Address correspondence to Stuart McLaughlin: Stuart.McLaughlin{at}StonyBrook.edu
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INTRODUCTION |
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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(979991), 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
Fig. 1 illustrates our model; the cartoons (Fig. 1, A and B) focus on the 40-residue JM domain (residues 645682 in ErbB1) between the helical transmembrane (TM) and structured PTK domains. Fig. 1 C shows the sequence of the ErbB1 JM domain using color coding to indicate the amino acids that can interact with the bilayer: basic (R and K) residues are blue, acidic residues (E) are red, and hydrophobic residues (F, L, I, and V) are green. The cytoplasmic leaflet of a mammalian cell plasma membrane typically contains 1530% monovalent acidic phospholipid, mainly phosphatidylserine (PS), which produces a negative surface potential of 30 to 40 mV (McLaughlin, 1989
). Hence the membrane bilayer attracts blue and green residues through electrostatic and hydrophobic interactions, respectively, and electrostatically repels red residues.
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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 645660 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 (
10100 s1).
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.
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MATERIALS AND METHODS |
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All peptides were obtained from American Peptide Co., Inc. Each peptide was blocked with an acetyl group at its NH2 terminus and an amide group at its COOH terminus. We performed binding measurements with peptides corresponding to the regions of ErbB receptors shown in Fig. S6 (available at http://www.jgp.org/cgi/content/full/jgp.200509274/DC1); the peptides had an extra Cys group at the NH2 terminus, which permitted covalent attachment of either a radioactive (NEM) or fluorescent (acrylodan for stop flow, Texas red for FRET, Alexa488 for FCS measurements) probes as described elsewhere (Wang et al., 2002, and references therein). We used peptides without Cys for the zeta potential and surface pressure measurements. Labeled peptides were purified by high pressure liquid chromatography and MALDI-time-of-flight mass spectroscopy. We obtained similar results (see Fig. 4; Table I) with bovine brain (Sigma-Aldrich and Calbiochem) and human brain (Calbiochem) calmodulin, although different samples varied approximately twofold in their affinity for a given peptide.
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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 lipidchloroform 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(645660) 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(645660) 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(645660) 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(645660) 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(645660) 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(645660) 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
The supplemental material for this paper comprises eight figures (available at http://www.jgp.org/cgi/content/full/jgp.200509274/DC1). Fig. S1 shows how the binding of the ErbB1 JM peptide depends on the mole fraction of acidic lipid in the membrane. Fig. S2 shows FRET between the ErbB1 JM peptide and PIP2. Fig. S3 shows the effect of the ErbB1 JM peptide on PLC-catalyzed PIP2 hydrolysis. Fig. S4 shows an atomic model of membrane, adsorbed ErbB1 JM peptide and calcium/calmodulin, and illustrates the predicted electrostatic potentials of the membrane and molecules. Fig. S5 shows the electrostatic potential adjacent to a complex of calmodulin and a peptide similar to the ErbB1 JM domain. Fig. S6 shows ErbB family members share a common basic/hydrophobic JM region. Fig. S7 shows the kinase domains of ErbB family members have a positively charged face in common. Fig. S8 shows the patterns of ErbB1 phosphorylation predicted by the model under different conditions.
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RESULTS |
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![]() | (1) |
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ErbB1(645660) Laterally Sequesters PIP2
When the 645660 region of ErbB1 is bound to the bilayer component of the plasma membrane, it produces a local positive electrostatic potential that will attract multivalent acidic lipids, even when monovalent acidic lipids are present in excess (see Fig. S4, available at http://www.jgp.org/cgi/content/full/jgp.200509274/DC1). FRET and PLC activity measurements demonstrate directly that membrane-bound ErbB1(645660) laterally sequesters PIP2, even in membranes comprising physiological levels of both PS and PIP2 (Figs. S2 and S3).
