induced by dynamic clustering of EGFR. Biochem. Biophys. Res. Commun. 324:1143–1149.
Jorissen, R.N., F. Walker, N. Pouliot, T.P.J. Garrett, C.W. Ward, and A.W. Burgess. 2003. Epidermal growth factor receptor: mechanisms of activation and signalling. Exp. Cell Res. 284:31–53.
Kholodenko, B.N., O.V. Demin, G. Moehren, and J.B. Hoek. 1999. Quantification of short term signaling by the epidermal growth factor receptor. J. Biol. Chem. 274:30169–30181.
Kim, S.A., K.G. Heinze, M.N. Waxham, and P. Schwille. 2004. Intracellular calmodulin availability accessed with two-photon cross-correlation. Proc. Natl. Acad. Sci. USA. 101:105–110.
King, C.R., I. Borrello, L. Porter, P. Comoglio, and J. Schlessinger. 1989. Ligand-independent tyrosine phosphorylation of EGF receptor and the erbB-2/neu proto-oncogene product is induced by hyperosmotic shock. Oncogene. 4:13–18.
Klapper, L.N., M.H. Kirschbaum, M. Sela, and Y. Yarden. 2000. Biochemical and clinical implications of the ErbB/HER signaling network of growth factor receptors. Adv. Cancer Res. 77:25–79.
Landau, M., S.J. Fleishman, and N. Ben-Tal. 2004. A putative mechanism for downregulation of the catalytic activity of the EGF receptor via direct contact between its kinase and C-terminal domains. Structure (Camb.). 12:2265–2275.
Li, H., M.J. Ruano, and A. Villalobo. 2004a. Endogenous calmodulin interacts with the epidermal growth factor receptor in living cells. FEBS Lett. 559:175–180.
Li, H., J. Sanchez-Torres, A. Del Carpio, V. Salas, and A. Villalobo. 2004b. The ErebB2/Neu/HER2 receptor is a new calmodulin-binding protein. Biochem. J. 381:257–266.
Li, W., L. Tsiokas, S.C. Sansom, and R. Ma. 2004c. Epidermal growth factor activates store-operated Ca2+ channels through an inositol 1,4,5-trisphosphate-independent pathway in human glomerular mesangial cells. J. Biol. Chem. 279:4570–4577.
Martin-Nieto, J., and A. Villalobo. 1998. The human epidermal growth factor receptor contains a juxtamembrane calmodulin-binding site. Biochemistry. 37:227–236.
Matilla, E., T. Pellinen, J. Nevo, K. Vuoriluoto, A. Arjonen, and J. Evaska. 2004. Negative regulation of EGFR signaling through integrin-11-mediated activation of protein tyrosine phosphatase TCPTP. Nat. Cell Biol. 7:78–85.
Matsuda, M., H.F. Paterson, R. Rodriguez, A.C. Fensome, M.V. Ellis, K. Swann, and M. Katan. 2001. Real time fluorescence imaging of PLC translocation and its interaction with the epidermal growth factor receptor. J. Cell Biol. 153:599–612.
McLaughlin, S. 1989. The electrostatic properties of membranes. Annu. Rev. Biophys. Biophys. Chem. 18:113–136.
Moehren, G., N. Markevich, O. Demin, A. Kiyatkin, I. Goryanin, J.B. Hoek, and B.N. Kholodenko. 2002. Temperature dependence of the epidermal growth factor receptor signaling network can be accounted for by a kinetic model. Biochemistry. 41:306–320.
Murasawa, S., Y. Mori, Y. Nozana, N. Gotoh, M. Shibuya, H. Masaki, K. Maruyama, Y. Tsutsumi, Y. Moriguchi, Y. Shibazaki, et al. 1998. Angiotensin II type 1 receptor-induced extracellular signal-regulated protein kinase activation is mediated by Ca2+/calmodulin-dependent transactivation of epidermal growth factor receptor. Circ. Res. 82:1338–1348.
Murray, D., A. Arbuzova, B. Honig, and S. McLaughlin. 2002. The role of electrostatic and nonpolar interactions in the associations of peripheral proteins with membranes. Curr. Top. Membranes. 52:277–307.
Nojiri, S., and J.B. Hoek. 2000. Suppression of epidermal growth factor-induced phospholipase C activation associated with actin rearrangement in rat hepatocytes in primary culture. Hepatology. 32:947–957.
Ogiso, H., R. Ishitani, O. Nureki, S. Fukai, M. Yamanaka, J.H. Kim, K. Saito, A. Sakamoto, M. Inoue, M. Shirouzu, and S. Yokoyama. 2002. Crystal structure of the complex of human epidermal growth factor and receptor extracellular domains. Cell. 110:775–787.
Osawa, M., M.B. Swindells, J. Tanikawa, T. Tanaka, T. Mase, T. Furuya, and M. Ikura. 1998. Solution structure of calmodulin-W-7 complex: the basis of diversity in molecular recognition. J. Mol. Biol. 276:165–176.
Paez, J.G., A.J. Pasi, J.C. Lee, S. Tracy, H. Grenlich, S. Gabriel, P. Herman, F.J. Kaye, N. Lindeman, T.J. Boggon, et al. 2004. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science. 304:1497–1500.
Papin, J.A., T. Hunter, B.O. Palsson, and S. Subramaniam. 2005. Reconstruction of cellular signaling networks and analysis of their properties. Nat. Rev. Mol. Cell Biol. 6:99–111.
Pandiella, A., L. Beguinot, T.J. Velu, and J. Meldolesi. 1988. Transmembrane signalling at epidermal growth factor receptors overexpressed in NIH 3T3 cells. Phosphoinositide hydrolysis, cytosolic Ca2+ increase and alkalinization correlate with epidermal-growth-factor-induced cell proliferation. Biochem. J. 254:223–228.
Persechini, A., and P.M. Stemmer. 2002. Calmodulin is a limiting factor in the cell. Trends Cardiovasc. Med. 12:32–37.
Prenzel, N., E. Zwick, H. Daub, M. Leserer, R. Abraham, C. Wallasch, and A. Ullrich. 1999. EGF receptor transactivation by G-protein-coupled receptors requires metalloproteinase cleavage of proHB-EGF. Nature. 402:884–888.
Rakhilin, S.V., P.A. Olson, A. Nishi, N.N. Starkova, A.A. Fienberg, A.C. Nairn, D.J. Surmeier, and P. Greengard. 2004. A network of control mediated by regulator of calcium/calmodulin-dependent signaling. Science. 306:698–701.
Reynolds, A.R., C. Tischeer, P.J. Verveer, O. Rocks, and P.I.H. Bastiaens. 2003. EGFR activation coupled to inhibition of tyrosine phosphatases causes lateral signal propagation. Nat. Cell Biol. 5:447–453.
Rodriguez, I., M. Kaszkin, A. Holloschi, K. Kabsch, M.M. Marques, X. Mao, and A. Alonso. 2002. Hyperosmotic stress induces phosphorylation of cytosolic phospholipase A2 in HaCaT cells by an epidermal growth factor receptor-mediated process. Cell. Signal. 14:839–848.
Roepstorff, K., P. Thomsen, K. Samndvig, and B. van Deurs. 2002. Sequestration of epidermal growth factor receptors in non-caveolar lipid rafts inhibits ligand binding. J. Biol. Chem. 277:18954–18960.
Rusu, L., A. Gambhir, S. McLaughlin, and J. Radler. 2004. Fluorescence correlation spectroscopy studies of peptide and protein binding to phospholipid vesicles. Biophys. J. 87:1044–1053.
Schlessinger, J. 2000. Cell signaling by receptor tyrosine kinases. Cell. 103:211–225.
Schlessinger, J. 2003. Signal transduction. Autoinhibition control. Science. 300:750–752.
Shapiro, M.S. 2004. Why biophysicists make models: quantifying modulation of the M current. J. Gen. Physiol. 123:657–662.
Simons, K., and W.L.C. Vaz. 2004. Model systems, lipid rafts, and cell membranes. Annu. Rev. Biophys. Biomol. Struct. 33:269–295.
Stamos, J., M.X. Sliwkowski, and C. Eigenbrot. 2002. Structure of the epidermal growth factor receptor kinase domain alone and in complex with a 4-anilinoquinazoline inhibitor. J. Biol. Chem. 277:46265–46272.
Sordella, R., D.W. Bell, D.A. Haber, and J. Settleman. 2004. Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways. Science. 305:1163–1167.
Tonks, N. 2003. PTP1B: from the sidelines to the front lines! FEBS Lett. 546:140–148.
Ullrich, A., L. Coussens, J.S. Hayflick, T.S. Dull, A. Gray, A.W. Tam, J. Lee, Y. Yarden, T.A. Liberman, J. Schlessinger, et al. 1984. Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. Nature. 309:418–425.
Yarden, Y., and M.X. Sliwkowski. 2001. Untangling the ErbB signalling network. Nat. Rev. Mol. Cell Biol. 2:127–137.
Wang, J., A. Gambhir, G. Hangyás-Mihályné, D. Murray, U. Golebiewska, and S. McLaughlin. 2002. 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. J. Biol. Chem. 277:34401–34412.
Wang, X.-J., H.-J. Liao, A. Chattopadhyay, and G. Carpenter. 2001. EGF-dependent translocation of green fluorescent protein-tagged PLC-1 to the plasma membrane and endosomes. Exp. Cell Res. 267:28–36.
Wang, Y., and Z. Wang. 2003. Regulation of EGF-induced phospholipase C-1 translocation and activation by its SH2 and PH domains. Traffic. 4:618–630.
Welsh, J.G., G.N. Gill, M.G. Rosenfeld, and A. Wells. 1991. A negative feedback loop attenuates EGF-induced morphological changes. J. Cell Biol. 114:533–543.
Westhover, E.J., D.F. Cover, H.L. Brockman, R.E. Brown, and L.J. Pike. 2003. Cholesterol depletion results in site-specific increases in epidermal growth factor receptor phosphorylation due to membrane level effects. J. Biol. Chem. 278:51125–51133.
Wiley, H.S., S.Y. Shvartsman, and D.A. Lauffenburger. 2003. Computational modeling of the EGF-receptor system: a paradigm for systems biology. Trends Cell Biol. 13:43–50.
Wong, W., and J.D. Scott. 2004. AKAP signaling complexes: focal points in space and time. Nat. Rev. Mol. Cell Biol. 5:959–970.
Wood, E.R., A.T. Truesdale, O.B. McDonald, D. Yuan, A. Hassell, S.H. Dickerson, B. Ellis, C. Pennisi, E. Horne, K. Lackey, et al. 2004. A unique structure for epidermal growth factor receptor bound to GW572016 (Lapatinib). Cancer Res. 64:6652–6659.
Zwick, E., P.O. Hackel, N. Prenzel, and A. Ullrich. 1999. The EGF receptor as central transducer of heterologous signalling systems. Trends Pharmacol. Sci. 20:408–412.(Stuart McLaughlin, Steven)
2 Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794
3 Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794
4 Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY 10021
We propose a new mechanism to explain autoinhibition of the epidermal growth factor receptor (EGFR/ErbB) family of receptor tyrosine kinases based on a structural model that postulates both their juxtamembrane and protein tyrosine kinase domains bind electrostatically to acidic lipids in the plasma membrane, restricting access of the kinase domain to substrate tyrosines. Ligand-induced dimerization promotes partial trans autophosphorylation of ErbB1, leading to a rapid rise in intracellular [Ca2+] that can activate calmodulin. We postulate the Ca2+/calmodulin complex binds rapidly to residues 645–660 of the juxtamembrane domain, reversing its net charge from +8 to –8 and repelling it from the negatively charged inner leaflet of the membrane. The repulsion has two consequences: it releases electrostatically sequestered phosphatidylinositol 4,5-bisphosphate (PIP2), and it disengages the kinase domain from the membrane, allowing it to become fully active and phosphorylate an adjacent ErbB molecule or other substrate. We tested various aspects of the model by measuring ErbB juxtamembrane peptide binding to phospholipid vesicles using both a centrifugation assay and fluorescence correlation spectroscopy; analyzing the kinetics of interactions between ErbB peptides, membranes, and Ca2+/calmodulin using fluorescence stop flow; assessing ErbB1 activation in Cos1 cells; measuring fluorescence resonance energy transfer between ErbB peptides and PIP2; and making theoretical electrostatic calculations on atomic models of membranes and ErbB juxtamembrane and kinase domains.
Abbreviations used in this paper: Ca/CaM, calcium/calmodulin; CD, circular dichroism; EGFR, epidermal growth factor receptor; FCS, fluorescence correlation spectroscopy; FRET, fluorescence resonance energy transfer; IP3, inositiol 1,4,5-trisphosphate; JM, juxtamembrane; LUV, large unilamellar vesicle; MLV, multilamellar vesicle; NEM, [ethyl-1,2-3H]N-ethylmaleimide; PC, phosphatidylcholine; PIP2, phosphatidylinositol 4,5-bisphosphate; PS, phosphatidylserine; PTK, protein tyrosine kinase; TM, transmembrane.
INTRODUCTION
The general mechanism by which binding of a ligand to the extracellular domain of a typical receptor tyrosine kinase activates its intracellular protein tyrosine kinase (PTK) domain is well understood: binding produces dimerization (or dimer rearrangement) that leads to trans autophosphorylation of tyrosines in the "activation loop" of the PTK domain (Hubbard and Till, 2000; Schlessinger, 2000, 2003; Huse and Kuriyan, 2002; Jorissen et al., 2003; Hubbard, 2004). The ErbB tyrosine kinase family (ErbB1/HER1/EGFR, ErbB2/HER2, ErbB3/HER3, ErbB4/HER4) appears to be the major exception to this rule because their PTK domains do not require phosphorylation for catalytic competency (Gotoh et al., 1992; Jorissen et al., 2003; Hubbard, 2004). The structure of the ErbB1 PTK domain suggests why this is so: the unphosphorylated activation loop is in an active conformation and the catalytic elements are "primed and ready for phospho-transfer," suggesting "the regulation of the vital cellular processes influenced by epidermal growth factor receptor (EGFR) signaling must be exerted by control of the delivery of the COOH-terminal substrate tyrosines to the active site" (Stamos et al., 2002). Understanding this control mechanism is important because members of the ErbB family are frequently overactive in solid tumors (Blume-Jensen and Hunter, 2001; Yarden and Sliwkowski, 2001; Gschwind et al., 2004; Paez et al., 2004; Sordella et al., 2004); ErbB2, for example, is overexpressed in 25% of breast cancers, and this overexpression correlates with poor prognosis (Klapper et al., 2000).
Several groups have recently proposed models to explain autoinhibition of the ErbB family. Landau et al. (2004) developed a computational model that suggests direct contact between a positively charged face of the kinase domain and a negatively charged segment of the COOH-terminal tail region of the receptor produces autoinhibition. Alternatively, Aifa et al. (2005) proposed that this negatively charged segment, ErbB1(979–991), interacts with a cluster of basic residues in the juxtamembrane (JM) region of an adjacent ErbB molecule. The ErbB1 basic JM region also plays an important role in the structural model of autoinhibition we propose.
In our model, basic residues in the ErbB JM and PTK domains interact electrostatically with acidic lipids in the membrane, inhibiting catalytic activity in the absence of ligand. This autoinhibitory hypothesis has an obvious corollary: conditions that decrease the electrostatic binding (e.g., an increase in the cytoplasmic salt concentration, exposure to an amphipathic weak base that decreases the negative fixed charge density on the membrane) should release the JM and PTK regions from the membrane, producing ligand-independent trans autophosphorylation. We have tested this corollary by comparing data from our experiments with model membranes and peptides to data in the literature from intact cells.