Biological Experiments Consistent with our Autoinhibition Hypothesis
A quantitative comparison of three results from earlier experiments on cell membranes and data from peptide/phospholipid vesicle studies indicate that electrostatic interactions can explain ErbB autoinhibition. First, hyperosmotic shock stimulates tyrosine phosphorylation of ErbB1 and ErbB2 in the absence of ligand (King et al., 1989; Rodriguez et al., 2002
). The model predicts this effect because high salt should reduce the electrostatic attraction of the JM + PTK domains for the membrane; binding measurements show that increasing the salt concentration threefold reduces ErbB1 (645660) binding
500-fold (online supplemental material, available at http://www.jgp.org/cgi/content/full/jgp.200509274/DC1). Second, 1 mM Mn2+ or 10 mM Mg2+ activates ErbB1 in a broken cell preparation (Carpenter et al., 1979
). Our model also predicts this effect because these divalent cations bind to membranes containing acidic lipids, reducing the magnitude of the negative electrostatic potential; binding measurements show adding 1 mM Mn2+ or 10 mM Mg2+ reduces binding of ErbB1(645660) to 2:1 PC/PS vesicles by
100-fold or
1,000-fold, respectively. Third, 25 µM sphingosine, an amphipathic, membrane-permeable weak base, activates ErbB1 in WI-38 fibroblasts, provided the receptor is in an intact membrane (Davis et al., 1988
); in contrast, EGF can activate ErbB1 both in membranes and in solubilized form. The model predicts that amphipathic weak bases should reduce the negative charge on the inner leaflet of the bilayer and thus its electrostatic attraction for the basic JM region; our data demonstrate that 2 µM sphingosine reverses the charge (sign of the zeta potential, direction of the electrophoretic movement) of a 2:1 PC/PS vesicle and causes 75% of the ErbB1(645660) peptide to desorb from PC/PS vesicles (online supplemental material).
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(645660); 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]
10100 µM in a mammalian cell). Adding CaM in the absence of free Ca2+ does not affect ErbB1(645660) binding to the vesicles (filled circles). These data indicate that Ca/CaM binds to ErbB1(645660) 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(645660) membrane binding, incorporating the assumption that Ca/CaM and the membrane compete for the peptide:
![]() | (2) |
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Phosphorylation of Thr654 also decreases the affinity of ErbB1(645660) for Ca/CaM. Experiments similar to those shown in Fig. 4, but conducted with an ErbB1(645660) 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.
How rapidly can Ca/CaM remove the ErbB1 645660 region from a plasma membrane? We approached this question by making fluorescence stop flow measurements to determine how rapidly Ca/CaM can remove acrylodan-labeled ErbB1(645660) from model membranes that have a physiologically relevant fraction of acidic lipid, i.e., 85:15 PC/PS vesicles. The vesicles had 1% NBD-PS to quench the fluorescence. If is the measured time constant for moving a peptide from a vesicle to Ca/CaM, 1/
increases linearly from 3 s1 to 30 s1 as [Ca/CaM] increases to 5 µM. Put another way, adding 5 µM Ca/CaM reduces the lifetime of the peptide on the vesicles 10-fold, from
0.3 to 0.03 s. The transfer rate constant, defined as the slope of the (1/
) vs. [CaM] data, is 5 x 106 M1s1 for 85:15 PC/PS vesicles and 5 x 107 M1s1 for 90:10 PC/PS vesicles, a value close to the diffusion limited rate (
108 M1s1) at which Ca/CaM combines with other proteins in solution (online supplemental material, available at http://www.jgp.org/cgi/content/full/jgp.200509274/DC1).
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(645660) desorbs from the vesicles, presumably by ripping the peptide directly from the surface; we have discussed this mechanism elsewhere (Arbuzova et al., 1997, 1998
).
Figs. S4 and S5 (available at http://www.jgp.org/cgi/content/full/jgp.200509274/DC1) show, respectively, calculations of the electrostatic potential for an atomic model of Ca/CaM approaching a membrane-bound ErbB1(645660) and calculations of the electrostatic potential adjacent to Ca/CaM bound to a basic peptide. The atomic models illustrate how electrostatic attraction may guide Ca/CaM (net charge z = 16) to the membrane-associated JM region (z = +8), how electrostatic interactions may help rip the peptide from the surface (Arbuzova et al., 1998), and how the Ca/CaM-JM region complex (z = 8) may then be repelled from the membrane surface.