Our model also suggests a novel positive feedback mechanism by which ligand-induced dimerization may contribute to activation. As discussed below, it is well established that ligand-induced dimerization of ErbB1 leads to a transient (10 min) increase in the level of free Ca2+ within a cell. We postulate that calcium/calmodulin (Ca/CaM) binds to the ErbB JM region very rapidly (100 ms when [Ca/CaM] = 1 μM), reversing its charge and repelling both it and the PTK domain from the membrane. This implies Ca/CaM binding will increase the initial rate of trans autophosphorylation over and above the rate due to the local concentration effect resulting from ligand-induced dimerization. Our postulated Ca/CaM-mediated activation mechanism will be important only when the [Ca2+] is high enough to produce a significant increase in Ca/CaM.
The model predicts that Ca/CaM can pull peptides corresponding to the ErbB JM region off a membrane rapidly and that CaM inhibitors will inhibit, but not completely block, the initial phase of EGF-mediated ErbB autophosphorylation in cells. We tested these predictions experimentally; while the results are consistent with the predictions, they neither prove that the model is correct nor rule out other potential activation mechanisms that may act in parallel (Jorissen et al., 2003; Schlessinger, 2003). For example, there is much evidence that phosphatases play an important role in controlling the trans autophosphorylation of ErbB (e.g., Reynolds et al., 2003; Tonks, 2003; Ichinos et al., 2004; Matilla et al., 2004); we return to the role of phosphatases in the concluding section of DISCUSSION.
Structural Model
We divide the JM domain into three sections (Fig. 1 C) and hypothesize that the 16-residue NH2-terminal region (Arg645 to Gln660 of ErbB1) acts as a reversible membrane anchor. Specifically, we follow the suggestions of Ullrich et al. (1984) and Hunter et al. (1984) and postulate that in a quiescent cell, i.e., when no ligands are present and the free [Ca2+] is low, the eight basic and five hydrophobic amino acids bind this region to the negatively charged inner leaflet of the bilayer. In our model, the 12-residue central region (Glu661 to Gly672 of ErbB1) acts as a flexible linker that joins the reversible membrane anchor to the COOH-terminal section of the JM domain. The flexible linker has a net charge of –2 (3 E, 1R); it should be repelled, albeit weakly, from the membrane bilayer. The 10-residue COOH-terminal region of the JM domain (Glu673 to Ile682) was identified in the structure of the ErbB1 PTK core (Stamos et al., 2002). Fig. 2 illustrates that one face of this COOH-terminal JM region + PTK structure is positively charged (blue), and thus should bind electrostatically to the negatively charged bilayer, as drawn in Fig. 1 A. Fig. 2 also shows that the PTK domain has a strong dipole character (i.e., one face is positive, while the remainder of the molecule surface is mainly negative); thus it will not be free to rotate when bound to the bilayer. A key assumption of our model is that binding of the PTK core to the membrane inhibits its activity; hindered rotation could, for example, limit its contact with, and thus ability to phosphorylate, tyrosines in the COOH-terminal tail of an ErbB family dimer or other substrate.
Under physiological conditions, a ligand such as EGF binds to the ErbB1 extracellular domain and induces dimerization. Recent structural studies of the ErbB1 extracellular domain with (Ogiso et al., 2002) and without (Ferguson et al., 2003) bound EGF indicate that ligand binding triggers a dramatic rearrangement of the four extracellular subdomains in each receptor monomer. A direct intramolecular interaction between cysteine-rich subdomains II and IV in the unactivated receptor is broken upon EGF binding, and loss of contact releases a dimerization arm on domain II that mediates direct ErbB1 monomer–monomer contacts (for reviews see Burgess et al., 2003; Ferguson, 2004).
In our model, the intracellular JM + PTK regions of each dimer pair exist in equilibrium between the membrane-bound (Fig. 1 A) and free (not shown) states, with the autoinhibited membrane-bound state predominating in the absence of Ca/CaM. Upon ErbB dimerization (not depicted in Fig. 1 for simplicity), the small fraction of dimeric receptors with membrane-free JM + PTK regions can trans autophosphorylate. Phosphorylation of tyrosine residues in the ErbB COOH-terminal tail leads to binding and activation of PLC-, hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) with concomitant production of inositiol 1,4,5-trisphosphate (IP3), and IP3-mediated release of Ca2+ from internal stores (for review see Jorissen et al., 2003). The transitory release of Ca2+ from intracellular stores mediated by IP3 is followed by a more sustained influx of Ca2+ across the plasma membrane (e.g., Pandiella et al., 1988; Cheyette and Gross, 1991; Hughes et al., 1991; Bezzerides et al., 2004; Li et al., 2004c). Adding 30 nM EGF to A431 cells, for example, increases intracellular [Ca2+] about fivefold, to 600 nM, in 5 min; [Ca2+] then declines over 10 min to a value only slightly above the basal level (Hughes et al., 1991).
The EGF-mediated transient increase in cytoplasmic [Ca2+] activates CaM, and our data, together with previous work on peptides (Martin-Nieto and Villalobo, 1998) and native ErbB1 (Li et al., 2004a), suggest the Ca/CaM complex can bind rapidly and strongly to residues 645–660 of ErbB1, as shown in Fig. 1 B. This binding reverses the charge on the region from +8 to –8, converting its strong electrostatic attraction to the membrane into a strong electrostatic repulsion. We hypothesize that the binding energy of Ca/CaM for the reversible membrane anchor region and the electrostatic repulsion of the resulting complex from the negatively charged bilayer provide the energy to move the PTK core of an ErbB family member off the bilayer. Thus we refer to this mechanism as an "electrostatic engine" that increases both the frequency at which the JM + PTK domain moves from its autoinhibitory membrane-bound conformation (Fig. 1 A) to a freely rotating active state (Fig. 1 B) and the duration of time it spends in this active state. We discuss below experiments that suggest this putative engine may cycle rapidly (10–100 s–1).
Our fluorescence resonance energy transfer (FRET) and PLC activity measurements also show that the cluster of basic residues on the JM domain can, when bound to the membrane, electrostatically sequester PIP2. Thus ErbB may function as a scaffolding protein with its JM domain rapidly concentrating and releasing PIP2 in the vicinity of PLC- and phophoinositide 3-kinase (PI3K), enzymes that are bound to the ErbB COOH-terminal region and use PIP2 as a substrate.
MATERIALS AND METHODS
Materials
1-palmitoyl-2-oleoyl-sn-PS, 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (PC), 1-palmitoyl-2-oleoyl-sn-phosphatidylglycerol (PG), and the ammonium salt of L--phosphatidyl-D-myo-inositol 4,5-bisphosphate (PIP2) were purchased from Avanti Polar Lipids. Labeled [dioleoyl-1-14C]L--dioleoylphosphatidylcholine and [ethyl-1,2-3H]N-ethylmaleimide (NEM) were from PerkinElmer Life Sciences. 6-Acryloyl-2-dimethylaminonaphthalene (acrylodan), Alexa488, and Texas red were from Molecular Probes, Inc. Bodipy-TMR-PIP2 was purchased from Echelon. Sphingosine was from Sigma-Aldrich.
We formed multilamellar vesicles (MLVs) for zeta potential measurements, 100-nm-diameter large unilamellar vesicles (LUVs) for FRET, FCS, and stop flow fluorescence measurements, and 100-nm sucrose-loaded LUVs for centrifugation binding measurements, as described previously (Wang et al., 2002).
Methods
Measurements of Peptide Binding to LUVs.
We measured the binding of [3H]NEM-labeled peptides to sucrose-loaded PC/PS LUVs using a centrifugation technique described previously (Wang et al., 2002; Gambhir et al., 2004). In brief, we mixed sucrose-loaded LUVs and a trace concentration (5 nM) of [3H]NEM-labeled peptide, and then centrifuged the mixture at 100,000 g for 1 h. We calculated the percentage of peptide bound from counts of the radioactive peptide in the supernatant and in the pellet.
Zeta Potential of MLVs.
We measured the electrophoretic mobility (velocity/field) of single MLVs and calculated the zeta potential, the electrostatic potential at the hydrodynamic plane of shear (0.2 nm from the surface), using the Helmholtz-Smoluchowski equation (Wang et al., 2002, and references therein).
Surface Pressure Measurements.
We deposited a stock lipid–chloroform solution onto the surface of a 15-ml aqueous solution in a 5-cm-diameter Teflon trough with a magnetic stirrer at the bottom. Once the chloroform had evaporated, we measured the surface pressure of the monolayer using a square piece of filter paper and a balance from Nima Technology Ltd. We then added the peptide to the subphase and measured the change in surface pressure as described previously (Wang et al., 2002).
FRET.
We monitored FRET between a Bodipy TMR label on PIP2 and a Texas red label attached to membrane-bound ErbB1(645–660) as described previously (Gambhir et al., 2004); the membrane contained 69.7% PC, 30% PS, and 0.3% PIP2.
Circular Dichroism (CD) Spectroscopy.
We obtained the CD spectrum of ErbB1(645–660) bound to isotropic bicelles on an Olis DSM CD spectrometer (Olis Instruments) with a 0.2-mm path-length cell. The bicelles were composed of a mixture of DMPC, DMPG, and DHPC in a 10:3:13 molar ratio. The buffered bicelle solution (20 mM sodium phosphate, pH 7.0) was 10% (wt/vol) lipid and the peptide:lipid ratio was 1:100. Measurements of a bicelle solution without peptide prepared in parallel were subtracted as background. The corrected CD spectrum exhibits the strong negative ellipticity at 200 nm characteristic of random-coil structures.
Fluorescence Correlation Spectroscopy (FCS).
We used a Carl Zeiss MicroImaging, Inc. Confocor II microscope to monitor the binding of Alexa488-labeled ErbB1(645–660) to 2:1 PC/PS 100-nm LUVs and to study the ability of Ca/CaM to remove the peptide from the membrane. The experimental techniques were similar to those described in detail in Rusu et al. (2004). In brief, the correlation times of the peptide bound to 100-nm LUVs and to Ca/CaM are 1,700 μs and 100 μs, respectively; hence we could distinguish the two correlation times easily. We determined the affinity of Ca/CaM for the peptide by plotting the fraction of membrane-bound peptide against the concentration of Ca/CaM in the solution, as shown for experiments using the centrifugation technique (see Fig. 4).
Stop Flow Kinetics.
We made fluorescence stop flow kinetic measurements to determine the rate at which Ca/CaM removes membrane-bound acrylodan-labeled ErbB1(645–660) from PC/PS vesicles; adding Ca/CaM increased the fluorescence approximately fourfold as the peptide moved from vesicle to Ca/CaM. Specifically, one solution contained 200 or 400 nM acrylodan-labeled ErbB1(645–660) bound to 100-nm 85:14:1 PC/PS/NBD-PS vesicles (100 μM accessible lipid; the 1% NBD-PS in these membranes quenches the acrylodan fluorescence), and the other solution contained 0.5, 1, 2, 4, or 7 μM CaM, and 50 μM CaCl2. Both solutions contained 100 mM KCl buffered to pH 7.0 with 1 mM MOPS. We measured the time constants of the exponential increase in fluorescence, , and determined the slope of 1/ vs [CaM]. This slope is equal to the transfer rate constant. The two peptide concentrations produced identical time constants, as expected. We repeated the stop flow measurements with vesicles containing 10, 12, and 18% PS. The results and experimental details are similar to those shown in Fig. 7 of Arbuzova et al. (1997) for a different basic/hydrophobic acrylodan-labeled peptide.
Calculation of Electrostatic Potentials.
We built atomic models of the 2:1 PC/PS bilayer (Wang et al., 2002) and ErbB1(645–660) in an extended conformation using the Insight Biopolymer and Discover modules (Accelrys); the atomic radii and partial charges assigned to the peptide were taken from the CHARMM forcefield. We solved the nonlinear Poisson-Boltzmann equation for atomic models of these systems in 100 mM KCl. The resulting potential maps, as well as the atomic coordinates for the peptide/membrane/CaM models, were displayed using GRASP.
Online Supplemental Material
RESULTS
ErbB1(645–660) Binds Strongly to Phospholipid Vesicles through Nonspecific Electrostatic Interactions
We first tested the postulate that the ErbB1 reversible membrane anchor region binds to the plasma membrane by determining if a peptide corresponding to this region, ErbB1(645–660), binds to phospholipid vesicles. The data in Fig. 3 show this is indeed the case: ErbB1(645–660) binds strongly to vesicles containing physiological (15–30%) mole fractions of the monovalent acidic lipid PS. We describe the binding using Eq. 1 (Arbuzova et al., 2000):
(1)
ErbB1(645–660) Laterally Sequesters PIP2
Biological Experiments Consistent with our Autoinhibition Hypothesis
Ca/CaM Removes ErbB JM Peptides from Membranes Rapidly
The mechanism shown in Fig. 1 B, i.e., Ca/CaM binds to the membrane anchor region and removes it and the PTK domain to facilitate EGF-mediated activation, is admittedly speculative, but peptide experiments provide evidence that it is feasible. We first tested whether Ca/CaM competes with phospholipid membranes for binding of ErbB1(645–660); Fig. 4 shows peptide binding to 2:1 PC/PS vesicles in the presence of increasing [Ca/CaM]. In the absence of Ca/CaM, 90% of the peptide binds to the vesicles, as expected from both theoretical calculations (unpublished data; see Murray et al., 2002, for methods) and the binding results in Fig. 3. The open circles in Fig. 4 illustrate the effect of increasing [Ca/CaM]: adding 0.1 μM Ca/CaM reduces binding by 50% (total [CaM] 10–100 μM in a mammalian cell). Adding CaM in the absence of free Ca2+ does not affect ErbB1(645–660) binding to the vesicles (filled circles). These data indicate that Ca/CaM binds to ErbB1(645–660) with high affinity (Kd = 10 nM), preventing the peptide from binding to a phospholipid vesicle. We used a modified version of Eq. 1 to describe the effect of Ca/CaM on ErbB1(645–660) membrane binding, incorporating the assumption that Ca/CaM and the membrane compete for the peptide:
(2)
where K is the molar partition coefficient of the peptide onto the 2:1 PC/PS vesicles (106 M–1 from Fig. 3) and KCaM is the association constant of the peptide with Ca/CaM (KCaM = 108 M–1 from the fit of Eq. 2 to data in Fig. 4).
We repeated the measurements shown in Fig. 4 using 3:1 rather than 2:1 PC/PS vesicles and obtained a similar value for KCaM. We also used an independent method, fluorescence correlation spectroscopy (FCS), to validate the centrifugation technique used to obtain the measurements shown in Fig. 4. FCS measures the correlation time (inversely proportional to diffusion constant) of a fluorescent label, in this case Alexa488 attached to ErbB1(645–660). The labeled peptide diffuses slowly when bound to the large (100 nm) vesicles and rapidly when bound to the smaller Ca/CaM; thus we can determine the fraction of membrane-bound peptide as a function of the concentration of Ca/CaM. Both centrifugation and FCS measurements with Alexa488-labeled ErbB1(645–660) indicate KCaM = 107 M–1 (U. Golebiewska, personal communication; unpublished data). Thus, we conclude that the FCS measurements validate the centrifugation technique. As expected from electrostatics, the negatively charged Alexa probe decreases the affinity of the peptide for Ca/CaM (and vesicles) 10-fold.