We performed binding measurements similar to those shown in Figs. 3 and 4 with peptides corresponding to the reversible membrane anchor region of other members of the ErbB family. The basic/hydrophobic nature of this region is moderately conserved (Fig. S6, available at http://www.jgp.org/cgi/content/full/jgp.200509274/DC1), and Table I shows they all bind to both membranes and Ca/CaM, suggesting that the electrostatic engine model may apply to activation of all four molecules. Moreover, the PTK core regions of all ErbB family members share a positively charged face (Fig. S7).
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 12 min, and then falls to 150 nM in the next 23 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, 2050 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.
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DISCUSSION |
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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 M1, but their Ca/CaM binding measurements employed 0.3 x 106 M of a GST-ErbB1(645660) construct; thus we interpret their measurements to mean KCaM > 3 x 106 M1, 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(645660) moves off a 85:15 PC/PS vesicle at a rate of 3 s1). 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 IIstimulated 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
Our model also suggests a new and potentially important function for ErbB in signal transduction. Binding of the reversible membrane anchor region to the negatively charged membrane should produce a local positive potential (see Fig. S4, available at http://www.jgp.org/cgi/content/full/jgp.200509274/DC1) that acts as a basin of attraction for multivalent acidic lipids such as PIP2. FRET and PLC hydrolysis experiments show that ErbB1(645660) laterally sequesters PIP2, even when the membrane includes a physiologically relevant 100-fold excess of monovalent acidic lipids (Figs. S2 and S3). PIP2 is the substrate of PLC- and PI3 kinase, enzymes that bind to the phosphorylated COOH-terminal regions of ErbB family members (Schlessinger, 2000
). Thus a corollary of our hypothesis is that ErbB family members act as scaffolding proteins (Wong and Scott, 2004
), binding both enzymes and their substrate. PLC cannot hydrolyze PIP2 sequestered by peptides corresponding to the ErbB JM basic cluster, however (Fig. S3); PIP2 must first be released from the basic cluster.
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(645660) peptides from PC/PS vesicles at rates of 10 and 100 s1 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 645660 JM region of ErbB1 and rip it off the plasma membrane is
100 s1.
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 M1/108 M1s1), 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 10100 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 108 cm2s1 (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 s1, 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 10100 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
Fig. S8 (available at http://www.jgp.org/cgi/content/full/jgp.200509274/DC1) illustrates how the model shown in Fig. 1 can be used to predict the time course of ErbB1 trans autophosphorylation after stimulation by EGF. For simplicity, we assume that only three factors affect phosphorylation: ligand-induced dimerization, which increases phosphorylation by a local concentration effect; phosphatases, which remove phosphates from ErbB1; and the calmodulin-dependent positive feedback mechanism shown in Fig. 1, which operates only when intracellular [Ca2+] is elevated. The plots show the predicted percent trans autophosphorylation as a function of time after addition of EGF in three different cases: permeabilized cells lacking both CaM and phosphatases, cells exposed to CaM inhibitors, and normal cells. Recent experimental results appear to agree well with the predictions. We stress, however, that the calculations in Fig. S8 represent a highly oversimplified scheme; for example, we make no attempt to incorporate the well documented endocytosis of activated ErbB1 and PLC-1 (Matsuda et al., 2001
; Wang et al., 2001
; Wang and Wang, 2003
). Endocytosis has been considered quantitatively in models for ErbB family activation by Lauffenburger and others (e.g., Wiley et al., 2003
; Hendriks et al., 2005
). We also acknowledge that other, more complex, quantitative models for short term signaling by ErbB1 (Kholodenko et al., 1999
; Moehren et al., 2002
) can account for the maximum in ErbB1 autophosphorylation by a different mechanism than the one we invoke in Fig. 1. The advantages of formulating quantitative models of signal transduction phenomena are discussed in a recent commentary entitled "Why biophysicists make models" (Shapiro, 2004
) and in Papin et al. (2005)
. In our view, the main advantage of these models (e.g., our Fig. 1; Kholodenko et al., 1999
; Moehren et al., 2002
; Wiley et al., 2003
; Landau et al., 2004
; Hendriks et al., 2005
), is that they make quantitative predictions and can thus be easily falsified or modified by future experiments.
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ACKNOWLEDGMENTS |
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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
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REFERENCES |
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