Phosphorylation of Thr654 also decreases the affinity of ErbB1(645–660) for Ca/CaM. Experiments similar to those shown in Fig. 4, but conducted with an ErbB1(645–660) peptide with a phosphorylated Thr654, show that phosphorylation reduces KCaM 20-fold: removal of 50% of the phosphopeptide from the vesicles requires 20-fold more Ca/CaM than shown in Fig. 4 under similar conditions, i.e., when 90% of the phosphopeptide is bound initially. Phosphorylation also decreases the membrane binding of the peptide 10-fold. These results agree well with previous measurements showing that phosphorylation "drastically hampers" (Martin-Nieto and Villalobo, 1998) or "totally inhibits" (Aifa et al., 2002) the ability of Ca/CaM to bind to this region of ErbB1. The model shown in Fig. 1 thus predicts that phosphorylation of Thr654 by PKC should significantly attenuate at least the early phase of ErbB1 autophosphorylation, a prediction consistent with experiment (Couchet et al., 1984; Hunter et al., 1984; Countaway et al., 1990; Welsh et al., 1991). However, phosphorylation of Thr654 also produces a less robust inhibition of ErbB1 autophosphorylation in model systems, such as A431 cell membranes (e.g., Couchet et al., 1984), which must be due to some other phenomenon.
The kinetics results are perhaps surprising: the simplest interpretation of our equilibrium measurements (Fig. 4) is that Ca/CaM acts as a passive peptide buffer. That is, adding it to a solution containing peptides bound to vesicles should merely decrease the equilibrium concentration of free peptide in the bulk aqueous phase, allowing peptide to desorb from the vesicles at its spontaneous rate until a new equilibrium is attained. The stop flow measurements, however, reveal that Ca/CaM increases the rate at which ErbB1(645–660) desorbs from the vesicles, presumably by ripping the peptide directly from the surface; we have discussed this mechanism elsewhere (Arbuzova et al., 1997, 1998).
CaM Inhibitors Reduce the Initial Rate of EGF-mediated Autophosphorylation in Cos1 Cells
A key prediction of the mechanism shown in Fig. 1 B is that exposing cells to membrane-permeable CaM inhibitors such as W-7 should inhibit the initial rate of EGF-mediated ErbB1 autophosphorylation. According to our model, CaM inhibitors should affect autophosphorylation only for a short time (<15 min) after EGF stimulation because the concomitant increase in cytoplasmic Ca2+ to values that activate CaM significantly is only transient. For example, Hughes et al. (1991) reported the intracellular [Ca2+] in A431 cells increases to 600 nM in 5 min, but falls to 150 nM by 15 min. Nojiri and Hoek (2000) show the intracellular [Ca2+] in hepatocytes increases to 450 nM in 1–2 min, and then falls to 150 nM in the next 2–3 min. Fig. 5 shows treating Cos1 cells with W-7 produces dose-dependent inhibition of ErbB1 tyrosine phosphorylation measured 10 min after addition of EGF; the concentration range used, 20–50 μM, was selected on the basis of the inhibitor's affinity for Ca/CaM (Osawa et al., 1998). EGF-stimulated ErbB1 autophosphorylation is maximal 10 min after adding EGF to Cos1 cells under our conditions (unpublished data). Recent detailed studies in two other cell types showed that the CaM inhibitors W-7, W-12, and W-13 inhibit the initial peak of ErbB1 autophosphorylation, but not the steady-state value observed for times >20 min (Li et al., 2004a), as expected from our model. Li et al. (2004a) report an important control experiment: W-7, W-12, and W-13 do not inhibit the tyrosine kinase activity of a purified ErbB1 preparation.
DISCUSSION
Membrane Binding of ErbB Intracellular Regions and Postulated Role of Ca/CaM
Our peptide experiments and/or theoretical calculations support the hypothesis that both the JM region and PTK domain of ErbB family members bind electrostatically to the inner leaflet of the plasma membrane. If our postulate that this binding produces autoinhibition is correct, factors that reduce these electrostatic interactions should stimulate cellular ErbB autophosphorylation in the absence of ligand. Specifically, we postulate that autophosphorylation depends on both the rate at which ErbB monomers collide in the plasma membrane and the probability the JM + PTK domain is in a membrane-dissociated active conformation. Our model membrane experiments allowed us to measure quantitatively the levels of salt, divalent cations, or the amphipathic weak base sphingosine required to disengage a peptide corresponding to the membrane anchor region from a lipid bilayer; when we compared the values to those reported for ligand-independent activation of ErbB1 in cells or broken membrane preparations (Carpenter et al., 1979; Davis et al., 1988; King et al., 1989), there was quantitative agreement in each case. Thus the mechanism illustrated in Fig. 1 A provides a plausible and experimentally testable explanation for autoinhibition, but it does not address the specific mechanism by which membrane binding inhibits tyrosine kinase activity. Possible mechanisms could include restricted rotation of the PTK domain that prevents interaction of the catalytic site and substrate, or structural effects on the activation loop within the PTK domain (Stamos et al., 2002; Wood et al., 2004); several recent reviews discuss autoinhibitory mechanisms for receptor tyrosine kinases in some detail (Jorissen et al., 2003; Schlessinger, 2003; Hubbard, 2004).
Our data show that Ca/CaM binds strongly to peptides corresponding to the membrane anchor region of ErbB1 (Fig. 4) and the corresponding JM regions in other ErbB family members (Table I). The results agree qualitatively with previous reports that Ca/CaM binds strongly to these regions of ErbB1 (Martin-Nieto and Villalobo, 1998; Li et al., 2004a) and ErbB2 (Li et al., 2004b). We note that Martin-Nieto and Villalobo (1998) reported KCaM = 3 x 106 M–1, but their Ca/CaM binding measurements employed 0.3 x 10–6 M of a GST-ErbB1(645–660) construct; thus we interpret their measurements to mean KCaM > 3 x 106 M–1, which agrees with our estimate. These data suggest that the activation mechanism illustrated in Fig. 1 B is feasible and could enhance the activation produced by ligand-induced dimerization, which presumably acts through a local concentration effect (Schlessinger, 2000).
The Electrostatic Engine Mechanism in ErbB Activation
Our model suggests the following process for EGF-mediated trans autophosphorylation. When EGF-stimulated dimerization occurs, the level of intracellular [Ca2+], and thus Ca/CaM, is initially low. The ErbB JM + PTK regions, however, move off the membrane spontaneously, albeit at a low rate (e.g., stop flow measurements demonstrate acrylodan-labeled ErbB1(645–660) moves off a 85:15 PC/PS vesicle at a rate of 3 s–1). The small fraction of receptors with dissociated JM + PTK domains will produce a low level of trans autophosphorylation even in the absence of Ca/CaM. When PLC- binds to a phosphorylated ErbB and hydrolyzes its substrate PIP2, it produces IP3, which in turn releases Ca2+ from internal stores, increasing the concentration of Ca/CaM (for review see Jorissen et al., 2003). Our measurements show that Ca/CaM increases both the rate at which peptides corresponding to the ErbB1 membrane anchor region leave the membrane and the fraction of membrane-dissociated peptide (Fig. 4). As the JM and kinase domains of an ErbB move off the membrane, the latter becomes catalytically active in our model, as illustrated for one member of the ErbB1 dimer in Fig. 1 B. Thus we postulate that Ca/CaM drives a positive feedback mechanism that produces maximal activation of ErbB. The mechanism functions only when the local [Ca2+] is sufficiently elevated to provide a source of Ca/CaM; intracellular [Ca2+] measurements indicate this occurs only for times <15 min after exposure to EGF (Hughes et al., 1991; Nojiri and Hoek, 2000). This electrostatic engine model predicts that CaM inhibitors (or agents that increase the intracellular Ca2+ buffering capacity) will diminish the transient increase in trans autophosphorylation observed within 15 min of EGF stimulation, but have little effect on the steady-state level of activity measured at later times. CaM inhibitors do indeed inhibit peak level of EGF-mediated ErbB1 autophosphorylation in Cos1 (Fig. 5) and two other cell types (Li et al., 2004a), but have no effect on the steady-state level of autophosphorylation (Li et al., 2004a). Agents that deplete intracellular stores of Ca2+, such as thapsigargin, should and do inhibit receptor autophosphorylation in A431 cells measured 5 min after addition of EGF (Friedman et al., 1989).
Less direct evidence in support of this hypothesis comes from studies of Ca2+-induced transactivation of ErbB1 (i.e., activation that occurs without addition of a ligand that binds directly to ErbB1). Transactivation can occur in response to activation of Gq-coupled receptors, opening of ion channels selective for Ca2+, or addition of Ca2+ ionophores (for review see Zwick et al., 1999). Much recent work on the transactivation of ErbB1 has focused on the unique triple-membrane-passing signal (Prenzel et al., 1999; for reviews see Gschwind et al., 2001; Blobel, 2005), but our model provides a clue as to how an increase in [Ca2+] and Ca/CaM could initiate this interesting phenomenon. If Ca2+ helps initiate transactivation by the mechanism shown in Fig. 1 B, both CaM inhibitors and Ca2+ buffers should inhibit transactivation. Murasawa et al. (1998) reported both the CaM inhibitor W-7 and the Ca2+ buffer BAPTA-AM inhibit angiotensin II–stimulated ErbB1 transactivation in cardiac fibroblasts.
Rigorous testing of the model will require extensive molecular and cell biological experiments as well as biophysical measurements on larger peptides corresponding to the ErbB TM + JM domains reconstituted into vesicles, which are in progress. If additional work supports our postulate that Ca/CaM may act in concert with dimerization to stimulate ErbB1 activation, the model shown in Fig. 1 can probably be extrapolated to the ErbB2 and ErbB4 (Carpenter, 2003) family members: peptides corresponding to their JM regions also bind with high affinity to both membranes and Ca/CaM (Table I).
ErbB as a Scaffolding Protein
How rapidly can Ca/CaM bind to the JM region and release the electrostatically sequestered PIP2 Our stop flow experiments reveal that 2 μM Ca/CaM can remove acrylodan-labeled ErbB1(645–660) peptides from PC/PS vesicles at rates of 10 and 100 s–1 for vesicles containing 15 and 10% PS, respectively. It is difficult to extrapolate these results using a model system to a living cell for several reasons. For example, ErbB1 may be in noncaveolar cholesterol- and PIP2-enriched "rafts" that have a different lipid composition than the bulk plasma membrane (e.g., Chen and Resh, 2002; Roepstorff et al., 2002; Westhover et al., 2003; Simons and Vaz, 2004, and references therein). We can state that if the Ca/CaM level rises to 1 μM in a cell, the maximum (diffusion limited) rate at which Ca/CaM could bind to the 645–660 JM region of ErbB1 and rip it off the plasma membrane is 100 s–1.
How long will the Ca/CaM remain bound to the JM region The lifetime of Ca/CaM bound to a solubilized ErbB1 molecule is probably 1 s (KCaM/kon = 108 M–1/108 M–1s–1), but theoretical considerations suggest the lifetime of Ca/CaM bound to ErbB1 in a membrane will decrease significantly (possibly to 0.01 s) as the mole fraction of acidic lipid in the membrane increases. This is because the acidic lipids repel the negatively charged Ca/CaM bound to the JM region.
Thus the electrostatic engine shown in Fig. 1 (A and B) could cycle 10–100 times a second when the [Ca/CaM] increases to 1 μM. Even if the JM region remains bound to the bilayer for only 0.01 s, this provides more than sufficient time, t, for PIP2 to equilibrate with the basic cluster through diffusion (from the Einstein relation, t = x2/4D, where x is the distance PIP2 must diffuse in the plasma membrane and D is its diffusion constant). FCS measurements show that the diffusion constant of Bodipy-PIP2 in a fluid phase PC phospholipid membrane has the expected value of D = 3 x 10–8 cm2s–1 (Golebiewska, U., personal communication). In a plasma membrane, D could be 10-fold lower because cholesterol increases the viscosity, and as much as 90% of the PIP2 could be sequestered such that x = distance between PIP2 free to diffuse = 30 nm; even under these conditions the diffusion time is only 0.001 s. The maximal rate at which PLCs can hydrolyze PIP2 is 102 s–1, so the JM region of ErbB can potentially cycle on and off the membrane at a frequency that could facilitate the hydrolysis of PIP2 by an adjacent PLC.
One caveat concerning this electrostatic engine mechanism: the free [Ca/CaM] in cytoplasm may be significantly lower than the total cellular [CaM] of 10–100 μM. Recent measurements suggest that much of the Ca/CaM in cells may be bound to target proteins (Persechini and Stemmer, 2002; Black et al., 2004; Kim et al., 2004; Rakhilin et al., 2004).
Predictions of the Model
ACKNOWLEDGMENTS
We thank B. Kholodenko, T. Miller, J. Pessin, and J. Staros for helpful discussions and G. Hangyás-Mihályné, I. Zaitseva, and I. Ischenko for excellent technical assistance.
This work was supported by National Institutes of Health grants GM24971 (to S. McLaughlin), GM69651 (to S.O. Smith), CA28146 (to M. Hayman), National Science Foundation/Molecular and Cellular Biosciences 0212362 (to D. Murray), and a Carol M. Baldwin Foundation Breast Cancer Research Award (to S. McLaughlin).
Olaf S. Andersen served as editor.
Submitted: 11 February 2005
Accepted: 19 May 2005
REFERENCES
Aifa, S., K. Johansen, U.K. Nilsson, B. Liedberg, I. Lundstrom, and S.P.S. Svensson. 2002. Interactions between the juxtamembrane domain of the EGFR and calmodulin measured by surface plasmon resonance. Cell. Signal. 14:1005–1013.
Aifa, S., J. Aydin, G. Nordvall, I. Lundstrom, S.P.S. Svensson, and O. Hermanson. 2005. A basic peptide within the juxtamembrane region is required for EGF receptor dimerization. Exp. Cell Res. 302:108–114.
Arbuzova, A., J. Wang, D. Murray, J. Jacob, D.S. Cafiso, and S. McLaughlin. 1997. Kinetics of interaction of the myristoylated alanine-rich C kinase substrate, membranes, and calmodulin. J. Biol. Chem. 272:27167–27177.
Arbuzova, A., D. Murray, and S. McLaughlin. 1998. MARCKS, membranes and calmodulin: kinetics of their interaction. Biochim. Biophys. Acta. 1376:369–379.
Arbuzova, A., L. Wang, J. Wang, G. Hangyàs-Mihàlynè, D. Murray, B. Honig, and S. McLaughlin. 2000. Membrane binding of peptides containing both basic and aromatic residues. Biochemistry. 39:10330–10339.
Bezzerides, V.J., I.S. Ramsey, S. Kotecha, A. Greka, and D.E. Clapham. 2004. Rapid vesicular translocation and insertion of TRP channels. Nat. Cell Biol. 6:709–720.
Black, D.J., Q.-K. Tran, and A. Persechini. 2004. Monitoring the total available calmodulin concentration in intact cells over the physiological range in free Ca2+. Cell Calcium. 35:415–425.
Blobel, C.P. 2005. Adams: key components in EGFR signaling and development. Nat. Rev. Mol. Cell Biol. 6:32–43.
Blume-Jensen, P., and T. Hunter. 2001. Oncogenic kinase signalling. Nature. 411:355–365.
Burgess, A.W., H.-S. Cho, C. Eigenbrot, K.M. Ferguson, T.P.J. Garrett, D.J. Leahy, M.A. Lemmon, M.X. Sliwkowski, C.W. Ward, and S. Yokoyama. 2003. An open-and-shut case Recent insights into the activation of EGF/ErbB receptors. Mol. Cell. 12:541–552.
Carpenter, G. 2003. ErbB-4: mechanism of action and biology. Exp. Cell Res. 284:66–77.
Carpenter, G., L. King Jr., and S. Cohen. 1979. Rapid enhancement of protein phosphorylation in A-431 cell membrane preparations by epidermal growth factor. J. Biol. Chem. 254:4884–4891.
Chen, X., and M.D. Resh. 2002. Cholesterol depletion from the plasma membrane triggers ligand-independent activation of the epidermal growth factor receptor. J. Biol. Chem. 277:49631–49637.
Cheyette, T.E., and D.J. Gross. 1991. Epidermal growth factor-stimulated calcium ion transients in individual A431 cells: initiation kinetics and ligand concentration dependence. Cell Regul. 2:827–840.
Couchet, C., G.N. Gill, J. Meisenhelder, J.A. Cooper, and T. Hunter. 1984. C-kinase phosphorylates the epidermal growth factor receptor and reduces its epidermal growth factor-stimulated tyrosine protein kinase activity. J. Biol. Chem. 259:2553–2558.
Countaway, J.L., P. McQuilkin, N. Girones, and R.J. Davis. 1990. Multisite phosphorylation of the epidermal growth factor receptor. J. Biol. Chem. 265:3407–3416.
Davis, R.J., N. Gironès, and M. Faucher. 1988. Two alternative mechanisms control the interconversion of functional states of the epidermal growth factor receptor. J. Biol. Chem. 263:5373–5379.
Ferguson, K.M. 2004. Active and inactive conformations of the epidermal growth factor receptor. Biochem. Soc. Trans. 32:742–745.
Ferguson, K.M., M.B. Berger, J.M. Mendrola, H.S. Cho, D.J. Leahy, and M.A. Lemmon. 2003. EGF activates its receptor by removing interactions that autoinhibit ectodomain dimerization. Mol. Cell. 11:507–517.
Friedman, B., J. van Amsterdam, H. Fujiki, and M.R. Rosner. 1989. Phosphorylation at threonine-654 is not required for negative regulation of the epidermal growth factor receptor by non-phorbol tumor promoters. Proc. Natl. Acad. Sci. USA. 86:812–816.
Gambhir, A., G. Hangyás-Mihályné, I. Zaitseva, D.S. Cafiso, J. Wang, D. Murray, S. Pentyala, S.O. Smith, and S. McLaughlin. 2004. Electrostatic sequestration of PIP2 on phospholipid membranes by basic/aromatic regions of proteins. Biophys. J. 86:2188–2207.
Gotoh, N., A. Tojo, M. Hino, Y. Yazaki, and M. Shibuya. 1992. A highly conserved tyrosine residue at codon 845 within the kinase domain is not required for the transforming activity of human epidermal growth factor receptor. Biochem. Biophys. Res. Commun. 186:768–774.
Gschwind, A., E. Zwick, N. Prenzel, M. Leserer, and A. Ullrich. 2001. Cell communication networks: epidermal growth factor receptor transactivation as the paradigm for interreceptor signal transmission. Oncogene. 20:1594–1600.
Gschwind, A., O.M. Fischer, and A. Ullrich. 2004. The discovery of receptor tyrosine kinases: targets for cancer therapy. Nat. Rev. Cancer. 4:361–370.
Hendriks, B.S., G. Orr, A. Well, H.S. Wiley, and D.A. Lauffenburger. 2005. Parsing ERK activation reveals quantitatively equivalent contributions from epidermal growth factor receptor and HER2 in human mammary epithelial cells. J. Biol. Chem. 280:6157–6169.
Holthuis, J.C.M., and T.P. Levine. 2005. Lipid traffic: floppy drives and a superhighway. Nat. Rev. Mol. Cell Biol. 6:209–220.
Hubbard, S.R. 2004. Juxtamembrane autoinhibition in receptor tyrosine kinases. Nat. Rev. Mol. Cell Biol. 5:464–470.
Hubbard, S.R., and J.H. Till. 2000. Protein tyrosine kinase structure and function. Annu. Rev. Biochem. 69:373–398.
Hughes, A.R., G.S. Bird, J.F. Obie, O. Thastrup, and J.W. Putney Jr. 1991. Role of inositol (1,4,5)trisphosphate in epidermal growth factor-induced Ca2+ signaling in A431 cells. Mol. Pharmacol. 40:254–262.
Hunter, T., N. Ling, and J.A. Cooper. 1984. Protein kinase C phosphorylation of the EGF receptor at a threonine residue close to the cytoplasmic face of the plasma membrane. Nature. 311:480–483.
Huse, M., and J. Kuriyan. 2002. The conformational plasticity of protein kinases. Cell. 109:275–282.
Ichinos, J., M. Murata, T. Yanagida, and Y. Sako. 2004. EGF signaling amplification induced by dynamic clustering of EGFR. Biochem. Biophys. Res. Commun. 324:1143–1149.
Jorissen, R.N., F. Walker, N. Pouliot, T.P.J. Garrett, C.W. Ward, and A.W. Burgess. 2003. Epidermal growth factor receptor: mechanisms of activation and signalling. Exp. Cell Res. 284:31–53.
Kholodenko, B.N., O.V. Demin, G. Moehren, and J.B. Hoek. 1999. Quantification of short term signaling by the epidermal growth factor receptor. J. Biol. Chem. 274:30169–30181.
Kim, S.A., K.G. Heinze, M.N. Waxham, and P. Schwille. 2004. Intracellular calmodulin availability accessed with two-photon cross-correlation. Proc. Natl. Acad. Sci. USA. 101:105–110.
King, C.R., I. Borrello, L. Porter, P. Comoglio, and J. Schlessinger. 1989. Ligand-independent tyrosine phosphorylation of EGF receptor and the erbB-2/neu proto-oncogene product is induced by hyperosmotic shock. Oncogene. 4:13–18.
Klapper, L.N., M.H. Kirschbaum, M. Sela, and Y. Yarden. 2000. Biochemical and clinical implications of the ErbB/HER signaling network of growth factor receptors. Adv. Cancer Res. 77:25–79.
Landau, M., S.J. Fleishman, and N. Ben-Tal. 2004. A putative mechanism for downregulation of the catalytic activity of the EGF receptor via direct contact between its kinase and C-terminal domains. Structure (Camb.). 12:2265–2275.
Li, H., M.J. Ruano, and A. Villalobo. 2004a. Endogenous calmodulin interacts with the epidermal growth factor receptor in living cells. FEBS Lett. 559:175–180.
Li, H., J. Sanchez-Torres, A. Del Carpio, V. Salas, and A. Villalobo. 2004b. The ErebB2/Neu/HER2 receptor is a new calmodulin-binding protein. Biochem. J. 381:257–266.
Li, W., L. Tsiokas, S.C. Sansom, and R. Ma. 2004c. Epidermal growth factor activates store-operated Ca2+ channels through an inositol 1,4,5-trisphosphate-independent pathway in human glomerular mesangial cells. J. Biol. Chem. 279:4570–4577.
Martin-Nieto, J., and A. Villalobo. 1998. The human epidermal growth factor receptor contains a juxtamembrane calmodulin-binding site. Biochemistry. 37:227–236.
Matilla, E., T. Pellinen, J. Nevo, K. Vuoriluoto, A. Arjonen, and J. Evaska. 2004. Negative regulation of EGFR signaling through integrin-11-mediated activation of protein tyrosine phosphatase TCPTP. Nat. Cell Biol. 7:78–85.
Matsuda, M., H.F. Paterson, R. Rodriguez, A.C. Fensome, M.V. Ellis, K. Swann, and M. Katan. 2001. Real time fluorescence imaging of PLC translocation and its interaction with the epidermal growth factor receptor. J. Cell Biol. 153:599–612.
McLaughlin, S. 1989. The electrostatic properties of membranes. Annu. Rev. Biophys. Biophys. Chem. 18:113–136.
Moehren, G., N. Markevich, O. Demin, A. Kiyatkin, I. Goryanin, J.B. Hoek, and B.N. Kholodenko. 2002. Temperature dependence of the epidermal growth factor receptor signaling network can be accounted for by a kinetic model. Biochemistry. 41:306–320.
Murasawa, S., Y. Mori, Y. Nozana, N. Gotoh, M. Shibuya, H. Masaki, K. Maruyama, Y. Tsutsumi, Y. Moriguchi, Y. Shibazaki, et al. 1998. Angiotensin II type 1 receptor-induced extracellular signal-regulated protein kinase activation is mediated by Ca2+/calmodulin-dependent transactivation of epidermal growth factor receptor. Circ. Res. 82:1338–1348.
Murray, D., A. Arbuzova, B. Honig, and S. McLaughlin. 2002. The role of electrostatic and nonpolar interactions in the associations of peripheral proteins with membranes. Curr. Top. Membranes. 52:277–307.
Nojiri, S., and J.B. Hoek. 2000. Suppression of epidermal growth factor-induced phospholipase C activation associated with actin rearrangement in rat hepatocytes in primary culture. Hepatology. 32:947–957.
Ogiso, H., R. Ishitani, O. Nureki, S. Fukai, M. Yamanaka, J.H. Kim, K. Saito, A. Sakamoto, M. Inoue, M. Shirouzu, and S. Yokoyama. 2002. Crystal structure of the complex of human epidermal growth factor and receptor extracellular domains. Cell. 110:775–787.
Osawa, M., M.B. Swindells, J. Tanikawa, T. Tanaka, T. Mase, T. Furuya, and M. Ikura. 1998. Solution structure of calmodulin-W-7 complex: the basis of diversity in molecular recognition. J. Mol. Biol. 276:165–176.
Paez, J.G., A.J. Pasi, J.C. Lee, S. Tracy, H. Grenlich, S. Gabriel, P. Herman, F.J. Kaye, N. Lindeman, T.J. Boggon, et al. 2004. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science. 304:1497–1500.
Papin, J.A., T. Hunter, B.O. Palsson, and S. Subramaniam. 2005. Reconstruction of cellular signaling networks and analysis of their properties. Nat. Rev. Mol. Cell Biol. 6:99–111.
Pandiella, A., L. Beguinot, T.J. Velu, and J. Meldolesi. 1988. Transmembrane signalling at epidermal growth factor receptors overexpressed in NIH 3T3 cells. Phosphoinositide hydrolysis, cytosolic Ca2+ increase and alkalinization correlate with epidermal-growth-factor-induced cell proliferation. Biochem. J. 254:223–228.
Persechini, A., and P.M. Stemmer. 2002. Calmodulin is a limiting factor in the cell. Trends Cardiovasc. Med. 12:32–37.
Prenzel, N., E. Zwick, H. Daub, M. Leserer, R. Abraham, C. Wallasch, and A. Ullrich. 1999. EGF receptor transactivation by G-protein-coupled receptors requires metalloproteinase cleavage of proHB-EGF. Nature. 402:884–888.
Rakhilin, S.V., P.A. Olson, A. Nishi, N.N. Starkova, A.A. Fienberg, A.C. Nairn, D.J. Surmeier, and P. Greengard. 2004. A network of control mediated by regulator of calcium/calmodulin-dependent signaling. Science. 306:698–701.
Reynolds, A.R., C. Tischeer, P.J. Verveer, O. Rocks, and P.I.H. Bastiaens. 2003. EGFR activation coupled to inhibition of tyrosine phosphatases causes lateral signal propagation. Nat. Cell Biol. 5:447–453.
Rodriguez, I., M. Kaszkin, A. Holloschi, K. Kabsch, M.M. Marques, X. Mao, and A. Alonso. 2002. Hyperosmotic stress induces phosphorylation of cytosolic phospholipase A2 in HaCaT cells by an epidermal growth factor receptor-mediated process. Cell. Signal. 14:839–848.
Roepstorff, K., P. Thomsen, K. Samndvig, and B. van Deurs. 2002. Sequestration of epidermal growth factor receptors in non-caveolar lipid rafts inhibits ligand binding. J. Biol. Chem. 277:18954–18960.
Rusu, L., A. Gambhir, S. McLaughlin, and J. Radler. 2004. Fluorescence correlation spectroscopy studies of peptide and protein binding to phospholipid vesicles. Biophys. J. 87:1044–1053.
Schlessinger, J. 2000. Cell signaling by receptor tyrosine kinases. Cell. 103:211–225.
Schlessinger, J. 2003. Signal transduction. Autoinhibition control. Science. 300:750–752.
Shapiro, M.S. 2004. Why biophysicists make models: quantifying modulation of the M current. J. Gen. Physiol. 123:657–662.
Simons, K., and W.L.C. Vaz. 2004. Model systems, lipid rafts, and cell membranes. Annu. Rev. Biophys. Biomol. Struct. 33:269–295.
Stamos, J., M.X. Sliwkowski, and C. Eigenbrot. 2002. Structure of the epidermal growth factor receptor kinase domain alone and in complex with a 4-anilinoquinazoline inhibitor. J. Biol. Chem. 277:46265–46272.
Sordella, R., D.W. Bell, D.A. Haber, and J. Settleman. 2004. Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways. Science. 305:1163–1167.
Tonks, N. 2003. PTP1B: from the sidelines to the front lines! FEBS Lett. 546:140–148.
Ullrich, A., L. Coussens, J.S. Hayflick, T.S. Dull, A. Gray, A.W. Tam, J. Lee, Y. Yarden, T.A. Liberman, J. Schlessinger, et al. 1984. Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. Nature. 309:418–425.
Yarden, Y., and M.X. Sliwkowski. 2001. Untangling the ErbB signalling network. Nat. Rev. Mol. Cell Biol. 2:127–137.
Wang, J., A. Gambhir, G. Hangyás-Mihályné, D. Murray, U. Golebiewska, and S. McLaughlin. 2002. 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. J. Biol. Chem. 277:34401–34412.
Wang, X.-J., H.-J. Liao, A. Chattopadhyay, and G. Carpenter. 2001. EGF-dependent translocation of green fluorescent protein-tagged PLC-1 to the plasma membrane and endosomes. Exp. Cell Res. 267:28–36.
Wang, Y., and Z. Wang. 2003. Regulation of EGF-induced phospholipase C-1 translocation and activation by its SH2 and PH domains. Traffic. 4:618–630.
Welsh, J.G., G.N. Gill, M.G. Rosenfeld, and A. Wells. 1991. A negative feedback loop attenuates EGF-induced morphological changes. J. Cell Biol. 114:533–543.
Westhover, E.J., D.F. Cover, H.L. Brockman, R.E. Brown, and L.J. Pike. 2003. Cholesterol depletion results in site-specific increases in epidermal growth factor receptor phosphorylation due to membrane level effects. J. Biol. Chem. 278:51125–51133.
Wiley, H.S., S.Y. Shvartsman, and D.A. Lauffenburger. 2003. Computational modeling of the EGF-receptor system: a paradigm for systems biology. Trends Cell Biol. 13:43–50.
Wong, W., and J.D. Scott. 2004. AKAP signaling complexes: focal points in space and time. Nat. Rev. Mol. Cell Biol. 5:959–970.
Wood, E.R., A.T. Truesdale, O.B. McDonald, D. Yuan, A. Hassell, S.H. Dickerson, B. Ellis, C. Pennisi, E. Horne, K. Lackey, et al. 2004. A unique structure for epidermal growth factor receptor bound to GW572016 (Lapatinib). Cancer Res. 64:6652–6659.
Zwick, E., P.O. Hackel, N. Prenzel, and A. Ullrich. 1999. The EGF receptor as central transducer of heterologous signalling systems. Trends Pharmacol. Sci. 20:408–412.(Stuart McLaughlin, Steven)